Construction And Optimization Of Ergothioneine-producing Escherichia Coli

Ergothioneine is a natural antioxidant derived from histidine.It has various physiological functions such as scavenging free radicals,anti-oxidation,and maintaining the normal growth of cells.It is widely used in food,medicine,cosmetics and other fields,and has become a research hotspot.According to published reports,the construction of high-production of ergothioneine engineered bacteria has become a trend.However,the engineered strain that have been reported to produce ergothioneine have a long production cycle and low production and it is difficult to achieve large-scale production.In response to the current problems,this study completed the construction of the ergothioneine synthesis pathway in E.coli BL21(DE3),which has a short growth cycle,and carried out the efficient synthesis of ergothioneine through the modification strategy of the precursor amino acid metabolism pathway.And through the 3 L tank fed-batch fermentation experiment to provide a reference for large-scale production of ergothioneine.The main findings are as follows:(1)In E.coli BL21(DE3),two synthetic pathways of thioneine from different sources were constructed to achieve the synthesis of ergothioneine.The plasmid pRSFDuet-1 was used as a vector to heterologously express the ergothioneine synthesis gene cluster egtBCDE derived from Mycobacterium smegmatis and the ergothioneine synthesis pathway genes egtl and egt2 derived from Schizosaccharomyces pombe.After shaking flask culture,the ergothioneine standard product and the fermentation product of the engineered strain were identified by LC-MS,and it was found that the ergothioneine synthesis pathway derived from M.smegmatis can realize the synthesis of ergothioneine in E.coli.Through the optimization of induction conditions,it was determined that the induction conditions were 0.2 mM IPTG for induction culture at 25?,which made the ergothione production reach(58.87 ± 2.31)mg·L-1;while heterologous expression the way from S.pombe without ergothioneine generate.(2)The synthetic pathway of ergothioneine was optimized to increase the production of ergothioneine.The two constructed ergothioneine synthesis pathways were combined and optimized.Based on the expression gene cluster egtBCDE,the genes egtl,egt2 and both were expressed.When the gene egtl was expressed,the ergothioneine production was increased to(64.74 ± 4.83)mg·L-1,and the expression of the gene egt2 led to a sharp decrease in production;the introduction of EgtA,the isoenzyme of glutamyl cysteine synthase GshA derived from M.smegmatis,promotes the synthesis of ?-glutamylcysteine,the precursor of ergothioneine synthesis,and made the yield of ergothioneine was increased to(84.55 ± 1.73)mg·L-1;the introduction of enzymes Egtl and EgtA at the same time increased the yield of ergothioneine to(95.58 ± 3.20)mg·L-1,which was 1.62 times the initial yield.(3)Balance the amino acid metabolism pathway of the precursor for the synthesis of ergothioneine.Up-regulate the metabolic pathways of the three precursor amino acids(histidine,methionine,and cysteine),and overexpress the key enzymes in the pathway(ATP phosphoribosyltransferase HisG,aspartate kinase ThrA,high Serine acyltransferase MetA,phosphoglycerate dehydrogenase enzyme SerA,serine acetyltransferase CysE,cysteine synthase NrdH and CysK).It was found that the overexpression of the enzymes HisGG233H,T235Q,ThrA,SerAT410STOP and CysET167A had a significant effect on increasing the production of ergothioneine,which were increased to(104.90 ± 2.91)mg·L-1,(134.83 ± 4.22)mg·L-1,(130.26±3.34)mg·L-1 and(110.41 ± 3.61)mg·L-1,respectively.Combining the gene thrA and serAT410STOP to enhance the expression,so that the production of ergothioneine was increased to(144.97±5.40)mg·L-1.Down-regulate the alternative metabolic pathway.When non-coding sRNA regulation was used to achieve post-transcriptional suppression of branch genes(DNA binding transcription repressor PurR in the histidine metabolic pathway,DNA binding transcription repressor MetJ and homoserine kinase ThrB for branch gene expression in the methionine metabolic pathway,branch in the cysteine metabolic pathway Gene expression of L-serine deaminase I SdaA,tryptophanase TnaA and L-cysteine desulfurase YhaM),the ergothioneine production has not been significantly increased;when MetJ was inhibited,the yield has increased from(144.97±5.40)mg·L-1 to(152.58 ± 2.40)mg·L-1.(4)The 3 L tank batch feeding experiment provides a reference for the large-scale production of ergothioneine.The optimal strain was selected,and the 3 L tank fed-batch experiment was carried out under the condition of two different media.The concentration of(548.75±21.6)mg·L-1 was obtained under the fermentation of inorganic salt medium.The growth of bacteria was better under rich medium fermentation,and a higher concentration of ergothioneine(710.53 ± 38.70)mg·L-1 was obtained.Increasing the nutrient content of the medium can obtain a higher concentration of ergothioneine.