Metabolic Pathway Remodelling And High Producing Strain Cultivation Of E.coli Pyruvate Producing Strains

As one of the most important organic acids,pyruvate acid has a wide range of uses in medicine,food,chemicals and other fields as well as in scientific research.At present,there is a large gap in the domestic market for high-quality pyruvate.Therefore,the prospect of industrialized production of pyruvate is very broad.This experiment focused on gene editing of E.coli K-12 MG1655 strain using CRISPR/Cas9 technology.Truncating the lactate synthesis pathway by knocking out the ldh A gene.Truncating the acetic acid synthesis pathway by knocking out the pox B,pta and ack A genes.Truncating the formic acid synthesis pathway by knocking out the pfl B gene.Reduced pyruvate production of phosphoenolpyruvate by knocking out the pps A gene.Knocking out the pps A gene to reduce the production of phosphoenolpyruvate from pyruvate.Knocking out the ppc gene to reduce the consumption of phosphoenolpyruvate.Knocking out the frd BC gene to weaken the tricarboxylic acid cycle pathway.Finally,a genetically engineered strain MG1655-GP7(E.coli K-12 G1655?ldh A?pfl B?ack A?pta?pox B?ppc?frd BC?pps A)that can initially accumulate pyruvate was obtained.The pyruvate yield of MG1655-GP7 reached32.07 g/L after 68 hours of fermentation in a 5 L fermentor,which shows that only the use of genetic engineering can make E.coli accumulate pyruvate.When E.coli uses glucose as a carbon source,glucose will produce excess ATP and NADH via glycolysis to inhibit the accumulation of pyruvate,which can be solved by using gluconic acid with a higher degree of oxidation as a carbon source.When glucose enters the Entner-Doudoroff(ED)pathway for metabolism rather than the EMP pathway,the amount of ATP and NADH produced is halved,which promotes the accumulation of pyruvate.So in order to further increase pyruvate production,the authors decided to inhibit the production of ATP with NADH by the EMP pathway by knocking out the pgi gene.To inhibit the consumption of 6-phosphate-gluconate by the pentose phosphate pathway by knocking out the gnd gene.and knocking out the glf gene of Zymomonas mobilis on top of knocking out the pts G and pts I genes to improve the glucose transport system and reduce the consumption of phosphoenolpyruvate during glucose transport in E.coli cells.In addition,we plan to up-regulate the glucose-6-phosphate dehydrogenase gene(zwf),gluconate-6-phosphate dehydratase gene(edd)and 2-keto-3-deoxygluconate-6-phosphate aldolase gene(eda),which can strengthen the ED pathway,balance the levels of ATP and NADH,and guide the carbon flow to the direction of pyruvate synthesis.The MG1655-GP12(E.coli K-12 MG1655?ldh A?pfl B?ack A?pta?pox B?ppc?frd BC?pps A?gnd?pgi?pts G?pts I::glf)strain has now been obtained by gene editing and is theoretically more capable of producing pyruvate than the MG1655-GP7 strain.The production capacity of this strain is being evaluated.However,after extensive gene editing,strain MG1655-GP12 grew slower than the wild type strain,so in this experiment,Adapted Evolution experiments were carried out and after several passages,a strain of MG1655-GP12-1 whose growth rate recovered to 75% of the wild-type strain was selected.