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 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 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 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: Disclosed is a method for producing L-citrulline using recombinant Corynebacterium crenatum cells as whole-cell biocatalysts. The present invention provides a recombinant C. crenatum that expresses an exogenous arginine deiminase gene from Lactobacillus brevis. The recombinant C. crenatum SDNN403 is used as biocatalysts for converting L-arginine to produce L-citrulline. Using the method of the invention, the concentration of L-citrulline reached 301.4 g/L after a 48 hr conversion reaction, and the molar conversion rate reached 99.9%.

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: Disclosed is a method for producing L-citrulline using recombinant Corynebacterium crenatum cells as whole-cell biocatalysts. The present invention provides a recombinant C. crenatum that expresses an exogenous arginine deiminase gene from Lactobacillus brevis. The recombinant C. crenatum SDNN403 is used as biocatalysts for converting L-arginine to produce L-citrulline. Using the method of the invention, the concentration of L-citrulline reached 301.4 g/L after a 48 hr conversion reaction, and the molar conversion rate reached 99.9%.