Metabolic Engineering Of Escherichia Coli For Efficient N-acetylneuraminic Acid Biosynthesis

N-Acetylneuraminic acid(Neu5Ac)is an important functional sugar that has been widely used in food,medical and other fields due to its important physiological functions,and the demand for it is constantly rising.The current production methods have some problems that are difficult to solve,such as expensive raw materials for natural product extraction,complicated separation process by chemical methods,high cost of enzymatic synthesis and cumbersome operation by whole cell catalysis,which are not conducive to the large-scale industrial production of Neu5 Ac.The establishment of a stable and efficient N-acetylneuraminic acid fermentation production system by means of metabolic engineering is a need to further expand the N-acetylneuraminic acid market.In this study,a previously constructed N-acetylneuraminic acid producing strain Escherichia coli NEU-2was used as the starting strain to enhance the supply of the precursor phosphoenolpyruvate(PEP)in various ways and to obtain a high-producing strain of N-acetylneuraminic acid.The main studies were as follows.(1)Enhanced the glucose uptake efficiency using a non-PTS system.The native glk gene in E.coli was over-expressed in NEU-2,constructing NEU-3.The results of the shake-flask fermentation showed that the rate of glucose uptake by NEU-3 was comparable to that of NEU-0 with native PTS system and the Neu5 Ac titer was increased by 127.88%to 11.28 g/L compared to that of NEU-2.(2)Enhanced the PEP synthesis during central carbon metabolism.The genes pps and pck were over-expressed individually or in combination.Shake-flask fermentation results showed that simultaneous overexpression of genes pps and pck had a positive effect on enhancing Neu5 Ac production per unit biomass.(3)Fine-tuned the pyruvate kinase expression using weak promoters.The natural promoter of the pyruvate kinase encoding gene pyk F of strain NEU-3 was replaced with the weak promoters BBa＿J23103,BBa＿J23109 and BBa＿J23116 to obtain NEU-8,NEU-9 and NEU-10,respectively.In addition,the pyk F and pyk A genes of strain NEU-3 were knocked out and used as controls,respectively.Finally,the pyk A knockout strain NEU-12 was selected as the most effective strain among them due to its production and growth advantages.Shake-flask fermentation results showed an 18.46% increase in Neu5 Ac titer was obtained in NEU-12,with no significant impairment of cell growth.(4)To reduce the accumulation of acetic acid during fermentation,a series of engineered bacteria using NEU-12 as the starting strain was constructed,from two perspectives of knocking out the genes related to acetic acid synthesis,pox B and ack A,and over-expressing the acetic acid utilization gene acs,respectively.The results of the shakeflask fermentation showed that the accumulation of acetic acid during fermentation was significantly lower in the strains with pox B and ack A genes knocked out than in the control bacteria.(5)The recombinant strain NEU-12 was verified to have good performance in shakeflask fermentation and was further cultured in a 5 L fermenter.The fermentation results showed that a titer of 27.41 g/L Neu5 Ac was produced in NEU-12 at 40 h,the titer and productivity were higher than the reported values in E.coli.In this study,to address the bottleneck problem of insufficient supply of the precursor PEP in the synthesis of Neu5 Ac in E.coli that limits the efficiency of the target product synthesis,we used a metabolic engineering strategy to reasonably modify the Neu5 Acproducing bacteria,and finally obtained an E.coli engineered strain capable of synthesizing Neu5 Ac with high efficiency,and the yield and productivity reached the highest value among the E.coli reported so far.