| Ergothioneine（ERG）is a kind of natural antioxidant derived from histidine,which has various unique physiological and pharmacological functions.ERG is already widely applied in medicine,cosmetics,functional food and other industries,which has broad market prospects.At present,engineered microorganism has become the most direct and economical way to obtain ergothioneine,but there remain common problems limiting its industrialization,such as long fermentation period and low yield.Therefore,it is particularly crucial to improve the production capacity and intensity of ergothioneine strains.In previous study,an engineered Escherichia coli BL21（DE3）strain has been constructed to realize the synthesis of ergothioneine,but the yield of shake flask was only 143.82±1.18 mg·L-1,the yield of 3 L fermenter was 710.53 mg·L-1,and there were problems of insufficient precursor supply,metabolic branch consumption and low production intensity,so it could not meet the large-scale production.In this study,the fermentation conditions were first optimized to enhance the production efficiency of ergothioneine,meanwhile,the expression of ergothioneine synthesis pathway genes in engineered strain was optimized.Moreover,the metabolic pathway genes were weakened and the sulfate transporters were strengthened through genome editing.Finally,the efficient production of ergothioneine was achieved.The main conclusions of this study are as follows:（1）Optimize the precursor supply to release ergothioneine synthesis limitation.Given that histidine deficiency was the most critical factor limiting the synthesis of ergothioneine,the production of ergothioneine reached 175.81±1.19 mg·L-1 by addition of 0.1 g·L-1 histidine.Then,A 3 L fermenter was used to scale up the culture and the content of relevant intracellular and extracellular amino acids was determined during the fermentation.Through orthogonal experimental design with optimized combination addition of 0.01 g·L-1 cysteine,0.01 g·L-1ammonium ferric citrate and 0.01 g·L-1 vitamin B6,the yield of ergothioneine increased to182.61±3.92 mg·L-1.Subsequently,methyl donors were added to the medium,while the optimal concentration of 1.5 g·L-1 methionine and 0.6 g·L-1 betaine were added,the yield was further increased to 199.33±4.96 mg·L-1.On this basis,by adding 0.2%Ca Cl2 to improve the permeability of E.coli cell membrane,the final production was 243.06±7.85 mg·L-1,which was 69%higher than initial.After removing the limitation of precursor amino acid supply through exogenous addition,the optimal fermentation condition of 3 L fermenter was determined.After fed-batch fermentation culture for 108 h,the yield of ergothioneine reached to 2.01 g·L-1 and the titer reached 18.61 mg·L-1·h-1,greatly improving the production capacity.（2）Screening and optimization of ergothioneine synthesis pathway genes from bacterial and fungal species to determine the optimal expression combination.Firstly,solubility-enhancing tag GST was fused at the N-terminus of egt B,which is the pathway enzyme derived from Mycobacterium smegmatis.The results showed that the ergothioneine yield reached to212.83±3.82 mg·L-1.After that,the RBS of egt B was replaced by RBS9 with a lower translation initiation rate,the yield was found to increase to 227.65±5.65 mg·L-1.Secondly,ergothioneine synthesis pathway genes egt1 and egt2 from different fungi were screened,including spegt1 and spegt2 from Schizosaccharomyces pombe,ncegt1 and ncegt2 from Neurospora crassa,tregt1 and tregt2 from Trichoderma reesei,cpegt1 and cpegt2 from Claviceps purpurea,gfegt1 and gfegt2 from Grifola frondosa,poegt2 from Pleurotus ostreatus.The results showed that ergothioneine production could reach to 230.21±10.32 mg·L-1by the additional introduction of tregt1.Therefore,the optimal expression combination was determined to be the gene cluster egt BDCE from Mycobacterium smegmatis and tregt1 from Trichoderma reesei.（3）Optimization of transport and supply of sulfur in ergothioneine synthesis by genome editing.When the methionine transcriptional repressor met J,serine deaminase sdaA and GTP3′,8-cyclase moaA were respectively knocked out in E.coli BL21（DE3）genome,the ergothioneine yield was 31.83±5.26 mg·L-1,244.38±18.25 mg·L-1,242.89±11.02 mg·L-1,respectively.The knockout of met J significantly inhibited the growth of the strains,so the yield decreased significantly.The superimposed deletion of sdaA and moaA resulted in a declined ergothioneine production and it was found that the cell growth was significantly affected.On the basis of single knockout of sdaA or moaA,the T7 promoter was genomically integrated to enhance the expression of two sulfate transporters Cys PUWA and Cys Z.The results showed that with the deletion of moaA and genomically strengthing of Cys PUWA,the yield was the highest at 288.73±2.51 mg·L-1.On this basis,the pathway genes egt A,thr A and ser AT410STOPwere combined overexpressed,moreover,the optimized fermentation medium mentioned before was used to increase precursor supply,the final ergothioneine production could reach to422.17±12.79 mg·L-1,which was 193.5%higher than the initial.（4）Production of ergothioneine by fed-batch fermentation.The optimal strain was selected for fed-batch fermentation,the yield of ergothioneine reached to 2.63 g·L-1,which was 270.1%higher than the initial,and the production intensity was increased to 27.40 mg·L-1·h-1,realizing the efficient synthesis of ergothioneine. |
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