Abstract: The present invention provides a recombinant Bacillus subtilis with improved 2?-fucosyllactose production, and a construction method thereof. In the present invention, a strain capable of efficiently synthesizing 2?-fucosyllactose is obtained by the fusion expression of the fucosyltransferase gene and the L-fucokinase/guanosine 5?-diphosphate-L-fucose pyrophosphorylase gene in Bacillus subtilis BSGL-FF, the fermentation supernatant of which comprises a cumulative amount of 2?-fucosyllactose as high as 1.62 g/L, which is 55% higher than the amount achieved with the control strain. The construction method of the recombinant Bacillus subtilis of the present invention is simple, and convenient to use, and thus has good application prospects.

Abstract: The present invention provides a recombinant Corynebacterium glutamicum producing N-acetylglucosamine and use thereof. The recombinant Corynebacterium glutamicum is obtained by overexpressing, in Corynebacterium glutamicum, the transcription factor SugR derived therefrom. The recombinant Corynebacterium glutamicum of the present invention increases the production of acetylglucosamine to up to 26 g/L, and lays a foundation for further metabolic engineering of Corynebacterium glutamicum to produce glucosamine.

Abstract: The present disclosure discloses a salt-reduced fermentation method for high-salt dilute-state fermented soy sauce, and belongs to the technical field of fermentation engineering. The present disclosure separates and screens Weissella paramesenteroides JL-5 and Bacillus amyloliquefaciens JDF-2 which inhibit the growth of spoilage bacteria from low-salt soy sauce mash, and by changing the microbial fermentation process of high-salt dilute-state soy sauce, the strains obtained by screening are used for the fermentation of low-salt soy sauce mash. Without changing the flavor and quality of soy sauce, low-salt fermentation of the soy sauce is enabled to proceed normally, and the biogenic amine content is less than 100 mg L?1, and the number of spoilage bacteria in the soy sauce are reduced. The prepared low-salt soy sauce has an amino acid nitrogen content of greater than 1.2 g·100 mL?1, contents of various spoilage bacteria of less than 1.

Abstract: The present invention provides a method for promoting N-acetylglucosamine synthesis by using the GlcN6P responsive element. In the present invention, Bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1 is used as a starting strain, in which a CRISPRi system regulated by GlcN6P responsive element is integrated into the genome to dynamically weaken the N-acetylglucosamine synthesis competitive pathway; a GlcN6P responsive promoter is used to regulate the expression of GNA1 on the plasmid to dynamically regulate the N-acetylglucosamine synthesis pathway; and the key gene alsSD involved in the acetoin synthesis pathway is knocked out. During fed-batch fermentation with this strain in a 15 L fermenter, the production of N-acetylglucosamine reaches 131.6 g/L and no by-product acetoin is accumulated, which lays a foundation for the production of GlcNAc by industrial fermentation.

Abstract: The present disclosure relates to a method for improving the yield and production intensity of Gluconobacter oxydans (G. oxydans) sorbose, and belongs to the technical field of fermentation engineering. By knocking out genes related to formation of D-sorbitol or L-sorbose metabolic by-products in G. oxydans, the formation of the by-products is reduced, and the efficiency of transforming D-sorbitol into L-sorbose is improved, thereby improving the yield and production intensity of L-sorbose. A recombinant strain G. oxydan-11 constructed by the present disclosure, compared with a control strain, has an L-sorbose transformation rate of 96.12%, which is 4.47% higher than that of a wild strain, has a production intensity of 14 g/L·h, which is 14.7% higher than that of the wild strain, and has a fructose by-product content of only 5.6 g/L, which is 45.6% lower than that of the wild strain.

Abstract: The present invention provides a pyruvate-responsive biosensor and a construction method and use thereof. In the present invention, pyruvate-responsive biosensors with different dynamic ranges are successfully constructed by optimizing the PdhR binding sequence inserted on the P43 promoter and optimizing the insertion site, wherein the minimum increase in dynamic range is by 0.6 time, and the maximum increase is by 30.7 times. The pyruvate-responsive biosensors are useful in the precise control of the expression of each gene in the cell. Since pyruvate is a key metabolite of central carbon in the cells, these biosensors are capable of dynamically regulating the expression level of intracellular genes according to changes in the content of pyruvate in the cells, thereby achieving the dynamic control of intracellular metabolic flux. The pyruvate biosensor obtained in the present invention has a good specificity, and a response range to pyruvate of 10-35 nmol/g DCW.

Abstract: The present invention provides an acid-tolerant Saccharomyces cerevisiae strain and use thereof. By using exogenously added malic acid as a stress, an acid-tolerant mutant S. cerevisiae strain MTPfo-4 is obtained by directed evolution screening in the laboratory, which tolerates a minimum pH of 2.44. The mutant strain MTPfo-4, tolerant to multiple organic acids, has an increased tolerance to exogenous malic acid of up to 86.6 g/L. The mutant strain MTPfo-4 obtained is further identified. The mutant strain grows stably and well, and can tolerate a variety of organic acids (lactic acid, malic acid, succinic acid, fumaric acid, citric acid, gluconic acid, and tartaric acid). It also has a strong tolerance to inorganic acids (HCl and H3PO4). This is difficult to achieve in the existing research and reports of S. cerevisiae. The strain is intended to be used as an acid-tolerant chassis cell factory for producing various short-chain organic acids.

Abstract: The present invention relates to a method for promoting acetylglucosamine synthesis of Bacillus subtilis, which belongs to the field of genetic engineering. The present invention adopts the recombinant Bacillus subtilis BSGNKAP2 as a starting strain, exogenously introducing pyruvate carboxylase BalpycA derived from Bacillus cereus, eliminating the central carbon metabolism overflow of the Bacillus subtilis and avoiding the synthesis of the by-product acetoin; further, five exogenous reducing force metabolic reactions are introduced to replace the reaction of generating NADH in glycolysis pathway and tricarboxylic acid cycle to reconstruct intracellular reducing force metabolism, which specifically comprise glyceraldehyde-3-phosphate ferredoxin dehydrogenase, isocitrate NAD+ dehydrogenase, a malate quinone dehydrogenase, a ketoacid ferredoxin oxidoreductase and a nitrogenase ferritin. In a shake-flask fermentation process using a complex medium, acetylglucosamine yield of the recombinant strain BSGNKAP8 is 24.

Abstract: The present disclosure provides a method for extracting alpha-ketoglutarate and pyruvate simultaneously from microbial fermentation broth or enzyme transformation solution, which is related to the technical field of biological separation and extraction.

Abstract: The present invention provides a recombinant Bacillus subtilis with improved 2?-fucosyllactose production, and a construction method thereof. In the present invention, a strain capable of efficiently synthesizing 2?-fucosyllactose is obtained by the fusion expression of the fucosyltransferase gene and the L-fucokinase/guanosine 5?-diphosphate-L-fucose pyrophosphorylase gene in Bacillus subtilis BSGL-FF, the fermentation supernatant of which comprises a cumulative amount of 2?-fucosyllactose as high as 1.62 g/L, which is 55% higher than the amount achieved with the control strain. The construction method of the recombinant Bacillus subtilis of the present invention is simple, and convenient to use, and thus has good application prospects.

Abstract: The invention provides a recombinant Bacillus subtilis, construction method and use thereof, wherein the cell's own FMMs are used as a space scaffold, and a multi-enzyme complex is constructed from specific marker proteins FloA and FloT, such that an artificial substrate channel is formed, and the cell metabolic burden is effectively reduced. The recombinant Bacillus subtilis of the invention can efficiently synthesize GlcNAc without affecting cell life activity, and can also limit the toxic intermediate metabolite GlcN-6-P near the plasma membrane to reduce or eliminate its inhibition on cell activity. In the process of shaking flask fermentation using complex medium, the yield of acetyl glucosamine of the control strain BSG-C was only 0.45 g·L?1, while that of BSG-AT, BSG-ATP, BSG-ATPB increased to 5.29 g·L?1, 6.22 g·L?1 and 8.48 g·L?1 respectively. The construction method of recombinant Bacillus subtilis is simple, easy to use and has a good application prospect.

Abstract: The present disclosure provides a recombinant Bacillus subtilis for increasing the yield of menaquinone 7 (MK-7) and application thereof, and belongs to the field of genetic engineering. In the present disclosure, 14 recombinant strains BS1-BS14 are constructed through the modification of genes related to the biosynthetic pathway of MK-7 on a chromosome of Bacillus subtilis, wherein BS6-BS14 significantly increase the yield of the MK-7, reaching up to 33.5 mg/L, which is 3.53 times the yield of the original strain of wild-type Bacillus subtilis 168. The present disclosure further provides a method for modifying the MK-7 biosynthetic pathway in microorganisms to increase the yield of the MK-7, providing a theoretical basis for constructing a high-yielding strain of the MK-7.

Abstract: The disclosure discloses Bacillus subtilis for producing N-acetylneuraminic acid and application thereof, and belongs to the field of genetic engineering. The disclosure optimizes the expression levels of key enzymes in N-acetylneuraminic acid synthesis pathways on genome through promoters of different strength, reduces the protein synthesis pressure caused by the expression of enzymes on cells, and further integrates the three N-acetylneuraminic acids in a same Bacillus subtilis engineering strain. Bacillus subtilis with improved N-acetylneuraminic acid production is obtained, and the production reaches 10.4 g/L at the shake flask level, laying a foundation for further improving the NeuAc production from Bacillus subtilis.

Abstract: The invention provides a high-throughput screening system based on multi-manipulators, and belongs to the field of biotechnology and detection equipment. A high-throughput screening system based on multi-manipulators, comprises of the first manipulator, sampler, pipette, plate washer, microplate reader, the second manipulator, centrifuge, deep-well plate library, waste shallow-well plate barrel, shallow-well plate library, waste needle plate barrel, needle library, waste deep-well plate barrel, collection box. The present invention is a combination of microbiology and mechanics. The aim of the invention is to realize the automation and intelligentization of the high throughput screening experiment, effectively improve the experimental accuracy, reliability and efficiency. It contributes to the development of high throughput screening technology for microorganisms and drugs.

Abstract: The disclosure discloses a method for preparing hyaluronan oligosaccharides with odd-numbered degrees of polymerization by hydrolysis, and belongs to the technical field of biological engineering. The disclosure takes macromolecular hyaluronic acid as the substrate, and by controlling the addition amount and reaction time of two hyaluronic acid hydrolases, leech hyaluronidase (LHase) and bovine testicular hyaluronidase (BTH), simultaneously prepares two different structures of hyaluronan oligosaccharides with odd-numbered degrees of polymerization (trisaccharide, pentasaccharide and heptasaccharide). The disclosure provides a new, simple and feasible method for preparing hyaluronan oligosaccharides with odd-numbered degrees of polymerization, thereby laying a foundation for the research on the functions and characteristics of the hyaluronan oligosaccharides with odd-numbered degrees of polymerization.

Abstract: The present disclosure provides a multicopper oxidase mutant with improved salt tolerance. Threonine at site 317 of wild-type multicopper oxidase WT was mutated to asparagine, leucine at site 386 was mutated to tyrosine, and serine at site 427 was mutated to glutamic acid by site-directed mutagenesis to obtain a mutant T317N-L386Y-S427E. Compared with WT, the tolerance of T317N-L386Y-S427E to 6%, 9%, 12%, 15% and 18% NaCl (W/V) is improved.

Abstract: The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCl buffer assisted with 3?-phosphoadenosine 5?-phophosulfate (PAPS). C4ST and C46ST came from bioengineered Escherichia coli or Pichia pastoris. Chondroitin came from bioengineered Bacillus subtilis 168.

Abstract: The present disclosure relates to a method for regulating expression of protein of interest in Bacillus subtilis, and belongs to the technical field of genetic engineering. The method comprises: using Bacillus subtilis as an expression host, adding the N-terminal nucleotide sequence coding the first 15 amino acids of the endogenous protein before the coding gene of the protein of interest or modifying the original N-terminal nucleotide sequence coding the first 15 amino acids, and performing free expression in plasmid, thereby regulating expression of the protein of interest in Bacillus subtilis, and even regulating the expression difference in different growth phases and the expression level.

Abstract: The disclosure discloses a recombinant Bacillus subtilis engineered bacterium capable of efficiently expressing keratinase, and belongs to the technical fields of genetic engineering and fermentation engineering. The disclosure successfully constructs a genetically engineered bacterium B. subtilis WB600-pP43NMK-ker capable of efficiently expressing keratinase by using a keratinase gene from Bacillus licheniformis (BBE11-1) as a target gene, pP43NMK as an expression vector and B. subtilis WB600 as an expression host; and meanwhile, the disclosure conducts in-depth research on fermentation media and fermentation conditions when the engineered bacteria are configured as a production strain to produce keratinase to obtain a fermentation medium capable of increasing a yield of keratinase and an optimum process for fermentation production of keratinase.

Abstract: The disclosure herein relates to construction and application of engineering bacteria capable of secreting and expressing diacetylchitobiose deacetylase, and belongs to the technical field of fermentation engineering. Firstly, recombinant B. subtilis capable of heterologously secreting and expressing a diacetylchitobiose deacetylase gene is constructed, and a signal peptide fragment yncM is added into the recombinant vector for the first time. The signal peptide can secrete the target protein diacetylchitobiose deacetylase outside the cells of the recombinant B. subtilis, and a mutant of the 5?-end untranslated region is acquired, thereby significantly increasing the expression level of the target protein, and greatly simplifying the subsequent enzyme separation and purification steps. When the acquired diacetylchitobiose deacetylase is fermented and cultured in a fermentation medium for 50-60 h, the enzyme activity reaches a maximum of 1,548.