Abstract: The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-CAS system for a filamentous fungal host cell.

Abstract: The present invention is based on the advantageous use of single-stranded oligonucleotides in the in vivo (within a cell) assembly of double-stranded oligonucleotides into a single double-stranded nucleic acid construct. The present invention relates to the use of at least a first and a second single-stranded oligonucleotide in the assembly within a cell of at least two double-stranded nucleic acid molecules into a single double-stranded nucleic acid construct of pre-determined sequence, wherein the first and second single-stranded oligonucleotide are essentially complementary to each other.

Abstract: The present invention relates relates to a CRISPR-CAS system for a host cell, in particular to a method to produce a circular vector comprising one or more guide-polynucleotide expression cassettes, wherein said one or more guide-polynucleotide expression cassettes comprise a polynucleotide encoding a guide-polynucleotide operably linked to one or more control sequences which direct the expression of said guide-polynucleotide in a host cell, wherein said guide-polynucleotide comprises a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and wherein the guide-polynucleotide can direct binding of a Cas protein at a target-polynucleotide in a host cell to form a CRISPR-Cas complex, in vivo.

Abstract: The invention relates to a method for modification of a host cell at a target locus, which method comprises: providing a host cell comprising, at a first locus, at least two site-specific recombination sites and a nucleic acid having an essential function or encoding a product having an essential function; introducing into the host cell, at the target locus, a further nucleic acid having the essential function or encoding for a product having the essential function; and carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional, thereby to modify the host cell at the target locus. The invention also relates to a cell obtainable by a method of the invention.

Abstract: The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-CAS system for a lipolytic yeast host cell.

Abstract: The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-CAS system for a yeast host cell.

Abstract: The present invention relates to a method based on the use of restriction enzyme digestion and ligation via the cleavage sites, thereby to prepare two or more standardized expression cassettes.

Abstract: The present invention relates to a method based on the use of restriction enzyme digestion and ligation via cleavage sites, thereby to prepare two or more standardized expression cassettes.

Abstract: The present invention relates to a recombinant yeast cell which is capable of producing one or more of 4-methyl itaconate, 1-methyl itaconate or 1,4-dimethyl itaconate. The invention also relates to a recombinant yeast cell which is capable of producing itaconic acid and which overexpresses: —a nucleic acid encoding a polypeptide having cis-aconitate decarboxylase activity; and —a nucleic acid encoding a polypeptide which catalyzes a reaction towards acetyl CoA. These recombinant yeast cells may be used in processes for the production of itaconic acid, 4-methyl itaconate, 1-methyl itaconate or 1,4-dimethyl itaconate.

Abstract: The present invention relates to a recombinant cell which is capable of producing one or more of itaconic acid, 4-methyl itaconate or 1-methyl itaconate, wherein the said recombinant microorganism has been modified in its genome such that it results in a deficiency in the production of a trans-aconitate methyltransferase. A recombinant yeast cell which is capable of producing itaconic acid and which overexpresses: —a nucleic acid encoding a polypeptide having cis-aconitate decarboxylase activity; and —one or more nucleic acids encoding polypeptides which separately or together catalyze a reaction towards acetyl CoA, wherein the said recombinant microorganism has been modified in its genome such that it results in a deficiency in the production of a trans-aconitate methyltransferase. These recombinant yeast cells may be used in processes for the production of itaconic acid, 4-methyl itaconate or 1-methyl itaconate.

Abstract: The present invention relates to a method for improving protein yield. The method comprises modifying the value of a set of relevant protein features to fall within an optimal range or to become more close to an optimal value for one or more protein features in the eukaryotic host.

Abstract: The invention relates to a polypeptide having carbohydrate material degrading activity which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Abstract: The present invention relates to a recombinant yeast cell which is capable of producing one or more of 4-methyl itaconate or 1-methyl itaconate. The invention also relates to a recombinant yeast cell which is capable of producing itaconic acid and which overexpresses: a nucleic acid encoding a polypeptide having cis-aconitate decarboxylase activity; and a nucleic acid encoding a polypeptide which catalyzes a reaction towards acetyl CoA. These recombinant yeast cells may be used in processes for the production of itaconic acid, 4-methyl itaconate or 1-methyl itaconate.

Abstract: The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

Abstract: The invention relates to a method for modification of a host cell at a target locus, which method comprises: providing a host cell comprising, at a first locus, at least two site-specific recombination sites and a nucleic acid having an essential function or encoding a product having an essential function; introducing into the host cell, at the target locus, a further nucleic acid having the essential function or encoding for a product having the essential function; and carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional, thereby to modify the host cell at the target locus. The invention also relates to a cell obtainable by a method of the invention.

Abstract: A method for the production of a polypeptide of interest in a host cell, which method comprises: a. providing a host cell which harbours a nucleic acid encoding a polypeptide of interest, wherein the polypeptide of interest is modified so that it comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence; b. cultivating the host cell under conditions suitable for production of the polypeptide; and, optionally, c. recovering the compound of interest. The invention also relates to a modified polypeptide which comprises fewer methionine and/or lysine residues than a reference polypeptide, excluding any initial methionine amino acid located at the N-terminal end of the polypeptide sequence.

Abstract: The present invention relates to a method based on the use of restriction enzyme digestion and ligation via cleavage sites, thereby to prepare two or more standardized expression cassettes.

Abstract: The present invention relates to a method for carrying out recombination at a target locus.

Abstract: The present invention relates to methods of optimization of a protein coding sequences for expression in a given host cell. The methods apply genetic algorithms to optimise single codon fitness and/or codon pair fitness sequences coding for a predetermined amino acid sequence. In the algorithm generation of new sequence variants and subsequent selection of fitter variants is reiterated until the variant coding sequences reach a minimum value for single codon fitness and/or codon pair fitness. The invention also relates to a computer comprising a processor and memory, the processor being arranged to read from and write into the memory, the memory comprising data and instructions arranged to provide the processor with the capacity to perform the genetic algorithms for optimisation of single codon fitness and/or codon pair fitness.

Abstract: The present invention relates to a method for the preparation of a library of host cells, a plurality of which comprise an assembled polynucleotide at a target locus, which method comprises: (a) providing a plurality of polynucleotides comprising two or more polynucleotide subgroups, wherein: (i) a plurality of polynucleotides in each polynucleotide subgroup comprises sequence encoding a peptide or polypeptide and/or a regulatory sequence; (ii) a plurality of peptides or polypeptides encoded by, or a plurality of regulatory sequences comprised within, each polynucleotide subgroup share an activity and/or function; (iii) at least one polynucleotide subgroup comprises at least two non-identical polynucleotide species; (iv) a plurality of polynucleotides of each polynucleotide subgroup comprises sequence enabling homologous recombination with a plurality of polynucleotides from one or more other polynucleotide subgroups; and (v) a plurality of polynucleotides in two polynucleotide subgroups comprise a nucleotide