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 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 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 method for enhancing N-acetylglucosamine production by usage of a recombinant Bacillus subtilis with a glcK knockout. This invention enhanced the production of GlcNAc by knocking out the glcK gene which encodes a glucokinase, thus eliminating the GlcNAc phosphorylation to GlcNAc-6-P. The specific growth rate and content of GlcNAc in the supernatant of the recombinant Bacillus subtilis with the glcK knockout were 0.15 h?1 and 3.0 g/L, respectively, which were 2.32 times and 2.14 times of those of the control strain without glcK knockout. The recombinant Bacillus subtilis of the present invention would be potentially useful for industrial production of GlcNAc.

Abstract: The present invention provides a mutant with enhanced secretion of L-asparaginase and its application, which relates to the field of enzyme engineering. This invention successfully constructed a mutant with enhanced secretion of L-asparaginase through N-terminal deletion on amino acid sequence of L-asparaginase, and then expressed the mutant via a recombinant strain. Compared with the wild L-asparaginase, the secretion ability of L-asparaginase mutant in present invention significantly improves by 3.14 times compared to that of wild type. The recombinant strain that constructed in this prevention has an L-asparaginase yield of 407.6 U/mL and a production efficiency of 9.26 U/(mL/h), which reaches to the highest yield been reported.

Abstract: The present invention relates to a mutant strain of Lactococcus lactis and its application, and belongs to the field of food biotechnology. The mutant strain, Lactococcus lactis WH101, was screened for its better tolerance to harsh environmental factors, especially higher acid tolerance. The OD600 of Lactococcus lactis WH101 was increased by 5.5 times than that of the parent strain when cultured at pH 4.5. The survival rate of the mutant strain was 22.4 times higher than that of the parent strain after 3 hr treatment in pH 4.0 culture medium. The survival rate of the mutant strain was 5.2, 2.0 and 1.9 times over that of the parent strain treated with 15% ethanol for 4 hr, 15% NaCl for 6 hr and 1 mM H2O2 for 3 hr, respectively.

Abstract: The invention provides an effective method for improving N-acetylglucosamine (GlcNAc) production by engineered B. subtilis Deletion of phosphoenolpyruvate carboxykinase encoding gene pckA and encoding pyruvate kinase gene pyK in recombinant GlcNAc-producing strain BSGNK-PxylA-glmS-P43-GNA1 (BSGNK) is first performed to enhance GlcNAc production, followed by overexpression of pyruvate carboxylase encoding gene pycA for facilitating cell growth. Finally, the GlcNAc production of the recombinant strain BPTS3 reached to 11.3 g/L, which was 1.84-fold of BSGNK. This method can be used for improve cellular property of engineered B. subtilis for GlcNAc production, which can be further applied to industrial production of GlcNAc.

Abstract: The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from Streptococcus zooepidemicus and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g·L?1. Moreover, HAs with a broad range of molecular weights (103 Da to 106 MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

Abstract: The present invention provides a method for scarless in vitro DNA assembly using thermostable exonucleases and ligase, which relates to the field of genetic engineering. The present invention provides a fast method for assembling DNA subfragments with homologous ends, which employs thermostable polymerases and ligase in a thermal cycle of denaturation, annealing, digestion and ligation. After denaturation, DNA subfragments are assembled together via annealing of the homologous end sequences, the unpaired single-stranded overhangs are digested by polymerases, and the resulting nicked gaps are sealed by a ligase. Using this method, 2-6 DNA subfragments were successfully assembled within two hours. This method can be used in conventional DNA recombination and be adapted to high throughput assembly operations. In addition, combinatorial mutations can be easily introduced into the assembled sequence by use of primers with mutated bases.

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 mutant with enhanced secretion of L-asparaginase and its application, which relates to the field of enzyme engineering. This invention successfully constructed a mutant with enhanced secretion of L-asparaginase through N-terminal deletion on amino acid sequence of L-asparaginase, and then expressed the mutant via a recombinant strain. Compared with the wild L-asparaginase, the secretion ability of L-asparaginase mutant in present invention significantly improves by 3.14 times compared to that of wild type. The recombinant strain that constructed in this prevention has an L-asparaginase yield of 407.6 U/mL and a production efficiency of 9.26 U/(mL/h), which reaches to the highest yield been reported.

Abstract: The invention provides an effective method for improving N-acetylglucosamine (GlcNAc) production by engineered B. subtilis Deletion of phosphoenolpyruvate carboxykinase encoding gene pckA and encoding pyruvate kinase gene pyK in recombinant GlcNAc-producing strain BSGNK-PxylA-glmS-P43-GNA1 (BSGNK) is first performed to enhance GlcNAc production, followed by overexpression of pyruvate carboxylase encoding gene pycA for facilitating cell growth. Finally, the GlcNAc production of the recombinant strain BPTS3 reached to 11.3 g/L, which was 1.84-fold of BSGNK. This method can be used for improve cellular property of engineered B. subtilis for GlcNAc production, which can be further applied to industrial production of GlcNAc.

Abstract: The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from Streptococcus zooepidemicus and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g·L?1. Moreover, HAs with a broad range of molecular weights (103 Da to 106 MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

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 present invention provides a method for enhancing N-acetylglucosamine production by usage of a recombinant Bacillus subtilis with a glcK knockout. This invention enhanced the production of GlcNAc by knocking out the glcK gene which encodes a glucokinase, thus eliminating the GlcNAc phosphorylation to GlcNAc-6-P. The specific growth rate and content of GlcNAc in the supernatant of the recombinant Bacillus subtilis with the glcK knockout were 0.15 h?1 and 3.0 g/L, respectively, which were 2.32 times and 2.14 times of those of the control strain without glcK knockout. The recombinant Bacillus subtilis of the present invention would be potentially useful for industrial production of GlcNAc.

Abstract: The present invention provides a method for decreasing pyruvate catabolism and increasing the accumulation of pyruvate in microbes. By overexpressing wild type dihydrolipoamide acetyltransferase or dihydrolipoamide acetyltransferase mutants which have mutations at conservative active sites, the present invention provide a method to decrease overall activity of pyruvate dehydrogenase complex and pyruvate catabolism, and thus increase the accumulation of extracellular pyruvate without killing the pyruvate-producing microbes. Overexpressing dihydrolipoamide acetyltransferase mutants is an effective way to increase pyruvate accumulation.

Abstract: The present invention provides a method of rapid directed DNA evolution based on single-stranded combinatorial DNA mutant library. The single- and double-stranded mutant library are constructed either separately or simultaneously using editing primers that contain mutated nucleotides and are targeted to different regions of the parent DNA sequence. The mutant library is then inserted into expression vectors and mutants with desired property are obtained by high throughput screening. Evolution of promoters, enzymes and metabolic pathways were successfully achieved using this method and mutants with excellent properties were obtained. The method of the present invention is simple, rapid, and efficient. It can be used for directed evolution of regulatory sequence such as promoters and ribosome binding sites, and is especially suitable for introducing diverse mutations into protein encoding genes, leading to rapid directed evolution of gene of interest.

Abstract: The present invention provides a genetically engineered Torulopsis glabrata with enhanced extracellular secretion of pyruvic acid. T. glabrata strain was obtained from China Center for Type Culture Collection with CCTCC No: M202019 and over-expressed the optimized CutA (SEQ ID NO:2) encoding stress protein. Both of the temperature tolerance of T. glabrata and extracellular concentration of pyruvate were improved by overexpressing the optimized CutA. The optimum growth temperature of genetically engineered T. glabrata was increased too. The present invention can be widely used to increase extracellular levels of pyruvate during the fermentation process.

Abstract: The present invention provides methods for enhancing ?-KG production in Yarrowia lipolytica, relates to the field of metabolic engineering. This invention successfully overexpresses the glutamate dehydrogenase in wild type strain Y. lipolytica WSH-Z06 to construct the recombinant Y. lipolytica WSH-Z06 which regulates the glutamate catabolism to synthesis ?-KG. L-methionine imine is added into the fermentation medium during the process to strengthen the supply of intracellular glutamate and inhibite the intracellular glutamine synthesis from glutamate metabolism and then enhance the accumunation of ?-KG. Therefor, the present invention provides an effective method for enhancing the accumunation of ?-KG through regulation of intracellular amino acid metabolism.

Abstract: The present invention provides a method for decreasing pyruvate catabolism and increasing the accumulation of pyruvate in microbes. By overexpressing wild type dihydrolipoamide acetyltransferase or dihydrolipoamide acetyltransferase mutants which have mutations at conservative active sites, the present invention provide a method to decrease overall activity of pyruvate dehydrogenase complex and pyruvate catabolism, and thus increase the accumulation of extracellular pyruvate without killing the pyruvate-producing microbes. Overexpressing dihydrolipoamide acetyltransferase mutants is an effective way to increase pyruvate accumulation.