Directed Evolution To Improve Formate Utilization In Genetically Engineered E.coli

In recent years,as one of the ideal raw materials in the chemical industry and the biological field,one-carbon compounds have received more and more attention.Researchers use excess energy to synthesize one-carbon compounds,and then use it as a carbon source to maintain the growth of microorganisms and the synthesis of chemicals.This will be a new trend in the field of biomanufacturing.Formate,as the next new one-carbon substrate,has huge application potential.It can be used to electrochemically or photochemically catalyze the generation of CO₂and be directly used by microorganisms as an organic carbon source.In theory,formate can be used as the only carbon source for microorganisms to produce value-added chemicals.Therefore,building an efficient formate assimilation pathway in microorganisms is crucial for the utilization of cheap,renewable one-carbon compounds.The content of this article is mainly divided into four parts:the first part is to construct the reducing glycine pathway in E.coli,knocking out ser A(L-serine hydroxymethyl transferase)to construct a serine auxotrophy strain,and then introduce three exogenous genes from Clostridium ljungdahlii:Fhs(formate-THF ligase),fol D(5,10-methylene-THF dehydrogenase)and fch A(5,10-methine-THF cyclization hydrolysis)to assimilate formate,which provides an important one-carbon unit:methylenetetrahydrofolate to synthesize serine to rescue the growth defects,verifying the successful construction of the reducing glycine pathway in E.coli.The second part is to adopt a rational metabolic engineering strategy,by knocking out genes involved in glycine and serine synthesis related pathways,so that one carbon unit is derived from assimilation of formate as much as possible;on the basis of ser A knockout,kbl and lta E are knocked out to block the production of glycine by threonine,while knocking out ace A inactivates the glyoxylic acid cycle to prevent the formation of isocitrate and subsequent glyoxylic acid.Glycine is produced by the recessive transaminase.Besides,because the gcv R gene encodes the negative regulator of the GCV operon in the glycine cleavage system,in order to enhance the activity of the reverse glycine cleavage system,gcv R was knocked out.So,we constructed an engineered strain named MG9 which contained formate assimilation gene and 5 genes was knocked out continuously.The third part is based on the evolutionary engineering strategy.In view of the poor growth of the MG9 strain and the low utilization rate of formate,laboratory evolution hs gradually reduced the strain's dependence on glycine until the amount of glycine added is zero.The maximum OD₆₀₀of the evolved strain increased by about47%.The fourth part is about the analysis of ¹³C-labeled metabolites.By adding¹³C-labeled formate to the medium,it is proved that formate is assimilated to central carbon metabolism through the reducing glycine pathway,and by comparing the starting strain and the,the amount of labeled serine from evolutionary strain increased by about 16%compared with the starting strains,which proved that evolutionary engineering increased the utilization of sodium formate.