The present invention provides a method for promoting N-acetylglucosamine synthesis by using the GlcN6P responsive element. In the present invention, Bacillus subtilis BSGNY-P.sub.veg-glmS-P.sub.43-GNA1 is used as a starting strain, in which a CRISPRi system regulated by GlcN6P responsive element is integrated into the genome to dynamically weaken the N-acetylglucosamine synthesis competitive pathway; a GlcN6P responsive promoter is used to regulate the expression of GNA1 on the plasmid to dynamically regulate the N-acetylglucosamine synthesis pathway; and the key gene alsSD involved in the acetoin synthesis pathway is knocked out. During fed-batch fermentation with this strain in a 15 L fermenter, the production of N-acetylglucosamine reaches 131.6 g/L and no by-product acetoin is accumulated, which lays a foundation for the production of GlcNAc by industrial fermentation.

A genetically engineered bacterial strain that produces N-acetylglucosamine, as well as a method of construction and use thereof. The genetically engineered bacterial strain can ferment N-acetylglucosamine under a condition of 40-50° C. Through knocking out genes for glucosamine 6-phosphate deaminase, N-acetylglucosamine-6-phosphate deacetylase and the N-acetylglucosamine transporter protein from a parental bacterium, an N-acetylglucosamine catabolism pathway is blocked. Moreover, overexpression genes for glucosamine 6-phosphate synthase and glucosamine 6-phosphate acetylase are introduced, enabling extra cellular accumulation of N-acetylglucosamine and high-temperature fermentation of N-acetylglucosamine at a temperature higher than 40° C.

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a cell metabolically engineered for production of a mixture of at least four different neutral non-fucosylated oligosaccharides. Furthermore, the disclosure provides a method for the production of a mixture of at least four different neutral non-fucosylated oligosaccharides by a cell as well as the purification of at least one of the oligosaccharides from the cultivation.

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a method for the production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end by a cell as well as the purification of the di- or oligosaccharide from the cultivation. Furthermore, the disclosure provides a cell metabolically engineered for production of a di- or oligosaccharide with an N-acetylglucosamine at the reducing end.

This disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. This disclosure provides a method for the production of a mixture of at least two different oligosaccharides by a cell as well as the purification of at least one of the oligosaccharides from the cultivation. In addition, this disclosure provides a method for the production of a mixture of at least two different oligosaccharides by a metabolically engineered cell as well as the purification of at least one of the oligosaccharides from the cultivation.

The disclosure is in the technical field of synthetic biology and metabolic engineering. More particularly, the disclosure is in the technical field of cultivation or fermentation of metabolically engineered cells. The disclosure describes a cell metabolically engineered for production of an alpha-1,3 glycosylated form of fucose-alpha1,2-galactose-R (Fuc-a1,2-Gal-R). Furthermore, the disclosure provides a method for the production of an alpha-1,3 glycosylated form of Fuc-a1,2-Gal-R by a cell as well as the purification of the alpha-1,3 glycosylated form Fuc-a1,2-Gal-R from the cultivation.

The disclosure discloses Bacillus subtilis for producing N-acetylneuraminic acid and application thereof, and belongs to the field of genetic engineering. The disclosure optimizes the expression levels of key enzymes in N-acetylneuraminic acid synthesis pathways on genome through promoters of different strength, reduces the protein synthesis pressure caused by the expression of enzymes on cells, and further integrates the three N-acetylneuraminic acids in a same Bacillus subtilis engineering strain. Bacillus subtilis with improved N-acetylneuraminic acid production is obtained, and the production reaches 10.4 g/L at the shake flask level, laying a foundation for further improving the NeuAc production from Bacillus subtilis.

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.Math.L.sup.−1, while that of BSG-AT, BSG-ATP, BSG-ATPB increased to 5.29 g.Math.L.sup.−1, 6.22 g.Math.L.sup.−1 and 8.48 g.Math.L.sup.−1 respectively. The construction method of recombinant Bacillus subtilis is simple, easy to use and has a good application prospect.

The disclosure discloses Bacillus subtilis for producing N-acetylneuraminic acid and application thereof, and belongs to the field of genetic engineering. The disclosure optimizes the expression levels of key enzymes in N-acetylneuraminic acid synthesis pathways on genome through promoters of different strength, reduces the protein synthesis pressure caused by the expression of enzymes on cells, and further integrates the three N-acetylneuraminic acids in a same Bacillus subtilis engineering strain. Bacillus subtilis with improved N-acetylneuraminic acid production is obtained, and the production reaches 10.4 g/L at the shake flask level, laying a foundation for further improving the NeuAc production from Bacillus subtilis.

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.sup.1, while that of BSG-AT, BSG-ATP, BSG-ATPB increased to 5.29 g.L.sup.1, 6.22 g.L.sup.1 and 8.48 g.L.sup.1 respectively. The construction method of recombinant Bacillus subtilis is simple, easy to use and has a good application prospect.