Abstract: The present disclosure discloses recombinant Escherichia coli and application thereof in screening erythritol-producing strains, and belongs to the technical field of microorganisms. The recombinant Escherichia coli used in a method for screening an erythritol-producing strain disclosed by the present disclosure can well perform positive correlation induction on erythritol with different concentrations, so that the method for screening the erythritol-producing strain has the advantage of high sensitivity.

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 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.

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 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 relates to a recombinant strain of Bacillus subtilis, wherein pyruvate carboxylase BalpycA, glyceraldehyde-3-phosphate ferredoxin dehydrogenase gor, isocitrate NAD+ dehydrogenase icd, malate quinone dehydrogenase mqo, pyruvate ferredoxin oxidoreductase porAB and nitrogenase ferritin cyh are integrated and expressed in the recombinant strain. The invention also discloses use of the recombinant strain in fermentation production of acetylglucosamine. The recombinant Bacillus subtilis of the invention eliminates the central carbon metabolism overflow of the Bacillus subtilis and balances the intracellular reducing force, and the fermentation yield of acetylglucosamine is greatly improved.

Abstract: The disclosure discloses recombinant Bacillus subtilis for synthesizing guanosine diphosphate fucose and a construction method and application thereof. The recombinant Bacillus subtilis is obtained by intensively expressing guanylate kinase and nucleotide diphosphokinase genes and expressing exogenous fucokinase and phosphate guanylyltransferase genes in a genome of Bacillus subtilis 168. According to the disclosure, a bacterial strain for synthesizing the guanosine diphosphate fucose is obtained by reconstructing the Bacillus subtilis 168, with a volume of intracellular accumulation up to 196.15 g/L. According to the disclosure, by intensively expressing the guanylate kinase and nucleotide diphosphokinase genes, and enhancing the supply of intracellular GDP-L-fucose composition cofactors, the synthesis of the guanosine diphosphate fucose is promoted. The construction method for the recombinant Bacillus subtilis of the disclosure is simple and convenient to use, thus having good application prospects.

Abstract: The disclosure discloses recombinant Bacillus subtilis for synthesizing e lacto-N-neotetraose yield. The recombinant Bacillus subtilis is obtained by integrating two ?-1,4-galactotransferase genes on a genome of a host bacterium Bacillus subtilis 168?amyE:P43-lacY, P43-lgtB, PxylA-comK and exogenously expressing a ?-1,3-N-glucosaminotransferase gene. Compared with a strain before transformation, the recombinant Bacillus subtilis of the disclosure improves the yield of the synthesized lacto-N-neotetraose from 720 mg/L to 1300 mg/L, laying a foundation for further metabolic engineering transformation of Bacillus subtilis for producing the lacto-N-neotetraose.

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 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 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 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 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 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 invention relates to a recombinant strain of Bacillus subtilis, wherein pyruvate carboxylase BalpycA, glyceraldehyde-3-phosphate ferredoxin dehydrogenase gor, isocitrate NAD+ dehydrogenase icd, malate quinone dehydrogenase mqo, pyruvate ferredoxin oxidoreductase porAB and nitrogenase ferritin cyh are integrated and expressed in the recombinant strain. The invention also discloses use of the recombinant strain in fermentation production of acetylglucosamine. The recombinant Bacillus subtilis of the invention eliminates the central carbon metabolism overflow of the Bacillus subtilis and balances the intracellular reducing force, and the fermentation yield of acetylglucosamine is greatly improved.

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 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 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 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 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.