Abstract: The present invention discloses a method for producing N-acetylglucosamine (GlcNAc) by co-utilizing glucose and xylose based on CRISPRi, and belongs to the field of genetic engineering. According to the method, Bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1 is used as an original strain, dCas9 induced by xylose and three sgRNA expression fragments targeting to genes zwf, pfkA and glmM respectively are integrated on the genome, and the strain is fermented in a shake flask, so that the titer of GlcNAc reaches 20.5 g/L, the yield of GlcNAc is 0.612 g/g glucose, at the same time, the efficient co-utilizing of glucose and xylose by the recombinant B.s subtilis is achieved, and the foundation for further metabolic engineering transformation of the B. Subtilis to produce GlcNAc and industrialization thereof is laid.

Abstract: The present disclosure discloses an asparaginase mutant with efficient expression, activity and stability, belonging to the technical fields of gene engineering and enzyme engineering. The amino acid sequence of the asparaginase mutant is set forth in SEQ ID NO: 99, SEQ ID NO: 100 or SEQ ID NO: 101; meanwhile, compared with the wild type asparaginase mutant, the extracellular enzyme activity of the asparaginase fusion enzyme mutant is increased by up to 2.25 times, the stability is increased by up to 3.56 times, and the specific enzyme activity is increased by up to 1.34 times.

Abstract: The invention discloses a promoter which can be induced to express in acidic conditions, and relates to the field of bioengineering technology. The promoters of the invention are separated from A. niger and can actuate and/or regulate the expression of the effectively connected nucleic acids in A. niger. In the invention the expression of the promoters is studied in A. niger, and it is indicated that some promoters show weak expression, and some show strong activity. The invention provides an effective method and new thought for organic acids production by fungi or other products produced by fermentation under acidic conditions.

Abstract: The invention discloses a method for improving the yield of Bacillus subtilis acetylglucosamine, which belongs to the technical field of genetic engineering. In the invention, the recombinant Bacillus subtilis S5 (S5-PxylA-glmS-P43-GNA1) is taken as a starting strain, and the glmS ribozyme is integrated into the mid of rbs and the promoter sequence of the glmM and pfkA gene, respectively. The ribozyme mutant has the advantage of prolonging the stability of the mRNA and integrated into the mid of rbs and the promoter sequence of the pgi gene. The yield of GlcNAc of the recombinant strain reaches 11.79-20.05 g/L. This laid the foundation for the further metabolic engineering of Bacillus subtilis to produce GlcNAc.

Abstract: The present invention relates to a multi-functional peptide benefiting expression, purification, stabilization and catalytic efficiency of recombinant proteins, which relates to the field of enzyme engineering and protein purification. The present invention provides a self-assembling amphipathic peptide, which fused with proteins including alkaline polygalacturonate lyase (PGL), lipoxygenase (LOX) and green fluorescent protein (GFP), which leads to successful purification by the nickel affinity chromatography with recovery rates were 47.01%, 39.01% and 56.1%, respectively. Furthermore, the expression quantity and thermostability of the three proteins were enhanced in different degree. Of which the half-life of the PGL-S1v1 and LOX-S1v1 were 2.3 and 3.8-fold as compared with the corresponding wild-type and the specific activity were 1.1 and 1.9-fold increase, respectively. The crude enzyme activity of PGL-S1v1 was 9-fold increase than the PGL.

Abstract: The present invention a method for simultaneously reducing urethane and its precursors levels during Chinese liquor production process, which relates to the field of fermented food production. The present invention is carried out through inoculating Bacillus amyloliquefaciens JP21 or combines with urease into the grains for fermentation. The content of EC and urea in the fermented grains after 5-day fermentation could reduce 30.16% and 50.05% respectively, With an addition of the B. amyloliquefaciens during grains fermentation, EC and urea levels are successfully controlled, and at the same time aroma and tastes of Chinese liquor hasn't been affected.

Abstract: The invention discloses a promoter which can be induced to express in acidic conditions, and relates to the field of bioengineering technology. The promoters of the invention are separated from A. niger and can actuate and/or regulate the expression of the effectively connected nucleic acids in A. niger. In the invention the expression of the promoters is studied in A. niger, and it is indicated that some promoters show weak expression, and some show strong activity. The invention provides an effective method and new thought for organic acids production by fungi or other products produced by fermentation under acidic conditions.

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 invention provides a high-throughput screening system based on a single manipulator, and belongs to the field of biotechnology and detection equipment. A high-throughput screening system based on single manipulator, comprises of one manipulator which is surrounded by deep well plate loading platform, shallow well plate loading platform, pipetting head loading platform, control cabinet, waste pipetting heads container platform, shallow well plate recycling platform, deep well plate recycling platform, Microplate Reader with oscillating mixer and items-placing platform in a counterclockwise or clockwise order. The present invention is a combination of microbiology and mechanics. It promotes the automation of microbial high-throughput screening system for screening microorganisms with specific properties. It effectively contributes to the development of microbial breeding technology and further promotes the development of microbial science.

Abstract: The present invention provides a production-line-type high-throughput screening system, which relates to the field of biotechnology and testing equipment. The system comprises of four manipulators, three parallel conveyor belts with fixed slots, 2-DOF slipway and fixed fixtures, 96-channel pipetting system, coloring device, oscillating mixing device, microplate reader, well plates loading platform and well plates recycling platform. Manual operation takes five minutes to detect one 96-well plate, while this system can handle 20 96-well plates per minute. It expands the number of screening targets, making the screening process more clearly and concisely and liberating manual labor. The system makes effective contributions to the development of microbial breeding technology.

Abstract: The present invention relates to a multi-functional peptide benefiting expression, purification, stabilization and catalytic efficiency of recombinant proteins, which relates to the field of enzyme engineering and protein purification. The present invention provides a self-assembling amphipathic peptide, which fused with proteins including alkaline polygalacturonate lyase (PGL), lipoxygenase (LOX) and green fluorescent protein (GFP), which leads to successful purification by the nickel affinity chromatography with recovery rates were 47.01%, 39.01% and 56.1%, respectively. Furthermore, the expression quantity and thermostability of the three proteins were enhanced in different degree. Of which the half-life of the PGL-S1v1 and LOX-S1v1 were 2.3 and 3.8-fold as compared with the corresponding wild-type and the specific activity were 1.1 and 1.9-fold increase, respectively. The crude enzyme activity of PGL-S1v1 was 9-fold increase than the PGL.

Abstract: The present invention provides a recombinant Bacillus subtilis for producing acetylglucosamine and construction method thereof. The recombinant Bacillus subtilis is obtained by deletion of glmS ribozyme of Bacillus subtilis for regulating expression of glucosamine synthase, and insertion of a terminator and a constitutive promoter. The method comprises constructing a deleting cassette of a glmS ribozyme encoding gene, which includes an upstream homologous fragment, a resistance gene, a terminator sequence, a constitutive promoter sequence and a downstream homologous fragment in sequence; and transforming the deleting cassette into Bacillus subtilis, to obtain the recombinant Bacillus subtilis. In the invention, glucosamine synthase gene (glmS) ribozyme is deleted by homologous recombination, and in host cells, GlcN6P feedback inhibition of expression of glucosamine synthase gene glmS is blocked, and the accumulation of acetylglucosamine is improved.

Abstract: The present invention provides a method for improving the quality of soy sauce using Bacillus amyloliquefaciens, which relates to the field of microbiology and food technology. The method comprises inoculating Bacillus amyloliquefaciens BBE JY06 during soy sauce fermentation. With Bacillus amyloliquefaciens addition during soy sauce fermentation, the aroma and tastes of soy sauce is improved and the ethyl carbamate content in soy sauce is decreased as well.

Abstract: The invention discloses a method for improving citric acid production by Aspergillus niger fermentation, which integrates Aspergillus niger GABA pathway succinate semialdehyde dehydrogenase SSD gene into Aspergillus niger genome to obtain recombinant Aspergillus niger strain, and uses recombinant black The Aspergillus strain ferments to produce citric acid; the expression of the succinate semialdehyde dehydrogenase SSD gene is regulated by the Pgas promoter. The method of the invention realizes the expression of succinate semialdehyde dehydrogenase SSD in Aspergillus niger to enhance the GABA pathway so as to strengthen the TCA cycle and promote the synthesis of citric acid.

Abstract: The invention discloses a method for increasing citrate production from genome reconstructed Aspergillus niger. The method is to insert a gene of low affinity glucose transporter, LGT1, to genome of A. niger. The expression level of LGT1 is under control of promoter Pgas. The genome reconstructed A. niger is tolerant to higher fermentation temperature and lower pH than that of the parental strain. Moreover, the production, yield and purity of product from reconstructed A. niger are higher than that of parental strain, and the fermentation time is shorter.

Abstract: The invention discloses a promoter which can be induced to express in acidic conditions, and relates to the field of bioengineering technology. The promoters of the invention are separated from A. niger and can actuate and/or regulate the expression of the effectively connected nucleic acids in A. niger. In the invention the expression of the promoters is studied in A. niger, and it is indicated that some promoters show weak expression, and some show strong activity. The invention provides an effective method and new thought for organic acids production by fungi or other products produced by fermentation under acidic conditions.

Abstract: The present invention provides a recombinant Bacillus subtilis for producing acetylglucosamine and construction method thereof. The recombinant Bacillus subtilis is obtained by deletion of glmS ribozyme of Bacillus subtilis for regulating expression of glucosamine synthase, and insertion of a terminator and a constitutive promoter. The method comprises constructing a deleting cassette of a glmS ribozyme encoding gene, which includes an upstream homologous fragment, a resistance gene, a terminator sequence, a constitutive promoter sequence and a downstream homologous fragment in sequence; and transforming the deleting cassette into Bacillus subtilis, to obtain the recombinant Bacillus subtilis. In the invention, glucosamine synthase gene (glmS) ribozyme is deleted by homologous recombination, and in host cells, GlcN6P feedback inhibition of expression of glucosamine synthase gene glmS is blocked, and the accumulation of acetylglucosamine is improved.

Abstract: The present invention provides a method for high-efficiency production of pinoresinol by use of an H2O2 auto-scavenging enzymatic cascade. It uses eugenol as the substrate, which is relatively inexpensive and is industrially available. It uses an enzymatic cascade to remove H2O2 produced in the process of pinoresinol synthesis, thereby reducing its inhibitory effect on the enzyme activity. In addition, the present invention uses whole cells as a catalyst, which can continuously regenerate cofactors needed by the enzyme, thus eliminating the need for exogenous addition of expensive cofactors during the reaction. The yield of the present invention can reach 7.12 g/L and the conversion rate is 61.55%.

Abstract: The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals field. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCI 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 invention provides a method for reducing EC precursor, citrulline, in soy sauce by inoculating T. halophilus BBE R23 during soy sauce fermentation. Compared to the control, citrulline and EC in soy sauce prepared with T. halophilus BBE R23 is decreased by 86% and 50%, respectively. Addition of 106 CFU/mL T. halophilus BBE R23 also increases volatile flavoring compounds and free fatty acids concentration in the soy sauce product. The present invention provides a method of making soy sauce that not only reduces accumulation of EC precursor citrulline but also improves soy sauce flavor.