Recombinant escherichia coli and construction method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and relates to construction of genetic engineering strains, in particular to recombinant escherichia coli and a construction method andapplication thereof. According to the recombinant escherichia coli, an undecylenyl phosphate glucose phosphotransferase gene wcaJ is knocked out, manB, manC, gmd, wcaG and srlD genes are recombined and expressed, a pntAB gene, an ndK gene and a gsK gene are further recombined and expressed, and a pfkA gene is knocked out, so that a synthesis path of forming fructose 6-phosphate is enhanced and theglycolysis process of 6-fructose phosphate is weakened, the accumulation of fructose 6-phosphate is facilitated, and thus more precursor substances are provided for the synthesis of GDP-L-fucose; inaddition, the regeneration of the cofactors NADPH and GTP is enhanced, so that the capacity of producing GDP-L-fucose by the recombinant escherichia coli strain is further improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the construction of genetically engineered bacterial strains, in particular to a recombinant Escherichia coli and its construction method and application. Background technique [0002] Guanosine 5'-diphosphate-L-fucose (GDP-L-fucose), as an L-fucose donor, is essential in the biosynthesis of fucosylated oligosaccharides substrate. However, the low production level and high cost of GDP-L-fucose limit its application in the large-scale production of fucosyl oligosaccharides. [0003] In the prior art scheme, two pathways have been identified for the biosynthesis of the donor substrate GDP-L-fucose: the de novo pathway and the salvage pathway. [0004] The de novo synthesis pathway (De novo pathway) mainly originates from the central metabolic activity of bacteria. The synthesis pathway of GDP-L-fucose mainly starts from the glycolysis intermediate fructose 6-phosphate, and p...

AI Technical Summary

Problems solved by technology

From the perspective of the synthesis route, the remedial synthesis route is relatively simple, but the required substrate L-fucose is extremely difficult to obtain, and the market price is relatively high, so this scheme is not yet suitable for large-scale GDP-L-fucose sugar preparation

[0006] In wild-type Escherichia coli, GDP-L-fucose can be synthesized through the catabolism of glucose, but its production level is limited by the effect of catabolite repression, so that the GDP-L-fucose produced by grape metabolism is only For wild-type E. coli itself, it is difficult to accumulate in large quantities

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