Processes Of Producing Ethanol Using A Fermentation Organism

Abstract

The invention relates to improved processes of producing ethanol from starch-containing material wherein saccharification and/or fermentation is done at a temperature below the initial gelatinization temperature in the presence of glucoamylase and alpha-amylase, and optionally a protease and/or a cellulolytic enzyme composition; wherein the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, and compositions comprising a Saccharomyces yeast strain of the invention and naturally occurring and/or non-naturally occurring components.

Description

TECHNICAL FIELD

The present invention relates to improved raw starch hydrolysis processes of producing ethanol from starch-containing materials using a fermenting organism. The present invention also relates to a Saccharomyces strain having improved ability to ferment sugars to ethanol, to methods for the production of Saccharomyces strains having improved ability to ferment sugars to ethanol, and the use of Saccharomyces yeast strains having improved ability to ferment sugars to ethanol in the production of ethanol. Finally the invention relates to compositions comprising a Saccharomyces yeast strain of the invention and a naturally occurring and/or a non-naturally occurring component.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND ART

Processes of producing ethanol from starch-containing material are well-known in the art and used commercially today. The production of ethanol as a bio-fuel has become a major industry, with in excess of 21 billion gallons of ethanol being produced worldwide in 2012.

When producing ethanol, starch is conventionally converted into dextrins using a liquefying enzyme (e.g., Bacillus alpha-amylase) at temperatures above the initial gelatinization temperature of starch. The generated dextrins are hydrolyzed into sugars using a saccharifying enzyme (e.g., glucoamylase) and fermented into the desired fermentation product using a fermenting organism such as a yeast strain derived from Saccharomyces cerevisiae. Typically hydrolysis and fermentation are done in a simultaneous saccharification and fermentation (SSF) step.

Another type of process is also used commercially today. Starch is converted into sugars by enzymes at temperatures below the initial gelatinization temperature of the starch in question and converted into ethanol by yeast, typically derived from Saccharomyces cerevisiae. This type of process is referred to as a raw starch hydrolysis (RSH) process, or alternatively a “one-step process” or “no cook” process.

Yeast which are used for production of ethanol for use as fuel, such as in the corn ethanol industry, require several characteristics to ensure cost effective production of the ethanol. These characteristics include ethanol tolerance, low by-product yield, rapid fermentation, and the ability to limit the amount of residual sugars remaining in the ferment. Such characteristics have a marked effect on the viability of the industrial process.

Yeast of the genus Saccharomyces exhibit many of the characteristics required for production of ethanol. In particular, strains of Saccharomyces cerevisiae are widely used for the production of ethanol in the fuel ethanol industry. Strains of Saccharomyces cerevisiae that are widely used in the fuel ethanol industry have the ability to produce high yields of ethanol under fermentation conditions found in, for example, the fermentation of corn mash. An example of such a strain is the yeast used in commercially available ethanol yeast product called Ethanol Red™.

Strains of Saccharomyces cerevisiae are used in the fuel ethanol industry to ferment sugars such as glucose, fructose, sucrose and maltose to produce ethanol via the glycolytic pathway. These sugars are obtained from sources such as corn and other grains, sugar juice, molasses, grape juice, fruit juices, and starchy root vegetables and may include the breakdown of cellulosic material into glucose.

Although strains of Saccharomyces cerevisiae currently used in the fuel ethanol industry are well suited to ethanol production, there is an increasing need for improvements in the efficiency of ethanol production owing to the increased demand for ethanol as a fuel, and the increased availability of starch in new strains of corn.

There is therefore a need for new strains of Saccharomyces capable of improving the efficiency of ethanol production in industrial scale fermentation.

Further, despite significant improvement of ethanol production processes over the past decade there is still a desire and need for providing further improved processes of producing ethanol from starch-containing material that, e.g., can provide a higher ethanol yield.

SUMMARY OF THE INVENTION

The invention concerns improved raw starch hydrolysis processes for producing ethanol using a fermenting organism. The invention also relates to yeast strains suitable for use in processes and methods of the invention as well as compositions comprising a yeast strain of the invention.

More specifically in a first aspect the invention relates to processes of producing ethanol from starch-containing material, such as granular starch, comprising:

• • (a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
  • (b) fermenting using a fermentation organism;
  • wherein
    • saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease; and
    • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

A raw starch hydrolysis process of the invention results in one or more, such as all, of the following improvements compared to a corresponding process carried out under the same conditions using Ethanol Red™ (“ER”) as the fermenting organism:

• • increased ethanol yield;
  • reduced glycerol level;
  • reduced lactic acid level;
  • faster fermentation kinetics.

Examples of suitable enzymes used, especially glucoamylases, alpha-amylases, proteases, cellulolytic enzyme compositions etc are described in the “Enzymes And Enzyme Blends Used In A Process Of The Invention” section below.

In a preferred embodiment the following enzymes are present and/or added in saccharification and/or fermentation: Trametes cingulata glucoamylase, preferably the one shown in SEQ ID NO: 12 herein and an alpha-amylase. In a preferred embodiment the alpha-amylase is a Rhizomucor pusillus alpha-amylase, preferably the Rhizomucor pusillus alpha-amylase with a linker and starch-binding domain, in particular the Aspergillus niger glucoamylase linker and starch-binding domain (SBD) shown in SEQ ID NO: 13 herein.

In a preferred embodiment the following enzymes are present and/or added in saccharification and/or fermentation: Gloeophyllum glucoamylase, in particular Gloeophyllum trabeum glucoamylase, preferably the one shown in SEQ ID NO: 18 herein, especially one having one or more of the following substitutions: S95P, A121P, especially S95P+A121P and an alpha-amylase. In a preferred embodiment the alpha-amylase is derived from Rhizomucor pusillus, preferably Rhizomucor pusillus alpha-amylase with a linker and starch-binding domain, in particular the Aspergillus niger glucoamylase linker and starch-binding domain (SBD), disclosed as V039 in Table 5 in WO 2006/069290 or shown as SEQ ID NO: 13 herein.

In another preferred embodiment the following enzymes are present and/or added in saccharification and/or fermentation: Gloeophyllum glucoamylase, in particular Gloeophyllum trabeum glucoamylase, preferably the one shown in SEQ ID NO: 18 herein, preferably one having one or more of the following substitutions: S95P, A121P, especially S95P+A121P, and an alpha-amylase, in particular one derived from Rhizomucor pusillus, preferably Rhizomucor pusillus alpha-amylase with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one shown in SEQ ID NO: 13 herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+143N.

In another preferred embodiment the following enzymes are present and/or added in saccharification and/or fermentation: Pycnoporus glucoamylase, in particular Pycnoporus sanguineus glucoamylase, preferably the one shown in SEQ ID NO: 17 herein and an alpha-amylase. In a preferred embodiment the alpha-amylase is derived from Rhizomucor pusillus, preferably with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290 or shown as SEQ ID NO: 13 herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.

In an embodiment a protease is present and/or added in saccharification and/or fermentation. In a preferred embodiment the protease is a metallo protease or a serine protease. In an embodiment the metallo protease is derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metallo protease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 herein.

In an embodiment a cellulolytic enzyme composition is present and/or added in saccharification and/or fermentation or simultaneous saccharification and fermentation (SSF).

In a preferred embodiment the cellulolytic enzyme composition is derived from Trichoderma reesei, preferably further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (e.g., SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 herein) and Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 8 herein), or a cellulolytic enzyme composition derived from Trichoderma reesei, preferably further comprising Penicillium emersonii GH61A polypeptide, e.g., the one disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, and Aspergillus fumigatus beta-glucosidase, e.g., the one disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, preferably a variant having one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y, Aspergillus fumigatus CBH1, e.g., the one disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein, and Aspergillus fumigatus CBH II, e.g., the one disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein.

In a preferred embodiment the glucoamylase to alpha-amylase ratio is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg EP glucoamylase: mg EP alpha-amylase).

In a preferred embodiment the total dose of glucoamylase and alpha-amylase is from 10-1,000 μg/g DS, such as from 50-500 μg/g DS, such as 75-250 μg/g DS.

In a preferred embodiment the total dose of cellulolytic enzyme composition added is from 10-500 μg/g DS, such as from 20-400 μg/g DS, such as 20-300 μg/g DS.

In an embodiment the dose of protease added is from 1-200 μg/g DS, such as from 2-100 μg/g DS, such as 3-50 μg/g DS.

In a preferred embodiment saccharification step (a) and fermentation step (b) are carried out simultaneously.

A second aspect provides a Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V14/004037 (Saccharomyces cerevisiae MBG4851), or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

A third aspect provides a method of producing a Saccharomyces strain having defining characteristics of strain V14/004037 (Saccharomyces cerevisiae MBG4851), comprising:

(a) providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is strain V14/004037 or a derivative of strain V14/004037;
(b) culturing the first yeast strain and the second yeast strain under conditions which permit combining of DNA between the first yeast strain and the second yeast strain;
(c) screening or selecting for a derivative of strain V14/004037;
(d) optionally repeating steps (b) and (c) with the screened or selected strain from step (c) as the first and/or second strain, until a derivative is obtained which exhibits defining characteristics of strain V14/004037.

A fourth aspect provides a Saccharomyces strain produced by the method of the third aspect.

A fifth aspect provides a method of producing ethanol, comprising incubating a strain of the second or fourth aspect with a substrate comprising a fermentable sugar under conditions which promote fermentation of the fermentable sugar to produce ethanol.

A sixth aspect provides use of a strain of the second or fourth aspect in the production of ethanol.

A seventh provides a method of producing distiller's grain, comprising:

(a) incubating a Saccharomyces strain of the second or fourth aspect with a substrate comprising fermentable sugar under conditions which allow fermentation of the fermentable sugar to produce ethanol and distiller's grains;
(b) isolating the distiller's grains.

An eighth aspect provides distiller's grain produced by the method of the seventh aspect.

A ninth aspect provides use of a strain of the second or fourth aspect in the production of distiller's grains.

A tenth aspect provides use of a strain of the second or fourth aspect in the production of a Saccharomyces strain which exhibits one or more defining characteristics of strain V14/004037.

An eleventh aspect provides a composition comprising a Saccharomyces strain of the second or fourth aspect.

A twelfth aspect provides processes of using a Saccharomyces strain of the second or fourth aspect in a process of the first aspect.

Finally the invention relates to compositions comprising a Saccharomyces yeast strain of the invention and naturally occurring and/or non-naturally occurring components.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the ethanol titers during RSH fermentation.

FIG. 2 shows the glycerol titers during RSH fermentation.

FIG. 3 shows the lactic acid levels during RSH Bioreactor Fermentations.

DETAILED DESCRIPTION OF THE INVENTION

The invention concerns improved raw starch hydrolysis processes of producing ethanol from starch-containing material using a fermenting organism of the invention. A process for producing ethanol according to the invention is carried out as a raw starch hydrolysis (RSH) process. A raw starch hydrolysis process is a process where starch, typically granular starch, is converted into dextrins/sugars by raw starch degrading enzymes at temperatures below the initial gelatinization temperature of the starch in question and converted into ethanol by yeast, typically Saccharomyces cerevisiae. This type of process is often alternatively referred to as a “one-step process” or “no cook” process.

Specifically, the invention relates to processes of ethanol production from starch-containing material, such as granular starch, comprising:

• • (a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
  • (b) fermenting using a fermentation organism;
  • wherein
    • saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease; and
    • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

A raw starch hydrolysis process of the invention results in an increased ethanol yield compared to a corresponding process where Ethanol Red™ (“ER”) is used under the same conditions. See for instance, Example 1, table 2; Example 2, table 6; and Example 3, table 10).

A raw starch hydrolysis process of the invention results in a reduced glycerol level compared to a corresponding process where Ethanol Red™ (“ER”) is used under the same conditions. See for instance, Example 1, table 3; Example 2, table 7; and Example 3, table 11).

A raw starch hydrolysis process of the invention results in a reduced lactic acid level compared to a corresponding process where Ethanol Red™ (“ER”) is used under the same conditions. See for instance, Example 1, table 4; Example 2, table 8; and Example 3, table 12).

The process conditions according to the invention may be as described in any of Examples 1-3.

In processes of the invention the starch does not gelatinize as the process is carried out at temperatures below the initial gelatinization temperature of the starch in question.

The term “initial gelatinization temperature” means the lowest temperature at which starch gelatinization commences. In general, starch heated in water begins to gelatinize between about 50° C. and 75° C. The exact temperature of gelatinization depends on the specific starch and depends on the degree of cross-linking of the amylopectin. The initial gelatinization temperature can readily be determined by the skilled artisan. The initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In context of this invention the initial gelatinization temperature of a given starch-containing material may be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C., Starch/Stärke, Vol. 44 (12) pp. 461-466 (1992).

Therefore, according to the process of the invention ethanol is produced from un-gelatinized (i.e., uncooked), preferably milled grains, such as corn, or small grains such as wheat, oats, barley, rye, rice, or cereals such as sorghum. Examples of suitable starch-containing starting materials are listed in the section “Starch-Containing Materials”-section below.

In a preferred embodiment the enzymes may be added as one or more enzyme blends. According to the invention the fermentation product, i.e., ethanol, is produced without liquefying the starch-containing material. The process of the invention includes saccharifying (e.g., milled) starch-containing material, especially granular starch, below the initial gelatinization temperature, in the presence of at least a glucoamylase and an alpha-amylase and optionally a protease and/or a cellulolytic enzyme composition. The dextrins/sugars generated during saccharification can may according to the invention be simultaneously fermented into ethanol by a suitable fermenting organism, especially Saccharomyces cerevisiae MBG4851 or a fermenting organism having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having the defining characteristics of strain V14/004037, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037. See the “Fermenting Organisms”-section below.

In a preferred embodiment step (a) and step (b) are carried out simultaneously (i.e., one-step fermentation). However, step (a) and step (b) may also be carried our sequentially.

Before step (a) an aqueous slurry of starch-containing material, such as granular starch, having 10-55 wt.-% dry solids (DS), preferably 25-45 wt.-% dry solids, more preferably 30-40% dry solids of starch-containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants. Because the process of the invention is carried out below the initial gelatinization temperature and thus no significant viscosity increase takes place, high levels of stillage may be used, if desired. In an embodiment the aqueous slurry contains from about 1 to about 70 vol.-%, preferably 15-60% vol.-%, especially from about 30 to 50 vol.-% water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side-stripper water from distillation, or process water from other fermentation product plants, or combinations thereof, or the like.

In an embodiment backset, or another recycled stream, is added to the slurry before step (a), or to the saccharification (step (a)), or to the simultaneous saccharification and fermentation steps (combined step (a) and step (b)).

After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids in the starch-containing material are converted into a soluble starch hydrolysate.

A process of the invention is conducted at a temperature below the initial gelatinization temperature, which means that the temperature at which a separate step (a) is carried out typically lies in the range between 25-75° C., such as between 30-70° C., or between 45-60° C.

In a preferred embodiment the temperature during fermentation in step (b) or simultaneous saccharification and fermentation in steps (a) and (b) is between 25° C. and 40° C., preferably between 28° C. and 36° C., such as between 28° C. and 35° C., such as between 28° C. and 34° C., such as around 32° C.

In an embodiment of the invention fermentation or SSF is carried out for 30 to 150 hours, preferably 48 to 96 hours.

In an embodiment fermentation or SSF is carried out so that the sugar level, such as glucose level, is kept at a low level, such as below 6 wt.-%, such as below about 3 wt.-%, such as below about 2 wt.-%, such as below about 1 wt.-%., such as below about 0.5%, or below 0.25% wt.-%, such as below about 0.1 wt.-%. Such low levels of sugar can be accomplished by simply employing adjusted quantities of enzymes and fermenting organism. A skilled person in the art can easily determine which doses/quantities of enzyme and fermenting organism to use. The employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 wt.-%, such as below about 0.2 wt.-%.

The process of the invention may be carried out at a pH from 3 and 7, preferably from 3 to 6, or more preferably from 3.5 to 5.0.

The term “granular starch” means raw uncooked starch, i.e., starch in its natural form found in, e.g., cereal, tubers or grains. Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to around 50° C. to 75° C. the swelling may be reversible. However, at higher temperatures an irreversible swelling called “gelatinization” begins. The granular starch may be a highly refined starch, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure, or it may be a more crude starch-containing materials comprising (e.g., milled) whole grains including non-starch fractions such as germ residues and fibers.

The raw material, such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure and allowing for further processing. Examples of suitable particle sizes are disclosed in U.S. Pat. No. 4,514,496 and WO2004/081193 (incorporated by reference). Two processes are preferred according to the invention: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in production of, e.g., syrups. Both dry and wet milling is well known in the art of starch processing.

In an embodiment the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen. In a preferred embodiment starch-containing material is prepared by reducing the particle size of the starch-containing material, preferably by milling, such that at least 50% of the starch-containing material has a particle size of 0.1-0.5 mm.

According to the invention the enzymes are added so that the glucoamylase is present in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.

According to the invention the enzymes are added so that the alpha-amylase is present or added in an amount of 0.001 to 10 AFAU/g DS, preferably from 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.

According to the invention the enzymes are added so that the cellulolytic enzyme composition is present or added in an amount 1-10,000 micro grams EP/g DS, such as 2-5,000, such as 3 and 1,000, such as 4 and 500 micro grams EP/g DS.

According to the invention the enzymes are added so that the cellulolytic enzyme composition is present or added in an amount in the range from 0.1-100 FPU per gram total solids (TS), preferably 0.5-50 FPU per gram TS, especially 1-20 FPU per gram TS.

In an embodiment of the invention the enzymes are added so that the protease is present in an amount of 0.0001-1 mg enzyme protein per g DS, preferably 0.001 to 0.1 mg enzyme protein per g DS. Alternatively, the protease is present and/or added in an amount of 0.0001 to 1 LAPU/g DS, preferably 0.001 to 0.1 LAPU/g DS and/or 0.0001 to 1 mAU-RH/g DS, preferably 0.001 to 0.1 mAU-RH/g DS.

In an embodiment of the invention the enzymes are added so that the protease is present or added in an amount in the range 1-1,000 μg EP/g DS, such as 2-500 μg EP/g DS, such as 3-250 μg EP/g DS.

In a preferred embodiment ratio between glucoamylase and alpha-amylase is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg EP glucoamylase: mg EP alpha-amylase).

In a preferred embodiment the total dose of glucoamylase and alpha-amylase is according to the invention from 10-1,000 μg/g DS, such as from 50-500 μg/g DS, such as 75-250 μg/g DS.

In a preferred embodiment the total dose of cellulolytic enzyme composition added is from 10-500 μg/g DS, such as from 20-400 μg/g DS, such as 20-300 μg/g DS.

In an embodiment the dose of protease added is from 1-200 μg/g DS, such as from 2-100 μg/g DS, such as 3-50 μg/g DS.

Starch-Containing Materials

According to the process of the invention any suitable starch-containing starting material, including granular starch (raw uncooked starch), may be used. The starting material is generally selected based on the desired fermentation product. Examples of starch-containing starting materials, suitable for use in processes of the present invention, include cereal, tubers or grains. Specifically the starch-containing material may be corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, oats, rice, peas, beans, or sweet potatoes, or mixtures thereof. Contemplated are also waxy and non-waxy types of corn and barley.

In a preferred embodiment the starch-containing starting material is corn.

In a preferred embodiment the starch-containing starting material is wheat.

In a preferred embodiment the starch-containing starting material is barley.

In a preferred embodiment the starch-containing starting material is rye.

In a preferred embodiment the starch-containing starting material is milo.

In a preferred embodiment the starch-containing starting material is sago.

In a preferred embodiment the starch-containing starting material is cassava.

In a preferred embodiment the starch-containing starting material is tapioca.

In a preferred embodiment the starch-containing starting material is sorghum.

In a preferred embodiment the starch-containing starting material is rice,

In a preferred embodiment the starch-containing starting material is peas.

In a preferred embodiment the starch-containing starting material is beans.

In a preferred embodiment the starch-containing starting material is sweet potatoes.

In a preferred embodiment the starch-containing starting material is oats.

Fermenting Organisms

According to the process of the invention Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, is used for converting saccharified starch-containing material, such as granular starch, into ethanol.

In an embodiment the fermenting organism strain has properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, as it provides an increase in ethanol yield compared to Ethanol Red™ (“ER”) under the same process conditions.

In an embodiment the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, produces reduced levels of lactic acid compared to Ethanol Red™ (“ER”) under the same process conditions.

In an embodiment the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, produces reduced levels of glycerol compared to Ethanol Red™ (“ER”) under the same process conditions.

In an embodiment the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, has faster fermentation kinetics compared to Ethanol Red™ (“ER”) under the same process conditions.

In an embodiment the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, has one or more, such as all, of the following properties and defining characteristics:

increases ethanol yield compared to Ethanol Red™ (“ER”) under the same process conditions;

reduces the level of lactic acid compared to Ethanol Red™ (“ER”) under the same process conditions;

reduces the level of glycerol compared to Ethanol Red™ under the same process conditions;

has faster fermentation kinetics compared to Ethanol Red™ (“ER”) under the same process conditions.

The process conditions according to the invention may be as described in any of Examples 1-3.

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides an ethanol yield boost/increase over Ethanol Red™ (“ER”) of more than 0.5% (after 72 hours fermentation) determined using the process set-up and conditions used in Example 1.

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides an ethanol yield boost/increase over Ethanol Red™ (“ER”) of more than 1.0% (after 88 hours fermentation) determined using the process set-up and conditions used in Example 1.

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides an ethanol yield boost/increase of more than 1.0%, such as more than 2.0%, such as more than 3.0%, such as more than 4.0% over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 2 (i.e., 95 hours fermentation).

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides a reduction in lactic acid of more than 50%, such as more than 60% (after 72 hours fermentation) over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 1.

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides a reduction in lactic acid of more than 20%, such as more than 30% (after 95 hours fermentation) over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 2.

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides a reduction in lactic acid of more than 30%, such as more than 40% (after 72 hours fermentation using PsAMG) over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 3.

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides a reduction in lactic acid of more than 20%, such as more than 25%, such as more than 30% (after 95 hours fermentation) over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 2.

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides a reduction in glycerol levels of more than 10.0%, such as more than 15.0%, such as more than 20.0% over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 1 (i.e., after 72 hours fermentation).

In an embodiment of the invention the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, provides a reduction in glycerol levels of more than 5.0%, such as more than 10.0%, such as more than 12.0%, such as more than 14.0% over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 2 (i.e., after 95 hours fermentation).

Fermentation Medium

The term “fermentation medium” refers to the environment in which fermentation is carried out and which includes the fermentable substrate, that is, a carbohydrate source (e.g., glucose) that can be metabolized by the fermenting organism(s).

The fermentation medium may comprise nutrients and/or growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; urea, vitamins; and minerals, or combinations thereof.

Recovery

Subsequent to fermentation, the fermentation product (i.e., ethanol) may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product (i.e., ethanol). Alternatively the desired fermentation product (i.e., ethanol) may be extracted from the fermentation medium by micro or membrane filtration techniques. The fermentation product (i.e., ethanol) may also be recovered by stripping or other method well known in the art.

Enzymes and Enzyme Blends Used in a Process of the Invention

According to the invention a glucoamylase and an alpha-amylase are present and/or added in saccharification step (a) and/or fermentation step (b) (e.g., simultaneous saccharification and fermentation). Optionally a protease and/or a cellulolytic enzyme composition is(are) also present and/or added. Other enzymes such as pullulanases, pectinases, and/or trehalases may also be present and/or added.

Suitable and specifically contemplated enzymes and enzyme combinations (e.g., blends) are described below.

In an embodiment the following enzymes are present and/or added during saccharification and/or fermentation: Trametes glucoamylase, in particular Trametes cingulata glucoamylase, preferably the one shown in SEQ ID NO: 12 herein and an alpha-amylase.

In an embodiment the glucoamylase is derived from Trametes cingulata, such as the one shown in SEQ ID NO: 12 herein, or a glucoamylase selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 12 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 12 herein.

In an embodiment the following enzymes are present and/or added during saccharification and/or fermentation: Gloeophyllum glucoamylase, preferably Gloeophyllum trabeum glucoamylase, especially the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein and an alpha-amylase.

In an embodiment the glucoamylase is derived from Gloeophyllum trabeum, such as the one shown in SEQ ID NO: 18 herein, or a glucoamylase selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 18 herein.

In a preferred embodiment the Gloeophyllum trabeum glucoamylase, such as the one shown in SEQ ID NO: 18, has one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P (using SEQ ID NO: 18 for numbering).

The alpha-amylase used in a process of the invention is typically a fungal alpha-amylase, such as an acid fungal alpha-amylase. In a preferred embodiment the alpha-amylase is derived from Rhizomucor pusillus, preferably Rhizomucor pusillus alpha-amylase with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 13 herein.

In an embodiment the alpha-amylase is a Rhizomucor pusillus alpha-amylase or the Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD) shown in SEQ ID NO: 13 herein, especially one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering).

In an embodiment the alpha-amylase is selected from the group consisting of:

(i) an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 13 herein;
(ii) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 13 herein.

In an embodiment the following enzymes are present and/or added in saccharification and/or fermentation: Trametes glucoamylase, in particular Trametes cingulata glucoamylase, especially the Trametes cingulata glucoamylase shown in SEQ ID NO: 12 herein and an alpha-amylase derived from Rhizomucor pusillus, preferably with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), especially the one disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 13 herein.

In an embodiment the following enzymes are present and/or added in saccharification and/or fermentation: Gloeophyllum glucoamylase, in particular Gloeophyllum trabeum glucoamylase, preferably the one shown in SEQ ID NO: 18 and an alpha-amylase derived from Rhizomucor pusillus, preferably with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), such as the one disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 13 herein.

In another preferred embodiment the enzymes present and/or added comprises the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein having one or more of the following substitutions: S95P, A121P, especially S95P+A121P (using SEQ ID NO: 13 herein for numbering) and the alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one shown in SEQ ID NO: 13 herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N (using SEQ ID NO: 13 for numbering).

In an embodiment the following enzymes are present and/or added in saccharification and/or fermentation: the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 and the Rhizomucor pusillus alpha-amylase with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), especially the one shown in SEQ ID NO: 13 herein.

In an especially preferred embodiment the enzymes present and/or added in saccharification and or fermentation comprises a Pycnoporus sanguineus glucoamylase, such as the one shown in SEQ ID NO: 17 herein and the alpha-amylase derived from Rhizomucor pusillus with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one shown in SEQ ID NO: 13 herein, especially one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.

The enzymes present and/or added in saccharification and/or fermentation in a process of the invention comprise i) glucoamylase and ii) alpha-amylase; and may optionally further comprise iii) a cellulolytic enzyme composition and/or iv) a protease.

In an embodiment the protease is a metallo protease, preferably derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metallo protease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 herein.

In an embodiment the protease is selected from the group consisting of:

(i) a protease comprising the mature polypeptide of SEQ ID NO: 3 herein;
(ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 3 herein.

In an especially preferred embodiment the enzymes present and/or added in saccharification and/or fermentation comprises a Trametes glucoamylase, in particular Trametes cingulata glucoamylase, especially the one shown in SEQ ID NO: 12 herein and the alpha-amylase, in particular one derived from Rhizomucor pusillus with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one shown in SEQ ID NO: 13 herein, preferably having one or more of the following substitutions: G128D, D143N, especially G128D+D143N, and further a cellulolytic enzyme composition, in particular one derived from Trichoderma reesei, preferably further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 8 herein); or a cellulolytic enzyme composition, in particular one derived from Trichoderma reesei, preferably further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, preferably a variant having one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y, Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein.

In an especially preferred embodiment the enzymes present and/or added in saccharification and/or fermentation comprises Gloeophyllum glucoamylase, in particular Gloeophyllum trabeum glucoamylase, preferably the one shown in SEQ ID NO: 18 herein, preferably having one or more of the following substitutions: S95P, A121P, especially S95P+A121P and the alpha-amylase, in particular one derived from Rhizomucor pusillus with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one shown in SEQ ID NO: 13 herein, preferably having one or more of the following substitutions: G128D, D143N, especially G128D+D143N, and further a cellulolytic enzyme composition derived from Trichoderma reesei, preferably further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 8 herein); or a cellulolytic enzyme composition, in particular one derived from Trichoderma reesei, preferably further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, preferably a variant having one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y, Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein.

In an especially preferred embodiment the enzymes present and/or added in saccharification and/or fermentation according to the invention comprises a Pycnoporus glucoamylase, in particular a Pycnoporus sanguineus glucoamylase, preferably the one shown in SEQ ID NO: 17 herein and the alpha-amylase, in particular one derived from Rhizomucor pusillus with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one shown in SEQ ID NO: 13 herein, preferably having one or more of the following substitutions: G128D, D143N, especially G128D+D143N, and further a cellulolytic enzyme composition, in particular one derived from Trichoderma reesei, preferably further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 8 herein); or a cellulolytic enzyme composition, in particular one derived from Trichoderma reesei, preferably further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, preferably a variant having one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y, Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein.

In a preferred embodiment a cellulolytic enzyme composition is one described below in the “Cellulolytic Enzyme Compositions”-section.

The cellulolytic enzyme composition may be added in the process of the invention at the same time as the glucoamylase and alpha-amylase. According to the invention the enzymes, e.g., in the form of an enzyme composition, are added to the saccharification and/or fermentation, preferably simultaneous saccharification and fermentation (i.e., one-step process). It should be understood that the enzymes may also be added individually or as two, three, four or more enzyme compositions. In an embodiment the glucoamylase and alpha-amylase are added as one blend composition and the optional cellulolytic enzyme composition and optional protease are added separately. In another embodiment the glucoamylase, the alpha-amylase, and the cellulolytic enzyme composition are added as one enzyme composition and the optional protease is added separately. All enzymes may also in one embodiment be added as one enzyme composition comprising a glucoamylase, an alpha-amylase, a cellulolytic enzyme composition and/or a protease, and optionally other enzymes including pullulanase, trehalase and/or pectinase, such as pectin lyase or polygalacturonase.

Other enzymes may also be present. Specifically contemplated enzymes are described further below.

Glucoamylase

The glucoamylase used in a process of the invention may be of any origin, such as of bacterial or fungal origin. Fungal glucoamylases are preferred.

In an embodiment the glucoamylase may be one derived from a strain of Trametes, such as a strain of Trametes cingulata (SEQ ID NO: 12 herein); or a strain of Pachykytospora, such as a strain of Pachykytospora papyracea; or a strain of Leucopaxillus, such as a strain of Leucopaxillus giganteus (all disclosed in WO 2006/069289).

In an embodiment the glucoamylase is derived from a strain of Tramates, such as a strain of Trametes cingulata.

In a preferred embodiment the glucoamylase, such as one derived from a strain of Trametes cingulata, is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 12 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 12 herein.

In an embodiment the glucoamylase is from a strain of Aspergillus, preferably Aspergillus niger, Aspergillus awamori, or Aspergillus oryzae; or a strain of Trichoderma, preferably Trichoderma reesei; or a strain of Talaromyces, preferably Talaromyces emersonii (SEQ ID NO: 11 herein).

In an embodiment the glucoamylase is derived from a strain of Talaromyces, such as a strain of Talaromyces emersonii.

In an embodiment the glucoamylase, such as one derived from a strain of Talaromyces emersonii, is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 11 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 11 herein.

In another embodiment the glucoamylase is derived from a strain of Penicillium, such as a strain of Penicillium oxalicum.

In an embodiment the glucoamylase, such as one derived from a strain of Penicillium oxalicum, is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 16 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 16 herein.

In an embodiment the glucoamylase is derived from a strain of Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, such as one disclosed in WO 2011/068803 as any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16. In a preferred embodiment the glucoamylase is SEQ ID NO: 2 in WO 2011/068803 or SEQ ID NO: 4 herein. In another embodiment the glucoamylase is SEQ ID NO: 18 in WO 2011/068803 (i.e., Gloeophyllum trabeum glucoamylase) or the mature part of the glucoamylase shown as SEQ ID NO: 3 in WO2014/177546 (hereby incorporated by reference) and SEQ ID NO: 18 herein.

In a preferred embodiment the glucoamylase, such as one derived from a strain of Gloeophyllum sepiarium, is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 4 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 4 herein.

In a further embodiment the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus, such as a strain described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6). In a preferred embodiment the glucoamylase is the one shown in SEQ ID NO: 4 in WO 2011/066576 or SEQ ID NO: 17 herein.

In a preferred embodiment the glucoamylase, such as one derived from a strain of Pycnoporus sanguineus, is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 17 herein.

Contemplated are also glucoamylases which exhibit a high identity to any of the above-mentioned glucoamylases, i.e., at least 60%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to any one of the mature parts of the enzyme sequences mentioned above.

In a preferred embodiment the glucoamylase, such as one derived from a strain of Gloeophyllum trabeum, is selected from the group consisting of:

(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 18 herein.

In a preferred embodiment the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 has one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P. In a preferred embodiment the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 has one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P (using SEQ ID NO: 18 herein for numbering). All Gloeophyllum trabeum glucoamylase variants disclosed in co-pending PCT application # PCT/EP2014/058692 is hereby incorporated by reference.

A glucoamylase variant may comprise an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 18.

Alpha-Amylase

The alpha-amylase used in a process of the invention may be of any origin, such as of fungal or bacterial origin. In a preferred embodiment the alpha-amylase is an acid alpha-amylase, such as an acid fungal alpha-amylase, i.e., having a pH optimum below pH 7.

In an embodiment the alpha-amylase may be derived from a strain of the genus Rhizomucor, preferably a strain the Rhizomucor pusillus, such as the one shown in SEQ ID NO: 3 in WO 2013/006756 (see e.g., Table 1 in Example 1 hereby incorporated by reference), or the genus Meripilus, preferably a strain of Meripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from a Rhizomucor pusillus, such as one with a linker and starch-binding domain (SBD), preferably Aspergillus niger glucoamylase linker and starch-binding domain (SBD), disclosed as V039 in Table 5 in WO 2006/069290 (incorporated by reference) or SEQ ID NO: 13 herein.

In a preferred embodiment the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), disclosed in WO 2013/006756 (incorporated by reference) or SEQ ID NO: 13 herein.

In an embodiment the Rhizomucor pusillus alpha-amylase or the Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD) has at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C, especially G128D+D143N (using SEQ ID NO: 13 herein for numbering).

In an embodiment the Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), is selected from the group consisting of:

(i) an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 13 herein;
(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 13 herein.

In a preferred embodiment the alpha-amylase is a variant of the Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), wherein the alpha-amylase variant comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, but less than 100% to the mature polypeptide of SEQ ID NO: 13 herein.

In a preferred embodiment the alpha-amylase variant has one of the above mentioned substitutions, such as: G128D, Y141W, D143W or K192R.

In a preferred embodiment the alpha-amylase (using SEQ ID NO: 13 herein for numbering) has the following substitutions: Y141W+D143N.

In a preferred embodiment the alpha-amylase has the following substitutions: G128D+Y141W+D143N.

In a preferred embodiment the alpha-amylase has the following substitutions: G128D+Y141W+D143N+K192R;

In a preferred embodiment the alpha-amylase has the following substitutions: G128D+D143N (using SEQ ID NO: 13 for numbering).

A variant may comprise an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 13.

Protease

The enzymes present and/or added to saccharification and/or fermentation may optionally further include a protease. The protease may be of any origin, such as fungal or bacterial origin.

In an embodiment the protease is of fungal origin.

In an embodiment the protease is a metallo protease derived from a strain of the genus Thermoascus, preferably a strain of Thermoascus aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670, such as the metallo protease disclosed as the mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature polypeptide of SEQ ID NO: 3 herein.

In an embodiment the protease, such as one derived from a strain of Thermoascus aurantiacus, is selected from the group consisting of:

(i) a protease comprising the mature polypeptide of SEQ ID NO: 3 herein;
(ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 3 herein.

In an embodiment the protease is of bacterial origin.

In an embodiment the protease is derived from a strain of Pyrococcus, such as a strain of Pyrococcus furiosus, such as the protease shown in SEQ ID NO: 1 in U.S. Pat. No. 6,358,726 or SEQ ID NO: 5 herein.

In an embodiment the protease, such as one derived from Pyrococcus furiosus, is selected from the group consisting of:

(i) a protease comprising the mature polypeptide of SEQ ID NO: 5 herein;
(ii) a protease comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 5 herein.

Cellulolytic Enzyme Compositions

The enzymes present and/or added to saccharification and/or fermentation may optionally further include a cellulolytic enzyme composition. The cellulolytic enzyme composition may consist of or comprise one or more cellulolytic enzymes. The cellulolytic enzyme composition may be of any origin. In a preferred embodiment the cellulolytic enzyme composition comprises cellulolytic enzymes of fungal origin.

In an embodiment the cellulolytic enzyme composition is derived from a strain of Trichoderma, such as Trichoderma reesei; or a strain of Humicola, such as Humicola insolens; or a strain of Chrysosporium, such as Chrysosporium lucknowense; or a strain of Penicillium, such as Penicillium decumbens. In a preferred embodiment the cellulolytic enzyme composition is derived from a strain of Trichoderma reesei.

The cellulolytic enzyme composition may comprise a beta-glucosidase, a cellobiohydrolase, and an endoglucanase.

In an embodiment the cellulolytic enzyme composition comprising one or more polypeptides selected from the group consisting of:

• • beta-glucosidase;
  • cellobiohydrolase I;
  • cellobiohydrolase II;
  • or a mixture thereof.

In a preferred embodiment the cellulolytic enzyme composition further comprises a GH61 polypeptide having cellulolytic enhancing activity. Cellulolytic enhancing activity is defined and determined as described in WO 2011/041397 (incorporated by reference).

The term “GH61 polypeptide having cellulolytic enhancing activity” means a GH61 polypeptide that enhances the hydrolysis of a cellulosic material by enzymes having cellulolytic activity. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in PCS (Pretreated Corn Stover), wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at 50° C. compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST™1.5 L (Novozymes A/S, Bagsværd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.

The cellulolytic enzyme composition comprises a beta-glucosidase, preferably one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 2002/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637 (see SEQ ID NOs: 74 or 76), or Aspergillus fumigatus, such as one disclosed in SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein; or an Aspergillus fumigatus beta-glucosidase variant disclosed in WO 2012/044915; or a strain of the genus a strain Penicillium, such as a strain of the Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei. In an embodiment the beta-glucosidase is from a strain of Aspergillus, such as a strain of Aspergillus fumigatus, such as Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 8 herein), or a variant thereof, which variant comprises one or more substitutions selected from the group consisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y; such as a variant thereof with the following substitutions:

• • F100D+S283G+N456E+F512Y;
  • L89M+G91L+I186V+I140V;
  • I186V+L89M+G91L+I140V+F100D+S283G+N456E+F512Y.

In an embodiment the parent beta-glucosidase has at least 60% identity, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to the mature polypeptide of SEQ ID NO: 8 herein.

In case the beta-glucosidase is a beta-glucosidase variant it has at least 60% identity, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, but less than 100% identity to the mature polypeptide of SEQ ID NO: 8 herein.

In case the cellulolytic enzyme composition comprises a GH61 polypeptide, it may be one derived from the genus Thermoascus, such as a strain of Thermoascus aurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO: 2 or SEQ ID NO: 9 herein; or one derived from the genus Thielavia, such as a strain of Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8 (hereby incorporated by reference); or one derived from a strain of Aspergillus, such as a strain of Aspergillus fumigatus, such as the one described in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2 (hereby incorporated by reference); or one derived from a strain from Penicillium, such as a strain of Penicillium emersonii, such as the one disclosed in WO 2011/041397 as SEQ ID NO: 2 or SEQ ID NO: 10 herein.

In a preferred embodiment the GH61 polypeptide, such as one derived from a strain of Thermoascus, is selected from the group consisting of:

(i) a GH61 polypeptide comprising the mature polypeptide of SEQ ID NO: 9 herein;
(ii) a GH61 polypeptide comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 9 herein.

In a preferred embodiment the GH61 polypeptide, such as one derived from a strain of Penicillium sp., is selected from the group consisting of:

(i) a GH61 polypeptide comprising the mature polypeptide of SEQ ID NO: 10 herein;
(ii) a GH61 polypeptide comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 10 herein.

In an embodiment the cellulolytic enzyme composition comprises a cellobiohydrolase I (CBH I), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the Cel7a CBHI disclosed in SEQ ID NO: 6 in WO 2011/057140 or SEQ ID NO: 6 herein, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.

In a preferred embodiment the cellobiohydrolase I, such as one derived from a strain of Aspergillus fumigatus, is selected from the group consisting of:

(i) a cellobiohydrolase I comprising the mature polypeptide of SEQ ID NO: 6 herein;
(ii) a cellobiohydrolase I comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 6 herein.

In an embodiment the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a cellobiohydrolase II (CBH II), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus; such as the one disclosed as SEQ ID NO: 7 herein or a strain of the genus Trichoderma, such as Trichoderma reesei, or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.

In a preferred embodiment cellobiohydrolase II, such as one derived from a strain of Aspergillus fumigatus, is selected from the group consisting of:

(i) a cellobiohydrolase II comprising the mature polypeptide of SEQ ID NO: 7 herein;
(ii) a cellobiohydrolase II comprising an amino acid sequence having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 7 herein.

In an embodiment the cellulolytic enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity and a beta-glucosidase.

In an embodiment the cellulolytic enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity derived from a strain of Penicillium, such as a strain of Penicillium emersonii, such as the one disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, and a beta-glucosidase.

In an embodiment the cellulolytic enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, and a CBHI.

In an embodiment the cellulolytic enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity derived from a strain of Penicillium, such as a strain of Penicillium emersonii, such as the one disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, a beta-glucosidase, and a CBHII.

In an embodiment the cellulolytic enzyme composition, comprised in an enzyme composition of the invention, comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, a CBHI, and a CBHII.

In an embodiment the cellulolytic enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity derived from a strain of Penicillium, such as a strain of Penicillium emersonii, such as the one disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, a beta-glucosidase, a CBHI, and a CBHII.

In an embodiment the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic composition further comprising Thermoascus aurantiacus GH61A polypeptide (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 herein), and Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637).

In an embodiment the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic composition further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 9 herein) and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 8 herein).

In an embodiment the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic composition further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, which variant has one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH1, e.g., the one disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillus fumigatus CBH II, e.g., the one disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein.

In an embodiment the cellulolytic enzyme composition comprises one or more of the following components

(i) an Aspergillus fumigatus cellobiohydrolase I;
(ii) an Aspergillus fumigatus cellobiohydrolase II;
(iii) an Aspergillus fumigatus beta-glucosidase or variant thereof.

In an embodiment the Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 8 herein), comprises one or more substitutions selected from the group consisting of L89M, G91L, F100D, I140V, I186V, S283G, N456E, and F512Y; such as a variant thereof, with the following substitutions:

F100D+S283G+N456E+F512Y;

L89M+G91L+I186V+I140V; or

I186V+L89M+G91L+I140V+F100D+S283G+N456E+F512Y (using SEQ ID NO: 8 for numbering).

In an embodiment the cellulolytic composition further comprises the Penicillium sp. GH61 polypeptide shown in SEQ ID NO: 10 herein; or a GH61 polypeptide comprising an amino acid sequence having at least 60%, such as at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 10 herein.

Pullulanase

The enzymes present and/or added to saccharification and/or fermentation may optionally further include a pullulanase. The pullulanase may be of any origin, such as fungal or bacterial origin.

In an embodiment the pullulanase is derived from a strain of Bacillus sp. such as the one shown in SEQ ID NO: 15 herein or a strain of Bacillus deramificans.

In an embodiment the pullulanase, such as one derived from Bacillus sp, is selected from the group consisting of:

(i) a pullulanase comprising the mature polypeptide of SEQ ID NO: 15 herein;
(ii) a pullulanase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 15 herein.

Trehalase

According to the invention the enzymes present and/or added to saccharification and/or fermentation may optionally further include a trehalase.

The trehalase may be of any origin, such as fungal or bacterial origin.

In an embodiment the trehalase is of fungal origin, such as derived from a strain of Trichoderma, such as Trichoderma reesei, such as the one shown in SEQ ID NO: 14 herein.

In an embodiment the trehalase, such as one derived from Trichoderma reesei, is selected from the group consisting of:

(i) a trehalase comprising the mature polypeptide of SEQ ID NO: 14 herein;
(ii) a trehalase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 14 herein.

Pectinase

According to the invention the enzymes present and/or added to saccharification and/or fermentation may optionally further include a pectinase, such as a pectin lyase (also known as pectolyase) and/or a polygalacturonase, or a combination thereof.

The pectinase may be of any origin, such as fungal or bacterial origin.

In a preferred embodiment the pectinase is a pectin lyase (EC 4.2.2.10).

In an embodiment the pectin lyase is derived from a strain of Aspergillus, such as Aspergillus niger.

In a preferred embodiment the pectinase is a polygalacturonase (EC. 3.2.1.15). In an embodiment the polygalacacturonase is derived from a strain of Aspergillus, such as Aspergillus aculeatus.

In an embodiment the pectinase is a combination of pectin lyase and polygalacturonase. In an embodiment the pectinase is a combination of pectin lyase derived from Aspergillus niger and polygalacturonase derived from Aspergillus aculeatus.

Examples of Enzymes (e.g., Blend) Suitable for Use in a Raw Starch Hydrolysis Process of the Invention

In an embodiment enzymes (e.g., blend) for use in a process of the invention comprise a glucoamylase and an alpha-amylase, and optionally a protease and/or cellulolytic enzyme composition. Other optional enzymes may also be used.

In a preferred embodiment the enzymes (e.g., blend) used in a process of the invention comprises or consists of a glucoamylase from Trametes cingulata and an alpha-amylase from Rhizomucor pusillus with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD).

In a preferred embodiment the enzymes (e.g. blend) used in a process of the invention comprises the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein having one or more of the following substitutions: S95P, A121P, preferably S95P+A121P and an alpha-amylase, preferably an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), shown in SEQ ID NO: 13 herein, having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

In another preferred embodiment the enzymes (e.g., blend) used in a process of the invention comprises the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein and an alpha-amylase, preferably one derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one shown in SEQ ID NO: 13 herein, preferably having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.

In a preferred embodiment the enzymes (e.g., blend) used in a process of the invention comprises the Gloeophyllum sepiarium glucoamylase shown in SEQ ID NO: 4 herein and an alpha-amylase, preferably an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), shown in SEQ ID NO: 13 herein, preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

In a preferred embodiment the enzymes (e.g., blend) used in a process of the invention comprises the Trametes cingulata glucoamylase shown in SEQ ID NO: 12 herein and an alpha-amylase, preferably an alpha-amylase derived from Rhizomucor pusillus with a linker and starch-binding domain (SBD), in particular an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), such as the one shown in SEQ ID NO: 13 herein, having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N.

In an embodiment the enzymes (e.g., blend) used in a process of the invention comprises

i) fungal glucoamylase;

ii) fungal alpha-amylase;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, beta-glucosidase, CBH I and CBH II;

iv) optionally a protease.

In an embodiment the enzymes (blend) used in a process of the invention comprises

i) Trametes cingulata glucoamylase;

ii) Rhizomucor pusillus alpha-amylase, or variant thereof;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising Penicillium emersonii GH61A polypeptide, Aspergillus fumigatus beta-glucosidase with the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II;

iv) optionally a protease from Thermoascus aurantiacus, or variant thereof.

In an embodiment the enzymes (e.g., blend) used in a process of the invention comprises a

i) Trametes cingulata glucoamylase;

ii) Rhizomucor pusillus alpha-amylase, or variant thereof;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising Penicillium emersonii GH61A polypeptide, Aspergillus fumigatus beta-glucosidase with the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II;

iv) optionally a protease from Pyropoccus furiosus.

In an embodiment the enzymes (e.g., blend) used in a process of the invention comprises

i) glucoamylase derived from Trametes cingulata;

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei;

iv) optionally a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus.

In an embodiment the enzymes (e.g., blend) used in a process of the invention comprises

i) fungal glucoamylase;

ii) fungal alpha-amylase;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, beta-glucosidase CBH I and CBH II;

iv) pectinase, preferably a pectin lyase or a polygalacturonase, or a combination thereof.

In an embodiment the pectinase is a combination of pectin lyase derived from Aspergillus niger and polygalacturonase derived from Aspergillus aculeatus.

In an embodiment the pectinase is a combination of pectin lyase and polygalacturonase. In an embodiment the pectinase is a combination of pectin lyase derived from Aspergillus niger and polygalacturonase derived from Aspergillus aculeatus.

In an embodiment the enzymes (e.g., blend) used in a process of the invention comprises

• • i) fungal glucoamylase;
  • ii) fungal alpha-amylase;
  • iii) pectinase, preferably a pectin lyase or a polygalacturonase, or a combination thereof;
  • iv) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, beta-glucosidase CBH I and CBH II;
  • v) protease.

In an embodiment the enzymes (e.g., blend) used in a process of the invention comprises a

i) fungal glucoamylase;

ii) fungal alpha-amylase;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising a GH61 polypeptide, beta-glucosidase, CBH I and CBH II;

iv) optionally a protease.

In an embodiment the enzymes (e.g., blend) used in a process of the invention comprises

i) Trametes cingulata glucoamylase;

ii) Rhizomucor pusillus alpha-amylase, or variant thereof;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising Penicillium emersonii GH61A polypeptide, Aspergillus fumigatus beta-glucosidase with the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II;

iv) pectin lyase derived from Aspergillus niger or polygalacturonase derived from Aspergillus aculeatus, or a combination thereof;

v) protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus.

In a preferred embodiment the enzymes (blend) used in a process of the invention comprises

i) Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein having one or more of the following substitutions: S95P, A121P, such as S95P+A121P;

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), shown in SEQ ID NO: 13 herein, having of the following substitutions: G128D+D143N;

iii) optionally a cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising Penicillium emersonii GH61A polypeptide, Aspergillus fumigatus beta-glucosidase with the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II;

optionally iv) protease from Thermoascus aurantiacus, or a variant thereof.

In a preferred embodiment the enzymes (e.g., blend) used in a process of the invention comprises

i) Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein;

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), shown in SEQ ID NO: 13 herein, having of the following substitutions: G128D+D143N;

iii) optionally a cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising Penicillium emersonii GH61A polypeptide, Aspergillus fumigatus beta-glucosidase with the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II;

optionally iv) protease from Thermoascus aurantiacus, or a variant thereof.

In a preferred embodiment the enzymes (e.g., blend) used in a process of the invention comprises

i) Gloeophyllum sepiarium glucoamylase shown in SEQ ID NO: 4 herein;

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), shown in SEQ ID NO: 13 herein, having of the following substitutions: G128D+D143N;

iii) optionally a cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising Penicillium emersonii GH61A polypeptide, Aspergillus fumigatus beta-glucosidase with the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II;

optionally iv) protease from Thermoascus aurantiacus, or a variant thereof.

In a preferred embodiment the enzymes (e.g., blend) used in a process of the invention comprises

i) Trametes cingulata glucoamylase shown in SEQ ID NO: 12 herein;

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), shown in SEQ ID NO: 13 herein, having of the following substitutions: G128D+D143N;

iii) optionally cellulolytic enzyme composition derived from a strain of Trichoderma reesei, further comprising Penicillium emersonii GH61A polypeptide, Aspergillus fumigatus beta-glucosidase with the following substitutions: F100D, S283G, N456E, F512Y, and optionally Aspergillus fumigatus CBH I and Aspergillus fumigatus CBH II;

optionally iv) protease from Thermoascus aurantiacus, or a variant thereof.

Examples of Processes of the Invention

A process of the invention of producing ethanol from starch-containing material comprises:

• • (i) saccharifying starch-containing material at a temperature below the initial gelatinization temperature; and
  • (ii) fermenting using a fermentation organism;
  • wherein
    • saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease; and
    • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, wherein using said fermenting organism results in one or more, such as all, of the following:
      • increased ethanol yield compared to a corresponding process where Ethanol Red™ (“ER”) is used under the same conditions;
      • reduced glycerol level compared to a corresponding process where Ethanol Red™ (“ER”) is used under the same conditions;
      • reduced lactic acid level compared to a corresponding process where Ethanol Red™ (“ER”) is used under the same conditions.

A process of the invention of producing ethanol from starch-containing material comprises:

• • (i) saccharifying starch-containing material at a temperature below the initial gelatinization temperature; and
  • (ii) fermenting using a fermentation organism;
  • wherein
    • saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease; and
    • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, wherein using said fermenting organism results in one or more, such as all, of the following:
      • an ethanol yield boost/increase over Ethanol Red™ (“ER”) of more than 0.5% (after 72 hours fermentation) determined using the process set-up and conditions used in Example 1;
      • an ethanol yield boost/increase over Ethanol Red™ (“ER”) of more than 1.0% (after 88 hours fermentation) determined using the process set-up and conditions used in Example 1;
      • an ethanol yield boost/increase of more than 1.0%, such as more than 2.0%, such as more than 3.0%, such as more than 4.0% over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 2 (i.e., 95 hours fermentation);
      • a reduction in lactic acid of more than 50%, such as more than 60% (after 72 hours fermentation) over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 1;
      • a reduction in lactic acid of more than 20%, such as more than 25%, such as more than 30% (after 95 hours fermentation) over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 2;
      • a reduction in glycerol levels of more than 10.0%, such as more than 15.0%, such as more than 20.0% over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 1 (i.e., after 72 hours fermentation);
      • a reduction in glycerol levels of more than 5.0%, such as more than 10.0%, such as more than 12.0%, such as more than 14.0% over Ethanol Red™ (“ER”) when determined using the process set-up and conditions used in Example 2 (i.e., after 95 hours fermentation).
        In a preferred embodiment the process of producing ethanol from starch-containing material of the invention comprises:
        (a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
        (b) fermenting using a fermentation organism;
        wherein
  • saccharification and/or fermentation is done in the presence of the following enzymes:
    • i) glucoamylase derived from Trametes cingulata, Gloeophyllum trabeum, Gloeophyllum sepiarium, or Pycnoporus sanguineus;
    • ii) alpha-amylase;
    • iii) optionally cellulolytic enzyme composition, in particular derived from Trichoderma reesei;
    • iv) optionally a protease, in particular one derived from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus; and
      wherein
  • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

In a preferred embodiment the process of producing ethanol from starch-containing material of the invention comprises:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein

• • saccharification and/or fermentation is done in the presence of the following enzymes:
    • i) glucoamylase derived from Trametes cingulata, Gloeophyllum trabeum, Gloeophyllum sepiarium, or Pycnoporus sanguineus;
    • ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof;
    • iii) optionally cellulolytic enzyme composition derived from Trichoderma reesei;
    • iv) optionally a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus; and
      wherein
  • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

In a preferred embodiment the process of producing ethanol from starch-containing material of the invention comprises:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein

• • saccharification and/or fermentation is done in the presence of the following enzymes:
    • i) glucoamylase derived from Gloeophyllum trabeum disclosed in SEQ ID NO: 18, with the following substitutions: S95P+A121P;
    • ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 herein, with the following substitutions: G128D+D143N;
    • iii) optionally cellulolytic enzyme composition derived from Trichoderma reesei;
    • iv) optionally a protease from Thermoascus aurantiacus, or a variant thereof; and
      wherein
  • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851 or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

In a preferred embodiment the process of producing ethanol from starch containing material of the invention comprises:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein

• • saccharification and/or fermentation is done in the presence of the following enzymes:
    • i) glucoamylase derived from Pycnoporus sanguineus shown in SEQ ID NO: 17;
    • ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 herein, with the following substitutions: G128D+D143N;
    • iii) optionally cellulolytic enzyme composition derived from Trichoderma reesei;
    • iv) optionally a protease from Thermoascus aurantiacus, or a variant thereof; and
      wherein
  • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

In a preferred embodiment the process of producing ethanol from starch-containing material of the invention comprises:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein

• • saccharification and/or fermentation is done in the presence of the following enzymes:
    • i) glucoamylase derived from Gloeophyllum sepiarium shown in SEQ ID NO: 4;
    • ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 herein, with the following substitutions: G128D+D143N;
    • iii) optionally cellulolytic enzyme composition derived from Trichoderma reesei;
    • iv) optionally a protease from Thermoascus aurantiacus, or a variant thereof;
      wherein
  • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

In a preferred embodiment the process of producing ethanol from starch-containing material of the invention comprises:

(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein

• • saccharification and/or fermentation is done in the presence of the following enzymes:
    • i) glucoamylase derived from Trametes cingulata shown in SEQ ID NO: 12;
    • ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof, shown in SEQ ID NO: 13 herein, with the following substitutions: G128D+D143N;
    • iii) optionally cellulolytic enzyme composition derived from Trichoderma reesei;
    • iv) optionally a protease from Thermoascus aurantiacus, or a variant thereof; and
      wherein
  • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

Yeast of the Invention

The invention relates in one embodiment to a strain of Saccharomyces cerevisiae deposited under the Budapest Treaty at the National Measurement Institute (NMI) having deposit accession no. V14/004037.

The majority of the world's fuel ethanol is produced by industrial scale fermentation of starch-based sugars, in substrates such as corn mash. During industrial scale fermentation, the yeast encounter various physiological challenges including variable concentrations of sugars, high concentrations of yeast metabolites such as ethanol, glycerol, organic acids, osmotic stress, as well as potential competition from contaminating microbes such as wild yeasts and bacteria. As a consequence, many Saccharomyces strains are not suitable for use in industrial fermentation. The most widely used commercially available industrial strain of Saccharomyces (i.e. for industrial scale fermentation) is the Saccharomyces cerevisiae strain used, for example, in the product Ethanol Red. This strain is well suited to industrial ethanol production, however improved strains of Saccharomyces cerevisiae are needed.

WO 2011/035392 describes strain NMI V09/024011, which is a strain of Saccharomyces cerevisiae which produces higher levels of ethanol from corn mash than strains of Saccharomyces cerevisiae used in the fuel ethanol industry such as Ethanol Red™. However, a limitation of strain NMI V09/024011 is that its fermentation kinetics are slower than those of Ethanol Red. Also, the higher levels of ethanol that V09/024011 produces relative to Ethanol Red were only found when corn mash has been heavily supplemented with exogenous sugar sources such as dextrin. Under such conditions, mash fermentations need to be run for extended periods, beyond what are normally encountered in the industrial process. As such, high concentration sugar supplementation is not necessarily of industrial relevance and may not be encountered at scale. The inventors have now produced strain no. V14/004037 which is capable of producing even higher ethanol yields from endogenously occurring corn sugar consumed under the conditions encountered in industrial scale fermentation, such as those encountered during fermentation of corn mash, than V09/024011 or commercially available industrial Saccharomyces cerevisiae strains used in the ethanol industry. Strain no. V14/004037 also exhibits faster fermentation kinetics than strain no. V09/024011. As described herein, the levels of ethanol produced by strain no. V14/004037 under the conditions encountered during industrial fermentation of corn mash are greater than that of the commercially available industrial yeast strains such as Ethanol Red, and that of strain V09/024011. Thus, strain no. V14/004037 has the necessary characteristics for industrial production of ethanol from substrates such as corn mash.

Strain no. V14/004037 is a non-recombinant Saccharomyces cerevisiae strain developed by breeding which:

• • (a) produces a higher titre of ethanol at 72 hrs fermentation than strains V09/024011 and Ethanol Red, under the same conditions in a corn mash fermentation;
  • (b) produces less glycerol than Ethanol Red and V09/024011 under the same conditions in a corn mash fermentation.
  • (c) leaves less glucose remaining following fermentation than Ethanol Red and V09/024011 under the same conditions in a corn mash fermentation;
  • (d) leaves less maltose remaining following fermentation than Ethanol Red and V09/024011 under the same conditions in a corn mash fermentation.

As used herein, a defining characteristics of strain no. V14/004037 is any one or more of the following characteristics:

• • (a) produces ethanol in an amount in the range from 15.0 to 16.5% w/v at 32° C. in 96 hours in a corn mash fermentation;
  • (b) produces glycerol in an amount in the range from 0.900 to 1.00% w/v at 32° C. in 72 hours in a corn mash fermentation;
  • (c) produces a ratio of % w/v ethanol produced to % w/v glycerol produced following fermentation of corn mash at 32° C. in the range from 15 to 18;
  • (d) produces a ratio of % w/v ethanol produced to % w/v glucose remaining following fermentation of corn mash at 32° C. for 96 hours in the range from 100 to 900, 200 to 850, 300 to 850, 400 to 850.

Typically, the ethanol produced from fermentation of corn mash is produced from fermentation of sugars that are endogenous to the corn mash. Sugars that are endogenous to the corn mash are sugars that are derived from the corn rather than sugars that are added from an exogenous source.

Strain V14/004037 is also capable of growth in media in which xylose is the sole carbon source. In this regard, strain V14/004037 produces about a 7-fold increase in biomass when grown under the conditions specified in Test T1. As a consequence, strain V14/004037 can be readily distinguished from:

• • (a) naturally occurring strains of Saccharomyces;
  • (b) contaminating strains of Saccharomyces that do not utilize xylose; and
  • (c) other strains used in the ethanol industry that do not have the ethanol producing capabilities of strain V14/004037 and/or do not exhibit about a 7-fold increase in biomass in Test T1.

As current wild type and industrial strains of Saccharomyces are not capable of growth on xylose at the rate at which strain V14/004037 grows on xylose, strain V14/004037 is readily differentiated from current wild type strains of Saccharomyces and strains of Saccharomyces that are used in the ethanol industry prior to the present invention such as Ethanol Red.

The invention also relates to a derivative of Saccharomyces strain V14/004037. As used herein, a “derivative of strain V14/004037” is a strain derived from strain V14/004037, including through mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. The strain derived from strain V14/004037 may be a direct progeny (i.e. the product of a mating between strain V14/004037 and another strain or itself), or a distant progeny resulting from an initial mating between V14/004037 and another strain or itself, followed by a large number of subsequent matings.

In one embodiment, a derivative of strain V14/004037 is a hybrid strain produced by culturing a first yeast strain with strain V14/004037 under conditions which permit combining of DNA between the first yeast strain and strain V14/004037.

In one embodiment, a derivative of strain V14/004037 may be prepared by:

• • (a) culturing a first yeast strain with a second yeast strain, wherein the second yeast strain is strain V14/004037 or a derivative of strain V14/004037, under conditions which permit combining of DNA between the first yeast strain and the second yeast strain; and
  • (b) isolating hybrid strains; and
  • (c) optionally repeating steps (a) and (b) using a hybrid strain isolated in step (b) as the first yeast strain and/or the derivative of strain V14/004037.

In one embodiment, the derivative of strain V14/004037 exhibits one or more defining characteristic of strain V14/004037. Derivatives of Saccharomyces which exhibit one or more defining characteristics of strain V14/004037 are produced using strain V14/004037. In this regard, strain V14/004037 forms the basis for preparing other strains having the defining characteristics of strain V14/004037. For example, strains of Saccharomyces which exhibit one or more defining characteristics of strain V14/004037 can be derived from strain V14/004037 using methods such as classical mating, cell fusion, or cytoduction between yeast strains, mutagenesis or recombinant DNA technology.

In one embodiment, a derivative of strain V14/004037 which exhibits one or more defining characteristics of strain V14/004037 may be produced by:

• • (a) culturing a first yeast strain with a second yeast strain, wherein the second yeast strain is strain V14/004037 or a derivative of strain V14/004037, under conditions which permit combining of DNA between the first yeast strain and the second yeast strain;
  • (b) screening or selecting for a derivative of strain V14/004037, such as screening or selecting for a derivative with increased ethanol production in corn mash compared to the first strain, and/or screening or selecting for a hybrid which produces less glycerol in corn mash compared to the first strain;
  • (c) optionally repeating steps (a) and (b) with the screened or selected strain as the first yeast strain and/or the second yeast strain, until a derivative of strain V14/004037 is obtained which exhibits one or more defining characteristics of strain V14/004037.

The first yeast strain may be any strain of yeast if the DNA of the strain can be combined with the second yeast strain using methods such as classical mating, cell fusion or cytoduction. Typically, the first yeast strain is a Saccharomyces strain. More typically, the first yeast strain is a Saccharomyces cerevisiae strain. Saccharomyces cerevisiae is as defined by Kurtzman (2003) FEMS Yeast Research vol 4 pp. 233-245. The first yeast strain may have desired properties which are sought to be combined with the defining characteristics of strain V14/004037. The first yeast strain may be, for example, any Saccharomyces cerevisiae strain, such as for example Ethanol Red, V09/024011. It will also be appreciated that the first yeast strain may be strain V14/004037 or a strain which exhibits one or more defining characteristics of strain V14/004037.

The first and second yeast strains are cultured under conditions which permit combining of DNA between the yeast strains. As used herein, “combining of DNA” between yeast strains refers to combining of all or a part of the genome of the yeast strains. Combining of DNA between yeast strains may be by any method suitable for combining DNA of at least two yeast cells, and may include, for example, mating methods which comprise sporulation of the yeast strains to produce haploid cells and subsequent hybridising of compatible haploid cells; cytoduction; or cell fusion such as protoplast fusion.

In one embodiment, culturing the first yeast strain with the second yeast, under conditions which permit combining of DNA between the first yeast strain and the second yeast strain, comprises:

• • (i) sporulating the first yeast strain and the second yeast strain;
  • (ii) germinating and hybridizing spores produced by the first yeast strain with spores produced by the second yeast strain.

In one embodiment, the method of producing a derivative of strain V14/004037 which exhibits one or more defining characteristics of strain V14/004037, comprises:

• • (a) providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is strain V14/004037 or a derivative of strain V14/004037;
  • (b) sporulating the first yeast strain and the second yeast strain;
  • (c) germinating and hybridising the spores of the first yeast strain with germinated spores of the second yeast strain;
  • (d) screening or selecting for a derivative of strain V14/004037, such as screening or selecting for a derivative with increased ethanol production in corn mash compared to the first strain, and/or screening or selecting for a hybrid which produces less glycerol in corn mash compared to the first strain;
  • (e) optionally repeating steps (b) to (d) with the screened or selected strain as the first and/or second yeast strain.

Methods for sporulating, germinating and hybridising yeast strains, and in particular, Saccharomyces strains, are known in the art and are described in, for example, Ausubel, F. M. et al., (1997) Current Protocols in Molecular Biology, Volume 2, pages 13.2.1 to 13.2.5 (John Willey & Sons Inc); Chapter 7, “Sporulation and Hybridisation of yeast” by R. R. Fowell, in “The Yeasts” vol 1, A. H. Rose and J. S. Harrison (Eds), 1969, Academic Press.

In one embodiment, the yeast strains may be cultured under conditions which permit cell fusion. Methods for the generation of intraspecific or interspecific hybrids using cell fusion techniques are described in, for example, Spencer et al. (1990) in, Yeast Technology, Spencer JFT and Spencer DM (Eds), Springer Verlag, New York.

In another embodiment, the yeast strains may be cultured under conditions which permit cytoduction. Methods for cytoduction are described in, for example, Inge-Vechymov et al. (1986) Genetika 22: 2625-2636; Johnston (1990) in, Yeast technology, Spencer JFT and Spencer DM (Eds), Springer Verlag, New York.

In one embodiment, screening or selecting for derivatives of strain V14/004037 comprises screening or selecting for a derivative with increased ethanol production in corn mash compared to the first strain, and/or screening or selecting for a hybrid which produces less glycerol in corn mash compared to the first strain.

In another embodiment, the yeast cells may be screened or selected for strains which have one or more of the following characteristics:

• • (a) produces an amount of ethanol that is in the range from an amount higher than that produced by strain Ethanol Red to the amount produced by strain V14/004037, under the same conditions in a corn mash fermentation;
  • (b) produces an amount of glycerol that is in the range from an amount that is less than the amount produced by Ethanol Red to the amount produced by strain V14/004037, under the same conditions in a corn mash fermentation
  • (c) produces a ratio of ethanol to glycerol that is in the range from a ratio higher than the ratio of ethanol to glycerol of Ethanol Red to a ratio that is about the same as the ratio of ethanol to glycerol of strain V14/004037, under the same conditions in a corm mash fermentation.
  • (d) produces a ratio of ethanol to glucose that is in the range from a ratio higher than the ratio of ethanol to glucose of Ethanol Red to a ratio that is about the same as the ratio of ethanol to glucose of strain V14/004037 under the same conditions in a corn mash fermentation;
  • (e) produces a ratio of ethanol to maltose that is in the range from a ratio higher than the ratio of ethanol to maltose of Ethanol Red to a ratio that is about the same as the ratio of ethanol to maltose of strain V14/004037 under the same conditions in a corn mash fermentation.

Methods for determining the amount of ethanol and glycerol produced by a strain are known in the art. For example, methods for testing for determining the amount of ethanol and glycerol produced by a strain during fermentation of corn mash are described in, for example, WO 2011/035392. Once the amount of ethanol and glycerol produced are known, the ratio of ethanol/glycerol can be readily determined. Accordingly, strains can be readily screened for production levels of ethanol and/or glycerol using known methods.

In one embodiment, a derivative of strain V14/004037 which exhibits one or more defining characteristics of strain V14/004037 may be a mutant of strain V14/004037. Methods for producing mutants of Saccharomyces yeast, and specifically mutants of Saccharomyces cerevisiae, are known in the art and described in, for example, Lawrence C. W. (1991) Methods in Enzymology, 194: 273-281.

In another embodiment, a derivative of strain V14/004037 which exhibits one or more defining characteristics of strain V14/004037 may be a recombinant derivative of strain V14/004037. A recombinant derivative of strain V14/004037 is a strain produced by introducing into strain V14/004037 a nucleic acid using recombinant DNA technology. Methods for the introduction of nucleic acid into Saccharomyces yeast cells, and in particular strains of Saccharomyces, are known in the art and are described in, for example, Ausubel, F. M. et al. (1997), Current Protocols in Molecular Biology, Volume 2, pages 13.7.1 to 13.7.7, published by John Wiley & Sons Inc.

The invention also relates to methods for the production of ethanol using the strain described herein. In one form, strain V14/004037 or a derivative strain which exhibits the defining characteristics of strain V14/004037 is incubated with a substrate comprising fermentable sugars under conditions that allow fermentation of the fermentable sugars. The fermentable sugars may be one or more of glucose, galactose, maltose, fructose and sucrose. Typically, the fermentable sugar is glucose. While strain V14/004037 is well suited to fermentation in corn mash, it is envisaged the strain may also be suitable for other fermentation processes. Accordingly, the source of the fermentable sugar in the substrate may be, for example, hydrolysed starch, hydrolysed cellulose, molasses, cane juice, grape juice, fruit juice, glucose, maltodextrins, raw sugar juice, galactose, sucrose, or any other forms of fermentable sugars. In one form, the source of fermentable sugar in the substrate is hydrolysed starch. Typically, the starch is obtained from a substrate such as corn mash. In preparing the substrate, the grain is typically ground and mixed with water and hydrolytic enzyme(s) under conditions which result in hydrolysis of the starch and release of fermentable sugars such as glucose. Typical enzymes for hydrolysis of the starch include alpha-amylase, amyloglucosidase (glucoamylase), pullulanase, beta-amylase, glucoamylase, protease, cellulose or mixtures thereof. Enzymes suitable for hydrolysis are available from, for example, Novozymes or Genencor Inc. In one form, substrate is provided in the form of corn mash. Corn mash is typically produced by: (a) grinding corn to form a meal; (b) mixing the meal with water; and (c) hydrolyzing the starch in the corn meal. Methods for preparation of corn mash are known in the art and described in, for example, Thomas, K. C. et al., (2001) Journal of Applied Microbiology, volume 90, pages 819-828. Methods for the preparation of other starch-based substrates including sorghum, starch streams and combinations thereof are also known in the art and described in, for example, Kwiatkowski J. R. et al. (2003) Industrial Crops and Products 23: 288-296 and Bothast R. J. and Schlicher M. A. (2005) Applied Microbial Biotechnology 67: 19-25

The fermentation is carried out at a temperature which permits fermentation of the fermentable sugars. Typically, the temperature at which the fermentation is carried out is from 25-34° C.

The fermentation results in an alcoholic mash comprising ethanol and residual sugars in solution, and a particulate portion comprising residual solids including yeast. Ethanol is isolated from the mash using methods know in the art such as distillation or filtration.

Methods for fermentation and distillation are known in the art and are described in, for example, Kwiatkowski J. R. et al. (2003) Industrial Crops and Products 23: 288-296 and Bothast R. J. and Schlicher M. A. (2005) Applied Microbial Biotechnology 67: 19-25

The invention further relates to a method of producing distiller's grain. Distiller's grains may be produced from the residual solids produced in the fermentation using methods known in the art and described in, for example, U.S. Pat. No. 7,572,353. Because Saccharomyces strain V14/004037 reduces the level of residual sugars remaining following fermentation, the distiller's grain which results from fermentation using strain V14/004037 has a lowered glucose content and is therefore more stable and less prone to charring, caramelisation or contamination with unwanted microorganisms.

Furthermore, lower glycerol content in distillers grains is a process advantage because less time is required for drying the distiller's grains. In addition, less glycerol in the distiller's grains results in improved flowability, and further results in distiller's grains which has a higher nutrient content (e.g. higher protein).

As used herein, the singular forms “a”, “an” and “the” include plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “a cell” includes a plurality of such cells. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As used herein, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Test T1

Step 1: Yeast strains are streaked onto 2% w/v D-glucose 1% bacteriological peptone and 0.5% yeast extract medium solidified with 2% agar using standard microbiological techniques.
Step 2: After incubation for 72 hours at 30° C., yeast cells are taken from plates using a sterile microbiological loop and inoculated to an OD₆₀₀ (Optical Density at 600 nm) of between 0.1 and 0.2 units (OD₆₀₀ at T₀) in 50 ml of broth containing xylose (5% w/v), Difco Yeast Nitrogen Base w/o amino acids (0.67%), citric acid (0.3%) and trisodium citrate (0.7%) in distilled water in a 250 ml Erlenmeyer flask. An OD₆₀₀ of 0.1 unit is equal to approximately 9×10⁵ yeast cells/mL. D-(+)-Xylose, minimum 99% can be obtained from Sigma-Aldrich.
Step 3: Cultures are incubated at 30 deg Celsius with shaking at 220 rpm (10 cm orbital diameter) for 48 hours.
Step 4: After 48 hours incubation, OD₆₀₀ of culture is measured (OD₆₀₀ at T₄₈).
Step 5: The fold increase in biomass is determined by the equation:

OD₆₀₀ at T₄₈/OD₆₀₀ at T₀.

Composition of the Invention

In this aspect the invention relates to a formulated Saccharomyces yeast composition comprising a yeast strain of the invention and naturally occurring and/or nonenaturally occurring components.

As mentioned above a Saccharomyces yeast strain, in particular Saccharomyces cerevisiae yeast strain, of the invention, may according to the invention may be in any viable form, including crumbled, dry, including active dry and instant, compressed, cream form etc. In a preferred embodiment the Saccharomyces cerevisiae yeast strain of the invention is dry yeast, such as active dry yeast or instant yeast. In a preferred embodiment the Saccharomyces cerevisiae yeast strain of the invention is crumbled yeast. In a preferred embodiment the Saccharomyces cerevisiae yeast strain is compressed yeast. In an embodiment the Saccharomyces cerevisiae yeast strain of the invention is cream yeast.

In an embodiment the invention relates to a composition comprising a Saccharomyces yeast of the invention, in particular Saccharmyces MBG4851 and one or more of the component selected from the group consisting of: surfactants, emulsifiers, gums, swelling agent, and antioxidants and other processing aids.

Surfactant

According to the invention the composition may comprise a Saccharomyces yeast of the invention, in particular Saccharmyces MBG4851 and any suitable surfactants. In an embodiment the surfactant(s) is/are an anionic surfactant, cationic surfactant, and/or nonionic surfactant.

Emulsifier

According to the invention the composition may comprise a Saccharomyces yeast of the invention, in particular Saccharmyces MBG4851 and any suitable emulsifier. In an embodiment the emulsifier is a fatty-acid ester of sorbitan. In an embodiment the emulsifier is selected from the group of sorbitan monostearate (SMS), citric acid esters of monodiglycerides, polyglycerolester, fatty acid esters of propylene glycol.

In an embodiment the composition of the invention comprises a Saccharomyces yeast of the invention, in particular Saccharmyces MBG4851, and Olindronal SMS, Olindronal SK, or Olindronal SPL including composition concerned in European Patent No. 1,724,336 (hereby incorporated by reference). These products are commercially available from Bussetti, Austria, for active dry yeast.

Gum

According to the invention the composition may comprise a Saccharomyces yeast of the invention, in particular Saccharmyces MBG4851 and any suitable gum. In an embodiment the gum is acacia gum, in particular for cream, compressed and dry yeast.

Swelling Agents

According to the invention the composition may comprise a Saccharomyces yeast of the invention, in particular Saccharmyces MBG4851 and any suitable swelling agent. In an embodiment the swelling agent is methyl cellulose or carboxymethyl cellulose.

Antioxidant

According to the invention the composition may comprise a Saccharomyces yeast of the invention, in particular Saccharmyces MBG4851 and any suitable anti-oxidant. In an embodiment the antioxidant is butylated hydroxyanisol (BHA) and/or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particular for active dry yeast.

As used herein, the singular forms “a”, “an” and “the” include plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “a cell” includes a plurality of such cells. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As used herein, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure, including definitions will be controlling.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Materials & Methods

Materials:

GtAMG: Glucoamylase derived from Gloeophyllum trabeum disclosed in SEQ ID NO: 18 herein, with the following substitutions: S95P+A121P.
PsAMG: Glucoamylase derived from Pycnoporus sanguineus disclosed as shown in SEQ ID NO: 4 in WO 2011/066576 and in SEQ ID NO: 17 herein.
TcAMG: Glucoamylase derived from Trametes cingulata shown in SEQ ID NO: 12 herein or SEQ ID NO: 2 in WO 2006/69289.
JA126: Alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD) shown in SEQ ID NO: 13 herein.
AAPE096: Alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD) shown in SEQ ID NO: 13 herein, with the following substitutions: G128D+D143N.
RSH Blend P: Blend of TcAMG and JA126 with a ratio between AGU (from TcAMG) and FAU-F (JA126) of about 10:1.
Cellulase VD: Cellulolytic composition derived from Trichoderma reesei further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, preferably a variant having one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y and Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein.

Yeast:

ETHANOL RED™ (“ER”): Saccharomyces cerevisiae yeast available from Fermentis/Lesaffre, USA.
MBG4851: Saccharomyces cerevisiae yeast (non-recombinant) deposited by Microbiogen Pty Ltd, Unit E2, Lane Cove Business Park, 16 Mars Road, Lane Cove, NSW 2066, Australia under the terms of the Budapest Treaty with the National Measurement Institute, Victoria, Australia) and given the following accession number:

Deposit	Accession Number	Date of Deposit
MBG4851	V14/004037	Feb. 17, 2014
ETHANOL RED ™	V14/007039	Mar. 19, 2014

The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposits represent substantially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of deposits do not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Methods:

Identity

The relatedness between two amino acid sequences or between two polynucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3, windows=5, and diagonals=5.

For purposes of the present invention, the degree of identity between two polynucleotide sequences is determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=3, gap penalty=3, and windows=20.

SIGMA Enzymatic Assay for Trehalase

One SIGMA unit will convert 1.0 micro mol of trehalose to 2.0 micro mol of glucose per minutes at pH 5.7 at 37° C. (liberated glucose determined at pH 7.5).

Glucoamylase Activity

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

Glucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucose dehydrogenase reagent so that any alpha-D-glucose present is turned into beta-D-glucose. Glucose dehydrogenase reacts specifically with beta-D-glucose in the reaction mentioned above, forming NADH which is determined using a photometer at 340 nm as a measure of the original glucose concentration.

AMG incubation:
Substrate:              maltose 23.2 mM
Buffer:                 acetate 0.1M
pH:                     4.30 ± 0.05
Incubation temperature: 37° C. ± 1
Reaction time:          5 minutes
Enzyme working range:   0.5-4.0 AGU/mL

Color reaction:
GlucDH:                 430 U/L
Mutarotase:             9 U/L
NAD:                    0.21 mM
Buffer:                 phosphate 0.12M; 0.15M NaCl
pH:                     7.60 ± 0.05
Incubation temperature: 37° C. ± 1
Reaction time:          5 minutes
Wavelength:             340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003 M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Acid Alpha-Amylase Activity

When used according to the present invention the activity of an acid alpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units) or FAU-F.

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 AFAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.

Acid alpha-amylase, an endo-alpha-amylase (1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzes alpha-1,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths. The intensity of color formed with iodine is directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.

blue/violet t=23 sec. decoloration

Standard Conditions/Reaction Conditions:

• • Substrate: Soluble starch, approx. 0.17 g/L
  • Buffer: Citrate, approx. 0.03 M
  • Iodine (12): 0.03 g/L
  • CaCl₂: 1.85 mM
  • pH: 2.50±0.05
  • Incubation temperature: 40° C.
  • Reaction time: 23 seconds
  • Wavelength: 590 nm
  • Enzyme concentration: 0.025 AFAU/mL
  • Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.

Determination of FAU-F

FAU-F Fungal Alpha-Amylase Units (Fungamyl) is measured relative to an enzyme standard of a declared strength.

Reaction conditions
	Temperature	37° C.
	pH	7.15
	Wavelength	405	nm
	Reaction time	5	min
	Measuring time	2	min

A folder (EB-SM-0216.02) describing this standard method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference.

Measurement of Cellulase Activity Using Filter Paper Assay (FPU Assay)

1. Source of Method

1.1 The method is disclosed in a document entitled “Measurement of Cellulase Activities” by Adney, B. and Baker, J. 1996. Laboratory Analytical Procedure, LAP-006, National Renewable Energy Laboratory (NREL). It is based on the IUPAC method for measuring cellulase activity (Ghose, T. K., Measurement of Cellulse Activities, Pure & Appl. Chem. 59, pp. 257-268, 1987.

2. Procedure

2.1 The method is carried out as described by Adney and Baker, 1996, supra, except for the use of a 96 well plates to read the absorbance values after color development, as described below.

2.2 Enzyme Assay Tubes:

• 2.2.1 A rolled filter paper strip (#1 Whatman; 1×6 cm; 50 mg) is added to the bottom of a test tube (13×100 mm).
• 2.2.2 To the tube is added 1.0 mL of 0.05 M Na-citrate buffer (pH 4.80).
• 2.2.3 The tubes containing filter paper and buffer are incubated 5 min. at 50° C. (±0.1° C.) in a circulating water bath.
• 2.2.4 Following incubation, 0.5 mL of enzyme dilution in citrate buffer is added to the tube. Enzyme dilutions are designed to produce values slightly above and below the target value of 2.0 mg glucose.
• 2.2.5 The tube contents are mixed by gently vortexing for 3 seconds.
• 2.2.6 After vortexing, the tubes are incubated for 60 mins. at 50° C. (±0.1° C.) in a circulating water bath.
• 2.2.7 Immediately following the 60 min. incubation, the tubes are removed from the water bath, and 3.0 mL of DNS reagent is added to each tube to stop the reaction. The tubes are vortexed 3 seconds to mix.

2.3 Blank and Controls

• 2.3.1 A reagent blank is prepared by adding 1.5 mL of citrate buffer to a test tube.
• 2.3.2 A substrate control is prepared by placing a rolled filter paper strip into the bottom of a test tube, and adding 1.5 mL of citrate buffer.
• 2.3.3 Enzyme controls are prepared for each enzyme dilution by mixing 1.0 mL of citrate buffer with 0.5 mL of the appropriate enzyme dilution.
• 2.3.4 The reagent blank, substrate control, and enzyme controls are assayed in the same manner as the enzyme assay tubes, and done along with them.

2.4 Glucose Standards

• 2.4.1 A 100 mL stock solution of glucose (10.0 mg/mL) is prepared, and 5 mL aliquots are frozen. Prior to use, aliquots are thawed and vortexed to mix.
• 2.4.2 Dilutions of the stock solution are made in citrate buffer as follows:
  G1=1.0 mL stock+0.5 mL buffer=6.7 mg/mL=3.3 mg/0.5 mL
  G2=0.75 mL stock+0.75 mL buffer=5.0 mg/mL=2.5 mg/0.5 mL
  G3=0.5 mL stock+1.0 mL buffer=3.3 mg/mL=1.7 mg/0.5 mL
  G4=0.2 mL stock+0.8 mL buffer=2.0 mg/mL=1.0 mg/0.5 mL
• 2.4.3 Glucose standard tubes are prepared by adding 0.5 mL of each dilution to 1.0 mL of citrate buffer.
• 2.4.4 The glucose standard tubes are assayed in the same manner as the enzyme assay tubes, and done along with them.

2.5 Color Development

• 2.5.1 Following the 60 min. incubation and addition of DA/S, the tubes are all boiled together for 5 mins. in a water bath.
• 2.5.2 After boiling, they are immediately cooled in an ice/water bath.
• 2.5.3 When cool, the tubes are briefly vortexed, and the pulp is allowed to settle. Then each tube is diluted by adding 50 microL from the tube to 200 microL of ddH2O in a 96-well plate. Each well is mixed, and the absorbance is read at 540 nm.

2.6 Calculations (Examples are Given in the NREL Document)

• 2.6.1 A glucose standard curve is prepared by graphing glucose concentration (mg/0.5 mL) for the four standards (G1-G4) vs. A₅₄₀. This is fitted using a linear regression (Prism Software), and the equation for the line is used to determine the glucose produced for each of the enzyme assay tubes.
• 2.6.2 A plot of glucose produced (mg/0.5 mL) vs. total enzyme dilution is prepared, with the Y-axis (enzyme dilution) being on a log scale.
• 2.6.3 A line is drawn between the enzyme dilution that produced just above 2.0 mg glucose and the dilution that produced just below that. From this line, it is determined the enzyme dilution that would have produced exactly 2.0 mg of glucose.
• 2.6.4 The Filter Paper Units/mL (FPU/mL) are calculated as follows:

FPU/mL=0.37/enzyme dilution producing 2.0 mg glucose

Protease Assay Method—AU(RH)

The proteolytic activity may be determined with denatured hemoglobin as substrate. In the Anson-Hemoglobin method for the determination of proteolytic activity denatured hemoglobin is digested, and the undigested hemoglobin is precipitated with trichloroacetic acid (TCA). The amount of TCA soluble product is determined with phenol reagent, which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU-RH) is defined as the amount of enzyme which under standard conditions (i.e. 25° C., pH 5.5 and 10 min. reaction time) digests hemoglobin at an initial rate such that there is liberated per minute an amount of TCA soluble product which gives the same color with phenol reagent as one milliequivalent of tyrosine.

The AU(RH) method is described in EAL-SM-0350 and is available from Novozymes A/S Denmark on request.

Protease Assay Method (LAPU)

1 Leucine Amino Peptidase Unit (LAPU) is the amount of enzyme which decomposes 1 microM substrate per minute at the following conditions: 26 mM of L-leucine-p-nitroanilide as substrate, 0.1 M Tris buffer (pH 8.0), 37° C., 10 minutes reaction time.

LAPU is described in EB-SM-0298.02/01 available from Novozymes A/S Denmark on request.

EXAMPLES

Example 1

Tube Scale RSH Fermentations Using MBG4851 and ER

Mash Preparation

Yellow dent corn (obtained from Lincolnway on 19 Sep. 2013; ground in-house) was mixed with tap water and the dry solids (DS) level was determined to be 33.78% by moisture balance. This mixture was supplemented with 3 ppm penicillin and 500 ppm urea. The slurry was adjusted to pH 4.5 with 40% H₂SO₄.

Yeast Strains and Preparation

The two yeast strains tested in these experiments were Ethanol Red™ (“ER”) (Fermentis) and MBG4851. Yeast were propagated in filter sterilized liquid media (2% w/v D-glucose, 1% peptone, and 0.5% yeast extract). Using a sterile loop under a UV hood, cells from a lawn were transferred into 25 mL of the liquid media in 50 mL sterile centrifuge tubes with a hole drilled in the top and incubated at 150 rpm in a 30° C. air shaker. Tubes were angled at approximately 30 degrees to increase aeration. Cells were harvested at 18 hours by spinning at 3000 rpm for 10 minutes and decanting the supernatant. Cells were washed once in 25 ml of water and the resulting cell pellet was resuspended in 1.5 ml tap water. Total yeast concentration was determined using the YC-100 in duplicate.

Simultaneous Saccharification and Fermentation (SSF)

Approximately 5 grams of mash was transferred to test tubes having a 1/64 hole drilled in the top to allow CO₂ release. PsAMG/AAPE096 (ratio of PsAMG to AAPE096 was 33.5) was dosed to each tube of mash at 0.85 AGU/gDS. Yeast was dosed at 10e6 cells/g mash. Milli-Q water was added to each tube so that a total volume of liquid added (enzyme+MQ water) to each tube would be equally proportionate to the mash weight. Fermentations took place in a 32° C. water bath for 88 hours. Samples were vortexed periodically (in the morning and in the evening) throughout the fermentation.

HPLC Analysis

Fermentation sampling took place after 72 and 88 hours of fermentation by sacrificing 3 tubes per treatment. Each tube was processed for HPLC analysis by deactivation with 50 μL of 40% v/v H2SO4, vortexing, centrifuging at 1460×g for 10 minutes, and filtering through a 0.45 μm Whatman PP filter. All samples were processed without further dilution. Samples were stored at 4° C. prior to and during HPLC analysis.

TABLE 1
HPLC System
HPLC   Agilent's 1100/1200 series with Chem station software
System Degasser, Quaternary Pump, Auto-Sampler,
       Column Compartment/w Heater
       Refractive Index Detector (RI)
Column Bio-Rad HPX-87H Ion Exclusion Column 300 mm × 7.8 mm
       part# 125-0140
       Bio-Rad guard cartridge Cation H part# 125-0129,
       Holder part# 125-0131
Method 0.005M H2SO4 mobile phase
       Flow rate: 0.6 ml/min
       Column temperature: 65° C.
       RI detector temperature: 55° C.

Samples were analyzed for sugars (DP4+, DP3, DP2, glucose, and fructose), organic acids (lactic and acetic), glycerol, and ethanol.

Increased Ethanol Results

Ethanol titers at 72 and 88 hours are shown in Table 2 below.

TABLE 2
72 and 88 hour ethanol titers
			%
		MBG4851	increase
	ER (g/L	(g/L	with
	Ethanol)	Ethanol)	MBG4851
	72	155.390	156.406	0.65
	Hours
	88	154.898	157.210	1.49
	Hours

Reduced Glycerol Results

Glycerol titers at 72 and 88 hours are shown in Table 3 below.

TABLE 3
72 and 88 hour glycerol results
	ER	MBG4851	% Reduction
	(g/L Glycerol)	(g/L Glycerol)	with MBG4851
72 Hours	9.456	7.459	21.11
88 Hours	9.676	7.601	21.45

Lactic Acid Results

Results are shown in Table 4 below.

TABLE 4
72 and 88 Hour Lactic Acid Results
	ER	MBG485	% Reduction
	(g/L Lactic Acid)	(g/L Lactic Acid)	with MBG4851
72 Hours	0.597775	0.227973	61.86%
88 Hours	0.504066	0.207138	58.91%

Example 2

Reactor Scale RSH Fermentations Using MBG4851 and ER

All fermentations were done in 2 L IKA bioreactors.

Mash Preparation

Yellow dent corn (obtained from Lincolnway on 19 Sep. 2013; ground in-house) was mixed with tap water and the dry solids (DS) level was determined to be 35.5% by moisture balance. This mixture was supplemented with 3 ppm Lactrol and 200 ppm urea. The slurry was adjusted to pH 4.5 with 40% H₂SO₄.

Yeast Strains and Preparation

The two yeast strains tested in this experiment were Ethanol Red (Fermentis) and MBG4851. Yeasts were rehydrated by weighing 2.75 g of dried yeast into 50 ml of 36.5° C. tap water in a 125 mL Erlenmeyer flask. The flasks were then covered with parafilm and allowed to incubate in a 36.5° C. water bath. After 15 minutes, the flasks were swirled, but no other agitation took place. After a total of 30 minutes, the flasks were removed from the water bath.

Simultaneous Saccharification and Fermentation (SSF) PsAMG/AAPE096 (ratio of PsAMG to AAPE096 was 33.5) was dosed to each reactor at 0.85 AGU/gDS. 12.8 ml of rehydrated yeast was added to each bioreactor. Fermentations took place at 32° C. for 95 hours.

HPLC Analysis

Fermentation sampling took place by sampling 5 grams of mash into 15 ml tubes at 16, 24, 40, 48, 64, 72, 88, and 95 hours of fermentation. Each tube was processed for HPLC analysis by deactivation with 150 μL of 40% v/v H₂SO₄, vortexing, centrifuging at 1460×g for 10 minutes, and filtering through a 0.45 μm Whatman PP filter. Samples were stored at 4° C. prior to and during HPLC analysis.

TABLE 5
HPLC System
HPLC   Agilent's 1100/1200 series with Chem station software
System Degasser, Quaternary Pump, Auto-Sampler, Column
       Compartment/w Heater
       Refractive Index Detector (RI)
Column Bio-Rad HPX-87H Ion Exclusion Column 300 mm ×
       7.8 mm part# 125-0140
       Bio-Rad guard cartridge Cation H part# 125-0129,
       Holder part# 125-0131
Method 0.005M H2SO4 mobile phase
       Flow rate: 0.6 ml/min
       Column temperature: 65° C.
       RI detector temperature: 55° C.

Samples were analyzed for sugars (DP4+, DP3, DP2, glucose, and fructose), organic acids (lactic and acetic), glycerol, and ethanol.

Increased Ethanol and Faster Kinetics Results

Ethanol titers are shown across the entire course of fermentation in FIG. 1 below. Levels at the end of fermentation are shown in Table 6 below.

TABLE 6
Ethanol Titers at 95 hours Fermentation
ER	MBG4851	% Increase
(g/L Ethanol)	(g/L Ethanol)	with MBG4851
157.71	165.53	4.96%

Reduced Glycerol Results

Glycerol titers across fermentation are shown in FIG. 2 below. Levels at the end of fermentation are shown in Table 7 below.

TABLE 7
Glycerol Levels at 95 hours fermentation
ER	MBG4851	% Reduction
(g/L Glycerol)	(g/L Glycerol)	with MBG4851
10.814	9.163	14.80%

Lactic Acid Results

Lactic Acid titers are shown across the entire course of the fermentation in FIG. 3 below. Levels at the end of fermentation are shown in Table 8 below.

TABLE 8
Lactic Acid Results at 95 Hours RSH Fermentation
ER	MBG4851	% Reduction
(g/L Lactic Acid)	(g/L Lactic Acid)	with MBG4851
1.056	0.690	34.70%

Example 3

Tube Scale RSH Fermentations with Varying RSH Enzyme Using MBG4851 and ER

Mash Preparation

Yellow dent corn (obtained from Lincolnway on 19 Sep. 2013; ground in-house) was mixed with tap water and the dry solids (DS) level was determined to be 34.30% by moisture balance. This mixture was supplemented with 3 ppm penicillin and 500 ppm urea. The slurry was adjusted to pH 4.5 with 40% H₂SO₄.

Yeast Strains and Preparation

The two yeast strains tested in this experiment were Ethanol Red (Fermentis) and MBG4851. Yeasts were rehydrated by weighing 2.75 g of dried yeast into 50 ml of 36.5° C. tap water in a 125 mL Erlenmeyer flask. The flasks were then covered with parafilm and allowed to incubate in a 36.5° C. water bath. After 15 minutes, the flasks were swirled, but no other agitation took place. After a total of 30 minutes, the flasks were removed from the water bath.

Simultaneous Saccharification and Fermentation (SSF)

Approximately 5 grams of mash was transferred to test tubes having a 1/64 hole drilled in the top to allow CO₂ release. PsAMG/AAPE096 (ratio of PsAMG to AAPE096 was 33.5) was dosed to each tube of mash at 0.85 AGU/gDS or RSH Blend P was dosed at 0.32 AGU/gDS. Yeast was dosed at 10e6 cells/g mash. Milli-Q water was added to each tube so that a total volume of liquid added (enzyme+MQ water) to each tube would be equally proportionate to the mash weight. Fermentations took place in a 32° C. water bath for 88 hours. Samples were vortexed periodically (in the morning and in the evening) throughout the fermentation.

HPLC Analysis

Fermentation sampling took place after 72 and 88 hours of fermentation by sacrificing 3 tubes per treatment. Each tube was processed for HPLC analysis by deactivation with 50 μL of 40% v/v H₂SO₄, vortexing, centrifuging at 1460×g for 10 minutes, and filtering through a 0.45 μm Whatman PP filter. All samples were processed without further dilution. Samples were stored at 4° C. prior to and during HPLC analysis.

TABLE 9
HPLC System
HPLC   Agilent's 1100/1200 series with Chem station software
System Degasser, Quaternary Pump, Auto-Sampler, Column
       Compartment/w Heater
       Refractive Index Detector (RI)
Column Bio-Rad HPX-87H Ion Exclusion Column 300 mm ×
       7.8 mm part# 125-0140
       Bio-Rad guard cartridge Cation H part# 125-0129,
       Holder part# 125-0131
Method 0.005M H2SO4 mobile phase
       Flow rate: 0.6 ml/min
       Column temperature: 65° C.
       RI detector temperature: 55° C.

Samples were analyzed for sugars (DP4+, DP3, DP2, glucose, and fructose), organic acids (lactic and acetic), glycerol, and ethanol.

Increased Ethanol Results

Ethanol titers at 72 and 88 hours are shown in Table 10 below.

TABLE 10
72 and 88 hour ethanol titers
		ER (g/L	MBG4851	% increase
Enzyme	Time	Ethanol)	(g/L Ethanol)	with MBG4851
Ps	72 Hours	161.766	162.584	0.51
AMG	88 Hours	162.761	163.797	0.64
RSH	72 Hours	156.676	159.102	1.55
Blend P	88 Hours	160.893	164.628	2.32

Reduced Glycerol Results

Glycerol titers at 72 and 88 hours are shown in Table 11 below.

TABLE 11
72 and 88 hour glycerol results
		ER	MBG4851	% Reduction
Enzyme	Time	(g/L Glycerol)	(g/L Glycerol)	with MBG4851
Ps	72 Hours	10.324	7.812	24.33
AMG	88 Hours	10.439	8.364	19.88
RSH	72 Hours	9.761	7.461	23.56
Blend P	88 Hours	10.269	7.702	25.00

Lactic Acid Results

Results are shown in Table 12 below.

TABLE 12
72 and 88 Hour Lactic Acid Results
			% Reduction
	ER	MBG4851	with
Enzyme	(g/L Lactic Acid)	(g/L Lactic Acid)	MBG4851
Ps	72 Hours	1.008	0.625	46.49
AMG	88 Hours	1.027	0.734	48.85
RSH	72 Hours	1.139	0.709	34.41
Blend P	88 Hours	1.086	0.628	48.38

Example 4

Production of Strain V14/004037 (MBG4851)

Strain V14/004037 was produced using the methods described in WO 2005/121337 and through matings with various strains of Saccharomyces cerevisiae combined with selection for characteristics including low glycerol and high ethanol production.

Strain V14/004037 was verified to be a Saccharomyces cerevisiae strain by its ability to sporulate and produce progeny when the germinated spores were mated with standard strains of Saccharomyces cerevisiae, including tester strains of Saccharomyces cerevisiae. One such haploid tester strain is W303-1A. Specifically, germinated spores of strain V14/004037 were able to produce hybrid progeny when mated with tester strain W303-1A.

In more detail, haploid strain W303-1A was obtained from the Yeast Genetic Stock Center at the ATCC, USA (ATCC #208352) Strain V14/004037 was cultured to form haploid Saccharomyces yeast as described in Ausubel, F. M. et al. (1997), Current Protocols in Molecular Biology, Volume 2, pages 13.2.1 to 13.2.5, published by John Wiley & Sons. Subsequently, the spores were germinated on a solid medium such as GYP containing 1% w/v D-glucose, 0.5% yeast extract, 1% w/v bacteriological peptone and 1.5% w/v agar and incubated at 30° C. for three to five days. The isolated germinated spores from strain V14/004037 were then mated together with haploid W303-1A using the method described in, for example, Ausubel, F. M. et al. (1997), Current Protocols in molecular Biology, Volume 2, pages 13.2.1 to 13.2.5, published by John Wiley & Sons. Formation of hybrid zygotes could be observed under a microscope demonstrating that strain V14/004037 is a Saccharomyces cerevisiae strain.

Strain V14/004037 was deposited on 17 Feb. 2014 at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia under the Budapest Treaty and was designated accession number V14/004037.

Example 5

Growth of Strain V14/004037 on Xylose Minimal Media

Growth of strain V14/004037 on xylose as a sole carbon source was determined using Test T1. Saccharomyces cerevisiae strain V14/004037 was streaked onto 2% w/v D-glucose 1% bacteriological peptone and 0.5% yeast extract medium (GYP) solidified with 2% agar using standard microbiological techniques. After incubation for 72 hours at 30 deg Celsius, yeast cells were taken from plates using a sterile microbiological loop and inoculated to an OD₆₀₀ (Optical Density at 600 nm) of between 0.1 and 0.2 units (OD₆₀₀ at T0) in 50 ml of broth. An OD₆₀₀ of 0.1 unit is equal to approximately 9×10⁵ yeast cells/mL. The broth contained xylose (5% w/v), Difco Yeast Nitrogen Base w/o amino acids (0.67%), citric acid (0.3%) and trisodium citrate (0.7%) in distilled water in a 250 ml Erlenmeyer flask. Citric acid and trisodium citrate were provided as buffering agents that are not able to be used as growth substrates by Saccharomyces. D-(+)-Xylose 99% pure was obtained from Sigma-Aldrich (catalogue number X1500-500G). Cultures were incubated at 30 deg Celsius with shaking at 220 rpm (10 cm orbital diameter) for 48 hours prior to measuring OD₆₀₀ (OD₆₀₀ at T₄₈ hrs). The fold increase in biomass was determined by the equation: OD₆₀₀ at T₄₈ hrs divided by OD₆₀₀ at T₀.

Strain V14/004037 was inoculated at an initial OD₆₀₀ of 0.149 and increased more than 7-fold in 48 hours. Under the same conditions biomass of Ethanol Red yeast increased less than 2-fold.

Example 6

Raw Starch Hydrolysis Fermentation By MBG4851

Method: Whole corn was hammer-milled and sieved through a 0.85 mm filter. Raw starch mash was prepared as follows: 202.5 g sieved corn (at 85% corn solids), plus 297.5 mL water, plus 0.25 g urea was adjusted to pH4.5 with sodium hydroxide or sulphuric acid as appropriate. Suitable hydrolytic enzymes as described herein were added. Yeast were inoculated to a density equivalent of 0.5 g dry yeast weight per litre of mash. Mashes were incubated at a temperature of 32° C. Samples were taken after 24, 48, 72 and 96 hrs and analysed by HPLC for glucose, fructose, glycerol and ethanol.

A representative sample of Ethanol Red (used in this example) was deposited on 19 Mar. 2014 under the Budapest Treaty at the National Measurement Institute, 1/153 Bertie Street, Port Melbourne, Victoria 3207 and designated accession no. V14/007039. Values are presented as percent weight per volume (% w/v).

Results

All figures in % w/v, except the ethanol/glycerol ratio.

24 hrs	maltose	glucose	glycerol	ethanol	ethanol/glycerol
Ethanol Red	0.112	0.825	0.78	10.267	13.2
Ethanol Red	0.086	0.871	0.781	10.332	13.2
V14/004037	0.106	0.907	0.648	10.069	15.5
V14/004037	0.137	1.017	0.641	10.063	15.7

48 hrs	maltose	glucose	glycerol	ethanol	ethanol/glycerol
Ethanol Red	0.184	1.51	0.932	13.795	14.8
Ethanol Red	0.172	1.333	0.928	13.422	14.5
V14/004037	0.111	0.778	0.806	14.116	17.5
V14/004037	0.107	0.872	0.81	14.287	17.6

72 hrs	maltose	glucose	glycerol	ethanol	ethanol/glycerol
Ethanol Red	0.307	2.418	1.052	14.045	13.4
Ethanol Red	0.255	2.001	0.979	14.305	14.6
V14/004037	0.024	0.125	0.926	15.766	17.0
V14/004037	0.036	0.165	0.906	15.648	17.3

96 hrs	maltose	glucose	glycerol	ethanol	ethanol/glycerol
Ethanol Red	0.33	2.718	1.048	14.141	13.5
Ethanol Red	0.317	2.353	1.02	14.065	13.8
V14/004037	0	0.029	0.941	16.06	17.1
V14/004037	0	0.035	0.943	16.143	17.1

The Invention is Further Described in the Following Numbered Paragraphs:

1. A process of producing ethanol from starch-containing material comprising:

• • (a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature; and
  • (b) fermenting using a fermentation organism;
  • wherein
    • saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease; and
    • the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having the defining characteristics of strain V14/004037.
      2. The process of paragraph 1, wherein the glucoamylase is a Gloeophyllum glucoamylase, preferably Gloeophyllum trabeum glucoamylase.
      3. The process of any of paragraphs 1 or 2, wherein the glucoamylase is the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18.
      4. The process of any of paragraphs 1-3, wherein the glucoamylase is the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 having one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P.
      5. The process of any of paragraphs 1-4, wherein the glucoamylase is a Trametes glucoamylase, preferably Trametes cingulata glucoamylase.
      6. The process of any of paragraphs 1-5, wherein the glucoamylase is the Trametes cingulata glucoamylase shown in SEQ ID NO: 12.
      7. The process of any of paragraphs 1-6, wherein the glucoamylase is selected from the group consisting of:
      (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 12 herein;
      (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 12 herein.
      8. The process of any of paragraphs 1-7, wherein the alpha-amylase is derived from Rhizomucor pusillus, preferably with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 13 herein.
      9. The process any of paragraphs 1-8, wherein the glucoamylase is the Trametes cingulata glucoamylase shown in SEQ ID NO: 12 and the alpha-amylase is Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD).
      10. The process of any of paragraphs 1-9, wherein the alpha-amylase is derived from Rhizomucor pusillus.
      11. The process of any of paragraphs 1-10, wherein the glucoamylase is selected from the group consisting of:
      (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 herein;
      (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 18 herein.
      12. The process of any of paragraphs 1-11, wherein the alpha-amylase is Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering).
      13. The process any of paragraphs 1-12, wherein the glucoamylase is the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 having one of the following substitutions: S95P+A121P and the alpha-amylase is Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one having the following substitutions G128D+D143N (using SEQ ID NO: 13 for numbering).
      14. The process of any of paragraphs 1-13, wherein the glucoamylase is the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein.
      15. The process of any of paragraphs 1-14, wherein the glucoamylase is selected from the group consisting of:
      (i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17 herein;
      (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 17 herein.
      16. The process of any of paragraphs 1-15, wherein the glucoamylase is the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein, and the alpha-amylase is the Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one disclosed as V039 in Table 5 in WO 2006/069290 or SEQ ID NO: 13 herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.
      17. The process of any of paragraphs 1-16, wherein the ratio between glucoamylase and alpha-amylase is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg EP glucoamylase: mg EP alpha-amylase).
      18. The process of any of paragraphs 1-17, wherein the total dose of glucoamylase and alpha-amylase added is from 10-1,000 μg/g DS, such as from 50-500 μg/g DS, such as 75-250 μg/g DS.
      19. The process of any of paragraphs 1-18, wherein the total dose of cellulolytic enzyme composition added is from 10-500 μg/g DS, such as from 20-400 μg/g DS, such as 20-300 μg/g DS.
      20. The process of any of paragraphs 1-19, wherein the dose of protease added is from 1-200 μg/g DS, such as from 2-100 μg/g DS, such as 3-50 μg/g DS.
      21. The process of any of paragraphs 1-20, wherein the fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037, has one or more, such as all, of the following properties and defining characteristics:

increases ethanol yield compared to Ethanol Red™ under the same process conditions;

produces reduced levels of lactic acid compared to Ethanol Red™ under the same process conditions;

produces reduced levels of glycerol compared to Ethanol Red™ under the same process conditions;

has faster fermentation kinetics compared to Ethanol Red™ under the same process conditions.

22. The process of any of paragraphs 1-21, wherein the fermenting organism is a non-recombinant Saccharomyces strain, preferably non-recombinant Saccharomyces cerevisiae strain.
23. The process of any of paragraphs 1-2, wherein the fermenting organism strain is a non-recombinant Saccharomyces strain preferably non-recombinant Saccharomyces cerevisiae strain produced using the method described and concerned in U.S. Pat. No. 8,257,959-BB.
24. The process of any of paragraphs 1-23, wherein saccharification and fermentation are done separately or simultaneously.

25. The process of any of paragraphs 1-24, wherein the ethanol (i.e., product) is recovered after fermentation.

26. The process of any of paragraphs 1-25, wherein the starch-containing material is plant material selected from the corn (maize), cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, oat, rice, peas, beans, sweet potatoes, or a mixture thereof, preferably corn.
27. The process of any of paragraphs 1-26, wherein the starch-containing material is granular starch.
28. The process of any of paragraphs 1-27, wherein the process is carried out at a pH in the range between 3 and 7, preferably from 3 to 6, or more preferably from 3.5 to 5.0.
29. The process of any of paragraphs 1-28, wherein the dry solid content (DS) lies in the range from 10-55 wt.-% (DS), preferably 25-45 wt.-%, more preferably 30-40% of starch-containing material.
30. The process of any of paragraphs 1-29, wherein the sugar concentration is kept at a level below about 6 wt.-%, preferably 3 wt.-%, during saccharification and fermentation, especially below 0.25 wt.-%.
31. The process of any of paragraphs 1-30, wherein a slurry comprising starch-containing material reduced in particle size and water, is prepared before step (a).
32. The process of any of paragraphs 1-31, wherein the starch-containing material is prepared by reducing the particle size of the starch-containing material, preferably by milling, such that at least 50% of the starch-containing material has a particle size of 0.1-0.5 mm.
33. The process of any of paragraphs 1-32, wherein the starch-containing plant material is reduced in particle size, such as by dry or wet milling or using particle size emulsion technology.
34. The process of any of paragraphs 1-33, wherein the fermentation is carried out for 30 to 150 hours, preferably 48 to 96 hours.
35. The process of any of paragraphs 1-34, wherein the temperature during fermentation in step (b) or simultaneous saccharification and fermentation in steps (a) and (b) is between 25° C. and 40° C., preferably between 28° C. and 36° C., such as between 28° C. and 35° C., such as between 28° C. and 34° C., such as around 32° C.
36. The process of any of paragraphs 1-35, wherein further a protease is present during saccharification and/or fermentation.
37. The process of any of paragraphs 1-36, wherein the glucoamylase is present and/or added in an amount of 0.001 to 10 AGU/g DS, preferably from 0.01 to 5 AGU/g DS, especially 0.1 to 0.5 AGU/g DS.

38. The process of any of paragraphs 1-37, wherein the glucoamylase is present and/or added in an amount of 10-1,000 micro grams Enzyme Protein/g DS

39. The process of any of paragraphs 1-38, wherein the alpha-amylase is present and/or added in an amount of 0.001 to 10 AFAU/g DS, preferably from 0.01 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, preferably 0.01 to 1 FAU-F/g DS.
40. The process of any of paragraphs 1-39, wherein the alpha-amylase is present and/or added in an amount of 10-1,000 micro grams Enzyme Protein/g DS.
41. The process of any of paragraphs 1-40, wherein a cellulolytic enzyme composition is present and/or added during saccharification, fermentation or simultaneous saccharification and fermentation.
42. The process of paragraph 41, wherein the cellulolytic enzyme composition is present and/or added in an amount 1-10,000 micrograms EP/g DS, such as 2-5,000, such as 3 and 1,000, such as 4 and 500 micrograms EP/g DS.
43. The process of any of paragraphs 41-42, wherein cellulolytic enzyme composition is present and/or added in an amount in the range from 0.1-100 FPU per gram total solids (TS), preferably 0.5-50 FPU per gram TS, especially 1-20 FPU per gram TS.
44. The process of any of paragraphs 1-43, wherein protease is present and/or added in an amount in the range 1-1,000 μg EP/g DS, such as 2-500 μg EP/g DS, such as 3-250 μg EP/g DS.
45. The process of any of paragraphs 1-44, wherein the fermenting organism, in particular yeast, is added to the fermentation, so that the count per mL of fermentation medium is in the range from 10⁵ to 10¹², preferably from 10⁷ to 10¹⁰, especially about 5×10⁷.
46. The process of any of paragraphs 1-45, comprising:
(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein saccharification and/or fermentation is done in the presence of the following enzymes:

i) glucoamylase derived from Trametes cingulata;

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof;

wherein the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

47. The process of any of paragraphs 1-46, comprising:
(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein saccharification and/or fermentation is done in the presence of the following enzymes:

i) glucoamylase derived from Trametes cingulata;

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), or a variant thereof;

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei;

optionally iv) a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus;

wherein the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having the defining characteristics of strain V14/004037.

48. The process of any of paragraphs 1-47, comprising:
(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein saccharification and/or fermentation is done in the presence of the following enzymes:

i) glucoamylase derived from Gloeophyllum trabeum shown in SEQ ID NO: 18, preferably having at least one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P (using SEQ ID NO: 18 for numbering);

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering);

wherein the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having the defining characteristics of strain V14/004037.

49. The process of any of paragraphs 1-48, comprising:
(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein saccharification and/or fermentation is done in the presence of the following enzymes:

i) glucoamylase derived from Gloeophyllum trabeum shown in SEQ ID NO: 18, preferably having at least one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P, especially S95P+A121P (using SEQ ID NO: 18 for numbering);

ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering);

iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei; preferably a cellulolytic enzyme composition derived from Trichoderma reesei further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, preferably a variant having one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y and optionally Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein;

optionally iv) a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus;

wherein the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having the defining characteristics of strain V14/004037.

50. The process of any of paragraphs 1-49, comprising:
(a) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein saccharification and/or fermentation is done in the presence of the following enzymes:

• • i) glucoamylase derived from Pycnoporus sanguineus shown in SEQ ID NO: 17 herein,
  • ii) alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering);
  • optionally iii) cellulolytic enzyme composition derived from a strain of Trichoderma reesei; preferably a cellulolytic composition derived from Trichoderma reesei further comprising Penicillium emersonii GH61A polypeptide disclosed as SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 10 herein, and Aspergillus fumigatus beta-glucosidase disclosed as SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 8 herein, or a variant thereof, preferably a variant having one of, preferably all of, the following substitutions: F100D, S283G, N456E, F512Y and optionally Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 in WO2011/057140 and SEQ ID NO: 6 herein and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011/057140 and as SEQ ID NO: 7 herein;

optionally iv) a protease from Thermoascus aurantiacus, or a variant thereof and/or Pyrococcus furiosus;

wherein the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces strain V14/004037 having defining characteristics of strain V14/004037.

51. The process of any of paragraphs 1-50, wherein the ratio between glucoamylase and alpha-amylase is between 99:1 and 1:2, such as between 98:2 and 1:1, such as between 97:3 and 2:1, such as between 96:4 and 3:1, such as 97:3, 96:4, 95:5, 94:6, 93:7, 90:10, 85:15, 83:17 or 65:35 (mg EP glucoamylase: mg EP alpha-amylase).
52. The process of paragraphs 1-51, wherein the saccharification and fermentation are carried out simultaneously.
53. The process of any of paragraphs 1-52, wherein an enzyme composition of paragraphs 1-61 is used as the enzymes in saccharification or fermentation or simultaneous saccharification and fermentation.
54. A Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V14/004037 or a derivative of strain V14/004037 which exhibits one or more defining characteristics of strain V14/004037.
55. The strain of paragraph 54, wherein the strain is strain V14/004037.
56. A method of producing a derivative of strain V14/004037 which exhibits the defining characteristics of strain V14/004037, comprising:
(a) providing:
(i) a first yeast strain; and
(ii) a second yeast strain, wherein the second yeast strain is strain V14/004037 or a derivative of strain V14/004037;
(b) culturing the first yeast strain and the second yeast strain under conditions which permit combining of DNA between the first and second yeast strains;
(c) screening or selecting for a derivative of strain V14/004037.
57. The method of paragraph 56, wherein step (c) comprises screening or selecting for a hybrid strain which exhibits one or more defining characteristic of strain V14/004037.
58. The method of paragraph 56, comprising the further step of:
(d) repeating steps (b) and (c) with the screened or selected strain from step (c) as the first and/or second strain, until a derivative is obtained which exhibits defining characteristics of strain V14/004037.
59. The method of paragraph 56 or 58, wherein the culturing step (b) comprises:
(i) sporulating the first yeast strain and the second yeast strain;
(ii) hybridizing germinated spores produced by the first yeast strain with germinated spores produced by the second yeast strain.
60. A Saccharomyces strain produced by the method of paragraph 56.
61. A method of producing ethanol, comprising incubating a strain of paragraph 54 or 60 with a substrate comprising a fermentable sugar under conditions which permit fermentation of the fermentable sugar to produce ethanol.
62. Use of a strain of paragraph 54 or 60 in the production of ethanol.
63. A method of producing distiller's grain, comprising:
(a) incubating a Saccharomyces strain of paragraph 54 or 60 with a substrate comprising fermentable sugar under conditions which allow fermentation of the fermentable sugar to produce ethanol and distiller's grains;
(b) isolating the distiller's grains.
64. Distiller's grain produced by the method of paragraph 63.
65. Use of a strain of paragraph 54 or 60 in the production of distiller's grains.
66. Use of strain V14/004037 (Saccharomyces cerevisiae MBG4851) in the production of a Saccharomyces strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851 or which exhibits one or more defining characteristics of strain V14/004037.
67. Use of strain V14/004037 (Saccharomyces cerevisiae MBG4851) or a strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851 or a derivative of strain V14/004037 in a process according to any of paragraphs 1-53.
68. A composition comprising a Saccharomyces yeast strain of any of paragraphs 54 or 60 and one or more naturally occurring and/or non-naturally occurring components.
69. A composition of paragraph 68, wherein the components are selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, and antioxidants.
70. The composition of paragraph 68 or 69, wherein the Saccharomyces yeast strain is Saccharomyces MBG4851.
71. The composition of paragraphs 68-70, wherein the Saccharomyces yeast strain is in a viable form, in particular in dry, cream or compressed form.

Claims

1: A process of producing ethanol from starch-containing material comprising:

(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature; and

(b) fermenting using a fermentation organism;

wherein saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase and alpha-amylase, and optionally protease; and the fermenting organism is Saccharomyces cerevisiae MBG4851 (deposited under Accession No. V14/004037 at National Measurement Institute, Victoria, Australia) or a fermenting organism having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces cerevisiae MBG4851 having the defining characteristics of strain V14/004037.

2: The process of claim 1, wherein the fermenting organism having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces cerevisiae MBG4851 having defining characteristics of strain V14/004037, has one or more, such as all, of the following properties and defining characteristics:

increases ethanol yield compared to Ethanol Red™ under the same process conditions;

produces reduced levels of lactic acid compared to Ethanol Red™ under the same process conditions;

produces reduced levels of glycerol compared to Ethanol Red™ under the same process conditions;

has faster fermentation kinetics compared to Ethanol Red™ under the same process conditions.

3: The process of claim 1, wherein saccharification and fermentation are done simultaneously (SSF).

4: The process of claim 1, wherein the ethanol is recovered after fermentation.

5: The process of claim 1, wherein wherein saccharification and/or fermentation is done in the presence of a protease.

6: The process of claim 1, wherein a cellulolytic enzyme composition is present and/or added during saccharification, fermentation or simultaneous saccharification and fermentation (SSF).

7: The process of claim 1,

wherein saccharification and/or fermentation is done in the presence of a glucoamylase derived from Trametes cingulata.

8: (canceled)

9: The process of claim 1,

wherein saccharification and/or fermentation is done in the presence of a glucoamylase derived from Gloeophyllum trabeum shown in SEQ ID NO: 18.

10: (canceled)

11: The process of claim 1,

wherein saccharification and/or fermentation is done in the presence of

a glucoamylase derived from Pycnoporus sanguineus shown in SEQ ID NO: 17.

12: A Saccharomyces yeast strain deposited under the Budapest Treaty and having NMI accession no. V14/004037 or a derivative of strain V14/004037 which exhibits one or more defining characteristics of strain V14/004037.

13: (canceled)

14: A method of producing ethanol, comprising incubating the Saccharomyces yeast strain of claim 12 with a substrate comprising a fermentable sugar under conditions which permit fermentation of the fermentable sugar to produce ethanol.

15: (canceled)

16: A method of producing distiller's grain, comprising:

(a) incubating a Saccharomyces yeast strain of claim 12 with a substrate comprising fermentable sugar under conditions which allow fermentation of the fermentable sugar to produce ethanol and distiller's grains;

(b) isolating the distiller's grains.

17: Distiller's grain produced by the method of claim 16.

18-19: (canceled)

20: A composition comprising the Saccharomyces yeast strain of claim 12 and one or more naturally occurring and/or non-naturally occurring components.

21: The Saccharomyces yeast strain of claim 12, wherein the strain having properties that are about the same as that of Saccharomyces cerevisiae MBG4851, or a derivative of Saccharomyces cerevisiae MBG4851 having defining characteristics of strain V14/004037, has one or more, such as all, of the following properties and defining characteristics:

increases ethanol yield compared to Ethanol Red™ under the same process conditions;

produces reduced levels of lactic acid compared to Ethanol Red™ under the same process conditions;

produces reduced levels of glycerol compared to Ethanol Red™ under the same process conditions;

reduces the level of acetaldehyde in fermentation compared to Ethanol Red™ under the same process condition;

increases the oil recovery level compared to Ethanol Red™ under the same process conditions;

has faster fermentation kinetics compared to Ethanol Red™ under the same process conditions.

22: The process of claim 9, wherein saccharification and/or fermentation is done in the presence of a glucoamylase derived from Gloeophyllum trabeum shown in SEQ ID NO: 18 having at least one of the following substitutions: V59A; S95P; A121P; T119W; S95P+A121P; V59A+S95P; S95P+T119W; V59A+S95P+A121P; or S95P+T119W+A121P (using SEQ ID NO: 18 for numbering).

23: The process of claim 1, wherein saccharification and/or fermentation is done in the presence of an alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and Aspergillus niger glucoamylase starch-binding domain (SBD).

24: The process of claim 23, wherein the alpha-amylase derived from Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and Aspergillus niger glucoamylase starch-binding domain (SBD) has at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C, especially G128D+D143N (using SEQ ID NO: 13 for numbering).

25: The process of claim 5, wherein saccharification and/or fermentation is done in the presence of a protease derived from Thermoascus aurantiacus or Pyrococcus furiosus.

26: The process of claim 6, wherein the cellulolytic enzyme composition is derived from a strain of Trichoderma reesei.