Abstract: The invention provides a glutamate decarboxylase mutant with improved pH tolerance and use thereof in synthesis of gamma-aminobutyric acid. The mutant is obtained by mutating glutamate decarboxylase having an amino acid sequence as shown in SEQ ID NO. 3. The enzyme activity of the mutant at pH 6.5 is improved to 178% of the original enzyme (SEQ ID NO. 3). The final yield of 1000 g of substrate fed in batches in a 5L tank for 12 h is up to 688.13 g/L, which is about 52% higher than the productivity of the original glutamate decarboxylase. The final molar conversion rate can reach 98.2%. The invention not only broadens the enzyme activity of GAD under the optimum pH, but also broadens the enzyme activity of GAD under the neutral pH, and enhances the capability of the GAD to synthesize gamma-aminobutyric acid, and therefore is more suitable for industrial production.

Abstract: The present disclosure provides a recombinant nucleic acid of Escherichia coli, a recombinant E. coli, a culturing method thereof, and a method for biosynthesizing L-threonine thereby, and relates to the technical field of bioengineering. The recombinant nucleic acid of E. coli of the present disclosure, including the gene encoding phosphoenolpyruvate carboxykinase (pck), the gene encoding pyruvate carboxylase (pyc) and the gene encoding threonine operon, is transformed into E. coli to obtain a recombinant E. coli LMT4 strain that takes glucose as a substrate. Using the LMT4 for fermentative production may significantly improve the L-threonine yield and glucose conversion rate, laying a foundation for the industrial production of L-threonine.

Abstract: The present disclosure provides a Bacillus subtilis strain, a recombinant B. subtilis strain and use thereof, belongs to the technical field of microbial fermentation. B. subtilis strain RF1-6 provided by the present disclosure is a mutant strain with the highest riboflavin yield screened by gene modification and mutagenesis using a high riboflavin-producing strain RF1 as a starting strain, deposited at China Center for Type Culture Collection (CCTCC) under accession number M 2022565. Riboflavin yield can be increased by 22.8% compared with that of the high riboflavin-producing strain RF1.

Abstract: The disclosure relates to a sucrose phosphorylase mutant with improved enzyme activity, and construction method thereof and use thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the mutant of the disclosure is as shown in SEQ ID NO: 1. The mutant of the disclosure is based on sucrose phosphorylase derived from Leuconostoc mesenteroides, and subjected to site-directed mutagenesis to improve the enzyme activity of sucrose phosphorylase. The mutant is expressed in Corynebacterium glutamicum and used as a whole cell catalyst to produce 2-O-?-D-glycerol glucoside. At a 5 L fermentation tank level, a large quantity of 2-O-?-D-glycerol glucoside can be produced efficiently in a short time, which is conducive to expanding the prospect of industrial application of sucrose phosphorylase for the production of 2-O-?-D-glycerol glucoside and realizing its large-scale industrial application.

Abstract: The disclosure discloses 17?-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17?-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17?-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17?-hydroxysteroid dehydrogenase (1.66 U/mg).

Abstract: The present invention discloses a method for producing a 1,2-amino alcohol compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microorganism engineering. According to the present invention, engineered Escherichia coli co-expresses epoxide hydrolase, alcohol dehydrogenase, ?-transaminase and glutamate dehydrogenase, is capable of realizing whole-cell catalysis of an epoxide in one step to synthesize a 1,2-amino alcohol compound, and meanwhile, can realize regeneration of coenzyme NADP+ and an amino doner L-Glu; alcohol dehydrogenase expressed by the engineered Escherichia coli is RBS optimized alcohol dehydrogenase, and such RBS optimization can control the expression quantity of alcohol dehydrogenase, so that the catalysis rate of alcohol dehydrogenase and transaminase can achieve an optimum ratio, to eliminate influence caused by a rate-limiting step in a catalyzing course.

Abstract: The present disclosure discloses a method for producing 9?-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion, and belongs to the fields of gene engineering and enzyme engineering. According to the present disclosure, oxidation subunit KshA, reduction subunit KshB and unknown active subunit KshC of 3-ketosteroid-9?-hydroxylase sourcing from Mycobacterium sp. Strain VKM Ac-1817D are successfully expressed in E. coli BL21, and KshC is identified as an oxidation subunit, the enzyme activity of which is far higher than that of KshA. BL21/pET-28a(+)-fdh constructed in the laboratory is used for expressing formate dehydrogenase (FDH), and by using crude enzyme liquid of KSH (KshB+KshC) and FDH engineering bacteria as a biocatalyst and a steroidal compound (AD) as a substrate, optimum reaction temperature is determined as 30° C. and optimum pH is determined as 7.0. In optimum conditions, AD is converted to produce a product 9-OH-AD, and within 20 hours, the output of 9-OH-AD is 4.

Abstract: The present disclosure discloses a thermophilic L-asparaginase mutant and screening and fermentation methods thereof, and belongs to the field of gene engineering, enzyme engineering and fermentation engineering. In Bacillus subtilis 168, a Pyrococcus yayanosii CH1-derived L-asparaginase encoding gene is used as a template, and a mutation library is constructed by an error-prone PCR (epPCR) technology. A mutant strain with improved specific enzyme activity is screened through a high-flux screening method of synchronous cell disruption and enzyme activity measurement. Mutated residues included in a positive mutant are analyzed to construct a composite mutant strain S17G/A90S/R156S/K272A with improved specific enzyme activity and specific enzyme activity of 3108 U/mg. An expression quantity of the composite mutant strain in the Bacillus subtilis 168 is increased through measures of a strong promoter P43 and RBS optimization.

Abstract: The disclosure relates to a sucrose phosphorylase mutant with improved enzyme activity, and construction method thereof and use thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the mutant of the disclosure is as shown in SEQ ID NO: 1. The mutant of the disclosure is based on sucrose phosphorylase derived from Leuconostoc mesenteroides, and subjected to site-directed mutagenesis to improve the enzyme activity of sucrose phosphorylase. The mutant is expressed in Corynebacterium glutamicum and used as a whole cell catalyst to produce 2-O-?-D-glycerol glucoside. At a 5L fermentation tank level, a large quantity of 2-O-?-D-glycerol glucoside can be produced efficiently in a short time, which is conducive to expanding the prospect of industrial application of sucrose phosphorylase for the production of 2-O-?-D-glycerol glucoside and realizing its large-scale industrial application.

Abstract: The disclosure discloses 17?-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17?-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17?-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17?-hydroxysteroid dehydrogenase (1.66 U/mg).

Abstract: The present invention provides an aspartase mutant, a recombinant expression vector and recombinant bacterium containing the aspartase mutant, and the use thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the aspartase mutant is as set forth in SEQ ID NO: 1. In the aspartase mutant of the present invention, on the basis of wild type aspartase (with an amino acid sequence as set out in SEQ ID NO: 3), glutamic acid at position 427 is mutated into glutamine. In the present invention, by mutating the amino acid residue at position 427 into glutamine, the polar environment near an active site is changed, and thus ammonia supply during substrate reaction is further facilitated, thereby improving an enzyme activity, enhancing the ability of the enzyme in synthesizing a ?-amino acid, and providing a practical and effective strategy for industrial production of the ?-amino acid.

Abstract: The present invention discloses a Mycobacterium neoaurum-derived steroid C27-monooxygenase and an application thereof, which belong to the technical fields of genetic engineering and enzyme engineering. By the method of gene knockout and intensive expression, the present invention screens out three isoenzymes of a key enzyme SMO in the process of degrading sterol side chains from Mycobacterium neoaurum. The three isoenzymes are intensively expressed respectively in the Mycobacterium neoaurum for the high yield of androsta-1,4-diene-3,17-dione (ADD), the yield of ADD is increased remarkably, wherein the effect of SMO2 is most remarkable. By overexpressing SMO2, the final ADD yield is increased from 5.2 g·L?1 to 7.3 g·L?1. The present invention provides a helpful guidance for the industrialization of the microbial fermentation method for increasing the ADD yield.

Abstract: The present invention discloses a formate dehydrogenase mutant with improved enzyme activity and stability and a construction method thereof, which belongs to the technical field of genetic engineering. The mutant of the present invention is obtained by mutating alanine at a 10th site to cysteine based on the amino acid shown in SEQ ID NO. 2. The specific enzyme activity of the mutant enzyme obtained by the present invention is improved by 1.3 times compared with that before the mutation, a half-life period (t1/2) at 60° C. is increased by 6.8 times compared with that in the mutation period, the copper ion tolerance is increased by 30 times compared with that before the mutation, and when pH is 4, the stability is improved by 2.0 times, and the catalytic efficiency is increased by 1.4 times.

Abstract: The present invention discloses a method for producing a 1,2-amino alcohol compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microorganism engineering. According to the present invention, engineered Escherichia coli co-expresses epoxide hydrolase, alcohol dehydrogenase, ?-transaminase and glutamate dehydrogenase, is capable of realizing whole-cell catalysis of an epoxide in one step to synthesize a 1,2-amino alcohol compound, and meanwhile, can realize regeneration of coenzyme NADP+ and an amino doner L-Glu; alcohol dehydrogenase expressed by the engineered Escherichia coli is RBS optimized alcohol dehydrogenase, and such RBS optimization can control the expression quantity of alcohol dehydrogenase, so that the catalysis rate of alcohol dehydrogenase and transaminase can achieve an optimum ratio, to eliminate influence caused by a rate-limiting step in a catalyzing course.

Abstract: The present invention discloses a single-cell factory for efficiently synthesizing ?-aminobutyric acid and construction and application thereof, which belong to the technical field of microorganisms. The present invention expresses an L-threonine deaminase gene, an L-amino acid dehydrogenase gene and a dehydrogenase gene for providing cofactor NADH cycle in tandem to construct a recombinant Escherichia coli single-cell factory which is used for efficiently synthesizing ?-aminobutyric acid. The expression level of the L-threonine deaminase is optimized and controlled by an RBS sequence, so that the problem of transformation inhibition caused by the rapid accumulation of an intermediate product ketobutyric acid is solved, moreover, the expression level of the dehydrogenase for providing cofactor NADH cycle is optimized and controlled by a promoter and an RBS sequence, consequently, the NADH regeneration rate is increased, and ultimately, yield is increased.

Abstract: The invention discloses a method for increasing the yield of L-arginine by knocking out flavin reductases, and belongs to the technical field of amino acid production by microbial fermentation. Genes frd1 and frd2 for encoding hypothetic NADPH-dependent FMN reductase in Corynebacterium crenatum SDNN403 are over-expressed in E. coli BL21 and are purified to form target proteins Frd181 and Frd188, and functions of the target proteins are identified to obtain a result showing that the proteins Frd181 and Frd188 both are NAD(P)H-dependent flavin reductases producing H2O2. By taking a genome of the Corynebacterium crenatum SDNN403 as a template, frd1 and frd2 gene deletion fragments are obtained by overlap extension PCR; connecting pK18mobsacB to obtain knockout plasmids pK18mobsacB-?frd1 and pK18mobsacB-?frd2; carrying out electric shock to transform the Corynebacterium crenatum SDNN403; and carrying out secondary screening to obtain recombinant strains 403?frd1 and 403?frd2.

Abstract: The present invention relates to application of a novel signal peptide in L-arginine and its derivatives production from konjac powder, which belongs to the field of gene engineering, enzyme engineering and metabolism engineering. The present invention fused the signal peptide set forth in SEQ ID NO.1 with the ?-mannanase of Bacillus subtilis CCTCC M 209200, and expressed the fused gene in the strain with high L-arginine yield. The recombinant strain Corynebacterium crenatum CGMCC 0890/p MSPman had advantages on utilizing cheaper konjac powder as substrate, and after fermenting for 96 hours in a 5 L bioreactor, the L-arginine yield reached 45 g/L. Another two recombinant strains were constructed based on Corynebacterium crenatum CGMCC 0890/pMSPman, and after fermenting for 96 hours in a 5 L bioreactor, the L-ornithine yield and L-citrulline reached 23.5 g/L and 26.3 g/L respectively.

Abstract: The present invention relates to application of a novel signal peptide in L-glutamate and its derivatives production from konjac powder, which belongs to the field of gene engineering, enzyme engineering and metabolism engineering. The signal peptide which mediated secretion of ?-mannanase was invented, and the recombinant strain with this signal peptide had advantages on utilizing konjac powder to produce related products, and its utilization efficiency of konjac powder, production efficiency, and yield were higher than other signal peptides. The recombinant strain possessing this new signal peptide had advantages on utilizing cheaper konjac powder as substrate to lower the process costs on L-glutamic acid and its high-value-added products.

Abstract: The present disclosure discloses a method for producing 9?-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion, and belongs to the fields of gene engineering and enzyme engineering. According to the present disclosure, oxidation subunit KshA, reduction subunit KshB and unknown active subunit KshC of 3-ketosteroid-9?-hydroxylase sourcing from Mycobacterium sp. Strain VKM Ac-1817D are successfully expressed in E. coli BL21, and KshC is identified as an oxidation subunit, the enzyme activity of which is far higher than that of KshA. BL21/pET-28a(+)-fdh constructed in the laboratory is used for expressing formate dehydrogenase (FDH), and by using crude enzyme liquid of KSH (KshB+KshC) and FDH engineering bacteria as a biocatalyst and a steroidal compound (AD) as a substrate, optimum reaction temperature is determined as 30° C. and optimum pH is determined as 7.0. In optimum conditions, AD is converted to produce a product 9-OH-AD, and within 20 hours, the output of 9-OH-AD is 4.

Abstract: The present disclosure discloses a thermophilic L-asparaginase mutant and screening and fermentation methods thereof, and belongs to the field of gene engineering, enzyme engineering and fermentation engineering. In bacillus subtilis 168, a Pyrococcus yayanosii CH1-derived L-asparaginase encoding gene is used as a template, and a mutation library is constructed by an error-prone PCR (epPCR) technology. A mutant strain with improved specific enzyme activity is screened through a high-flux screening method of synchronous cell disruption and enzyme activity measurement. Mutated residues included in a positive mutant are analyzed to construct a composite mutant strain S17G/A90S/R156S/K272A with improved specific enzyme activity and specific enzyme activity of 3108 U/mg. An expression quantity of the composite mutant strain in the bacillus subtilis 168 is increased through measures of a strong promoter P43 and RBS optimization.