| N-acetylneuraminic acid(NeuAc)is a common sialic acid found in a wide variety of plants,animals and microorganisms in nature and can be used to synthesise other sialic acids or derivatives of sialic acid.It is found mainly at the end of the glycoprotein or glycolipid on the cell surface.So that it is able to maintain the structure of biological macromolecules and is important for the biological recognition of cells.In addition,NeuAc has a high commercial value as a health-care food and pharmaceutical product.Currently,NeuAc is produced mainly by enzymatic synthesis and whole-cell catalysis,but these methods require the addition of expensive precursors.Therefore,it is necessary to develop innovative engineering strategies to de novo synthesise NeuAc by using cheap carbon sources such as glucose.Previously,we constructed a pathway for the production of NeuAc in E.coli using glucose as the sole carbon source,and optimized the NeuAc production by rational modification,replacing the source and improving the solubility of key enzymes,but there is still a problem of large accumulation of the intermediate product N-acetylglucosamine(GlcNAc)and a deficiency of the precursor phosphoenolpyruvate(PEP)during fermentation.GlcNAc is catalyzed by acetylglucosamine 2-isomerase(Age)to generate precursor N-acetylmannose amine(ManNAc).In order to improve the production of NeuAc and avoid the accumulation of intermediate GlcNAc,we performed co-mutation of three key catalytic site(Alal72,Glu218,Asn236),and saturated mutation of CYS370 of Age successively.The result showed that some mutants such as C370A,C370N,and C370M effectively increased the production of NeuAc,the NeuAc yield of C370A was increased by 34%compared to the wild type.However,there was still a significant accumulation of the intermediates GlcNAc.To further eliminate the accumulation of GlcNAc,we tried overexpressing the UDP-N-acetylglucosamine 2-epimerase(NeuC)to accumulate ManNAc via a dual pathway,but it didn’t work well.We considered that another bottleneck in NeuAc production may be the supply of precursor PEP.Previous study in our laboratory knocked out the key genes of the PEP metabolic pathway,resulted in much higher yields.But when we continued to knock out other key genes on this basis,a large amount of pyruvate was accumulated,which affected the normal growth of the strain,we presume that it due to the rigid modification of the strain’s metabolism resulting in severe imbalance in the metabolism of the bacterium.We also attempted a rational modification of another pyruvate kinase gene pykF:performing point mutation of Lys382 and Arg431 of pykF to de-activate the feedforward activation by fructose-1,6-bisphosphate(FBP),and the fermentation results showed that although the yield was not significantly different from that of the starting strain,the mutant had higher glucose utilisation during fermentation than the starting strain.In order to increase the supply of PEP,we need to decrease the accumulation of pyruvate,there is a good strategy to regulate the PEP metabolic pathway through a dynamic regulation.Therefore,we constructed a positive-response biosensor responding to the central metabolite pyruvate in E.coli,and performed a series of optimizations and adjustments.We constructed a negative-response pyruvate biosensor by antisense transcription,and then dynamically repress relevant genes such as pykF、ptsG、gltA、zwf、ppc、pck in the PEP metabolic pathway.The dynamically repressed ptsG strain was obtained 25.4 g/L NeuAc after 144h of shake flask batch replenishment fermentation,and it is the highest yield of NeuAc denovo synthesis using glucose as the carbon source so far.In summary,this paper performed rational modification of Age to improve its catalytic ability.And then constructed a dynamic regulation system based on the pyruvate biosensor,which solved the accumulation of byproduct and boosted the production of NeuAc.We provides a new ideas to solve how to balance the growth and production in the NeuAc production. |
