Construction And Optimization Of Cell Factories For Escherichia Coli Citric Acid Production

Citric acid is widely used in food,medicine and chemical industry.With the development of technology and economy,the market demand of citric acid is increasing day by day.Traditional citric acid fermentation uses Aspergillus Niger strain,although the yield of acid is high,but there are still problems to be solved.Aspergillus niger has a long fermentation cycle,the fermentation conditions need to be strictly controlled,it is difficult to transform the metabolic pathway of strains,and the selected strains are not pure,which is manifested as strain degradation,acid production capacity fluctuating.And improper use and preservation process,external factors and internal factors can lead to Aspergillus niger back mutation or spontaneous mutation.E.coli has a short growth cycle,simple fermentation conditions and easy control,has a clear genetic background,and is easy to be modified at the gene level.It is widely used in the production of organic acids such as succinic acid,malic acid and fumaric acid.The PC recombinase activity from corynebacterium glutamate was the highest and reached 104 U/mg prot.The acetic acid kinase gene(ackA)was knocked out by Red homologous recombination method,and the pyruvate dehydrogenase complex gene(aceE-aceF-lpd)with gapA as the promoter was replaced,and the recombinant strain DH5αΔackA was obtained.The activity of pyruvate dehydrogenase in the recombinant strain was increased by 51%,citric acid production reached 6.18 g/L,citric acid-glucose conversion rate reached 41.25%,and the yield was increased by 36.4%compared with the original strain,without the accumulation of acetic acid.E.coli gltA,ppc,pc and pykF genes with gapA promoter were overexpressed in the knockout strain DH5αΔackA,we get the recombinant E.coli DH5αΔackA(pET-gapAgltA),DH5αΔackA(pET-gapA-ppc),DH5αΔackA(pET-gapA-pc)and DH5αΔackA(pET-gapA-pykF)four strains,The enzyme activities of citrate synthase CS,phosphoenolpyruvate carboxylase PPC and pyruvate kinase pykF were increased by 2 times,33.9%and 171.1%,respectively.Citric acid production reached 7.77 g/L,8.27 g/L,8.63 g/L and 7.82 g/L,respectively.Four recombinant strains,DH5αΔackA(pET-gltA-ppc),DH5αΔackA(pET-gltAppc-pykF)and DH5αΔackA(pET-gltA-ppc-pykF),were constructed by overexpressing gltA,ppc,pc and pykF genes in tandem.The citric acid production of DH5αΔackA was increased by 29.1%,56.0%,12.3%and 78.4%,respectively.Citric acid production of DH5αΔackA(pET-gltA-ppc-pc-pykf)reached 11.03 g/L and saccharide-acid conversion ratio reached 73.5%.This study laid a foundation for the production of citric acid and citric acid from Escherichia coli,and also provided theoretical support for the development of other strains with high citric acid production.