Systematic Metabolic Engineering Of Escherichia Coli For Biotechnological Production Of Pyruvate

As a new type of pharmaceutical,chemical and food intermediates,pyruvate has gradually expanded its demand in the domestic market.There have been many researches on pyruvic acid production by biotechnology.Some pyruvate synthetic strains have been initially constructed.However,at present,the pyruvic acid production by microorganisms requires the addition of vitamins and excessive levels of intracellular NADH/NAD+.In this dissertation,the method of systematic metabolic engineering was used to transform E.coli and further strengthen the production of pyruvic acid.The original strain E.coli YP211 used in this paper had deleted several pyruvate metabolic pathways.The transcriptome analysis of YP211 and wild-type revealed that the pathway for the metabolism of pyruvate was weakened and the TCA cycle gene was up-regulated.This would produce a large amount of NADH,inhibit the progress of glycolysis and is not conducive to the synthesis of pyruvate.In order to reduce intracellular NADH/NAD+ levels,a high copy plasmid was used to express exogenous NADH oxidase NoxE from L.lactis to construct strain YP301.The growth of YP301 increased by 42.6%,the concentration of pyruvate increased by 17.4%,and the yield did not change substantially.Next,the effect of NADH/NAD+ level regulation on pyruvate synthesis based on different reduced carbon sources was explored.It was found that pyruvic acid production with sodium gluconate as the carbon source was 22.9%higher than that with glucose as the carbon source.From the results of transcriptome analysis,intracellular metabolism of gluconate activated the ED pathway,allowing the carbon metabolism flowed to the ED pathway that formed pyruvate with fewer reaction steps and less NADH.However,the cost of gluconate sodium was too high to be a fermentation substrate.Therefore,through the transformation of the central carbon metabolism of E.coli,the ED pathway in the case of activating glucose as a carbon source would further reduce the fermentation cost.First,pfkA was deleted(weakened EMP pathway),and high-copy plasmid expressed edd eda(enhanced ED pathway)to construct strain YP203.Compared with YP211,the yield increased by 39.7%and the production rate increased by 67.4%.In order to maintain the stability of strains,the ribosome binding site of edd,a key gene of the ED pathway,was used to enhance the ED pathway by using synthetic biology methods,and the initiation rate of translation was increased by 100 times to construct strain YP501.Compared with YP211,the output increased by 35.7%and the production rate increased by 34.8%.Compared with strain YP401,which overexpressed edd only by high-copy plasmid,the yield increased by 17.3%and the production rate increased by 47.6%...