Abstract: The present invention provides a bacterial continuous evolution system, an orthogonal error-prone DNA polymerase, and a continuous evolution method. In the present invention, by combining an orthogonal DNA replication system and an orthogonal error-prone DNA polymerase, a continuous evolution method that includes all mutant types, enables long-DNA-fragment mutation, and is good in continuity and simple and convenient to operate is obtained. By inducing the opening and closing of DNA polymerase expression, switching between a linear plasmid error-prone mutation process and a high-fidelity replication process is realized, so as to achieve the efficient and continuous evolution of a target DNA sequence.

Abstract: The invention provides a Saccharomyces cerevisiae strain for producing a human milk lipid substitute. By integrating a heterologous lysophosphatidic acid acyltransferase into Saccharomyces cerevisiae and knocking out its own natural lysophosphatidic acid acyltransferase, the content of palmitic acid (C16:0) at Sn-2 position of triacylglycerol produced by Saccharomyces cerevisiae is increased, to synthesize a human milk lipid substitute. On this basis, a metabolic pathway related gene is knocked out, to further increase the content of human milk lipid substitute in the product. In the present invention, a human milk lipid substitute is de novo synthesized by Saccharomyces cerevisiae for the first time, in which the total fatty acid is 15% or more, and the relative content of C16:0 at Sn-2 position reaches about 60%.

Abstract: The invention provides a method for increasing the yield of 7-dehydrocholesterol in yeast by using compartmentalization. The method includes steps of: by taking yeast as a starting strain, expressing heterologous sterol delta 24-reductase and cholestenol delta-isomerase, wherein the yield of 7-DHC in Saccharomyces cerevisiae S288C is detected to be 10.15 mg/L. According to the method of the invention, partial enzymes in a 7-DHC synthesis path are positioned in compartments in Saccharomyces cerevisiae by using a peroxisome and a mitochondrial positioning tag, and a relatively independent 7-DHC synthesis path is formed. Meanwhile, the storage space of precursor substances needed by 7-DHC synthesis is increased, the feedback effect is reduced, the conversion efficiency between enzymes is improved in the same compartment, the loss of the acting substrate is reduced, and finally the yield of the 7-DHC is improved by 4 times and reaches 53.31 mg/L.

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 provides a pyruvate-responsive biosensor and a construction method and use thereof. In the present invention, pyruvate-responsive biosensors with different dynamic ranges are successfully constructed by optimizing the PdhR binding sequence inserted on the P43 promoter and optimizing the insertion site, wherein the minimum increase in dynamic range is by 0.6 time, and the maximum increase is by 30.7 times. The pyruvate-responsive biosensors are useful in the precise control of the expression of each gene in the cell. Since pyruvate is a key metabolite of central carbon in the cells, these biosensors are capable of dynamically regulating the expression level of intracellular genes according to changes in the content of pyruvate in the cells, thereby achieving the dynamic control of intracellular metabolic flux. The pyruvate biosensor obtained in the present invention has a good specificity, and a response range to pyruvate of 10-35 nmol/g DCW.

Abstract: The present invention provides an acid-tolerant Saccharomyces cerevisiae strain and use thereof. By using exogenously added malic acid as a stress, an acid-tolerant mutant S. cerevisiae strain MTPfo-4 is obtained by directed evolution screening in the laboratory, which tolerates a minimum pH of 2.44. The mutant strain MTPfo-4, tolerant to multiple organic acids, has an increased tolerance to exogenous malic acid of up to 86.6 g/L. The mutant strain MTPfo-4 obtained is further identified. The mutant strain grows stably and well, and can tolerate a variety of organic acids (lactic acid, malic acid, succinic acid, fumaric acid, citric acid, gluconic acid, and tartaric acid). It also has a strong tolerance to inorganic acids (HCl and H3PO4). This is difficult to achieve in the existing research and reports of S. cerevisiae. The strain is intended to be used as an acid-tolerant chassis cell factory for producing various short-chain organic acids.

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 present invention provides a recombinant Corynebacterium glutamicum producing N-acetylglucosamine and use thereof. The recombinant Corynebacterium glutamicum is obtained by overexpressing, in Corynebacterium glutamicum, the transcription factor SugR derived therefrom. The recombinant Corynebacterium glutamicum of the present invention increases the production of acetylglucosamine to up to 26 g/L, and lays a foundation for further metabolic engineering of Corynebacterium glutamicum to produce glucosamine.

Abstract: The present invention discloses a RamA transcription factor mutant for promoting the production of N-acetylglucosamine and use thereof. The mutant is obtained by mutating lysine at position 90 to asparagine and serine at position 92 to lysine in a parent having an amino acid sequence as shown in SEQ ID NO: 2. The present invention provides a genetically engineered strain that overexpresses the RamA transcription factor mutant and increases the production of N-acetylglucosamine. By overexpressing the transcription factor RamA that is involved in the regulation of carbon metabolism, the extracellular accumulation of N-acetylglucosamine is increased, with a maximum concentration reaching 31.5 g/L, which lays a foundation for further metabolic engineering of Corynebacterium glutamicum to produce glucosamine. The method for constructing recombinant Corynebacterium glutamicum of the invention is simple, and convenient in use, and thus has good application prospects.

Abstract: The present invention provides a recombinant Bacillus subtilis with improved 2?-fucosyllactose production, and a construction method thereof. In the present invention, a strain capable of efficiently synthesizing 2?-fucosyllactose is obtained by the fusion expression of the fucosyltransferase gene and the L-fucokinase/guanosine 5?-diphosphate-L-fucose pyrophosphorylase gene in Bacillus subtilis BSGL-FF, the fermentation supernatant of which comprises a cumulative amount of 2?-fucosyllactose as high as 1.62 g/L, which is 55% higher than the amount achieved with the control strain. The construction method of the recombinant Bacillus subtilis of the present invention is simple, and convenient to use, and thus has good application prospects.

Abstract: The present invention provides a recombinant Corynebacterium glutamicum producing N-acetylglucosamine and use thereof. The recombinant Corynebacterium glutamicum is obtained by overexpressing, in Corynebacterium glutamicum, the transcription factor SugR derived therefrom. The recombinant Corynebacterium glutamicum of the present invention increases the production of acetylglucosamine to up to 26 g/L, and lays a foundation for further metabolic engineering of Corynebacterium glutamicum to produce glucosamine.

Abstract: The present invention provides a pyruvate-responsive biosensor and a construction method and use thereof. In the present invention, pyruvate-responsive biosensors with different dynamic ranges are successfully constructed by optimizing the PdhR binding sequence inserted on the P43 promoter and optimizing the insertion site, wherein the minimum increase in dynamic range is by 0.6 time, and the maximum increase is by 30.7 times. The pyruvate-responsive biosensors are useful in the precise control of the expression of each gene in the cell. Since pyruvate is a key metabolite of central carbon in the cells, these biosensors are capable of dynamically regulating the expression level of intracellular genes according to changes in the content of pyruvate in the cells, thereby achieving the dynamic control of intracellular metabolic flux. The pyruvate biosensor obtained in the present invention has a good specificity, and a response range to pyruvate of 10-35 nmol/g DCW.

Abstract: The present invention provides an acid-tolerant Saccharomyces cerevisiae strain and use thereof. By using exogenously added malic acid as a stress, an acid-tolerant mutant S. cerevisiae strain MTPfo-4 is obtained by directed evolution screening in the laboratory, which tolerates a minimum pH of 2.44. The mutant strain MTPfo-4, tolerant to multiple organic acids, has an increased tolerance to exogenous malic acid of up to 86.6 g/L. The mutant strain MTPfo-4 obtained is further identified. The mutant strain grows stably and well, and can tolerate a variety of organic acids (lactic acid, malic acid, succinic acid, fumaric acid, citric acid, gluconic acid, and tartaric acid). It also has a strong tolerance to inorganic acids (HCl and H3PO4). This is difficult to achieve in the existing research and reports of S. cerevisiae. The strain is intended to be used as an acid-tolerant chassis cell factory for producing various short-chain organic acids.

Abstract: The present invention provides a recombinant Bacillus subtilis with improved 2?-fucosyllactose production, and a construction method thereof. In the present invention, a strain capable of efficiently synthesizing 2?-fucosyllactose is obtained by the fusion expression of the fucosyltransferase gene and the L-fucokinase/guanosine 5?-diphosphate-L-fucose pyrophosphorylase gene in Bacillus subtilis BSGL-FF, the fermentation supernatant of which comprises a cumulative amount of 2?-fucosyllactose as high as 1.62 g/L, which is 55% higher than the amount achieved with the control strain. The construction method of the recombinant Bacillus subtilis of the present invention is simple, and convenient to use, and thus has good application prospects.

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 provides a recombinant Bacillus subtilis for increasing the yield of menaquinone 7 (MK-7) and application thereof, and belongs to the field of genetic engineering. In the present disclosure, 14 recombinant strains BS1-BS14 are constructed through the modification of genes related to the biosynthetic pathway of MK-7 on a chromosome of Bacillus subtilis, wherein BS6-BS14 significantly increase the yield of the MK-7, reaching up to 33.5 mg/L, which is 3.53 times the yield of the original strain of wild-type Bacillus subtilis 168. The present disclosure further provides a method for modifying the MK-7 biosynthetic pathway in microorganisms to increase the yield of the MK-7, providing a theoretical basis for constructing a high-yielding strain of the MK-7.

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 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 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.