Pathway Construction And Metabolic Engineering For Fermentative Production Of Ectoine In Escherichia Coli

Ectoine is a protective agent and stabilizer which can alleviate toxic action of high osmotic pressure,high temperature,freeze-thawing,drying,radiation and chemical reagent on proteins,nucleic acids,biofilm and the whole cells.The synthesis pathway of ectoine just exists at some moderately halophiles.A novel process called "bacterial milking" was established for efficient ectoine production in the moderately halophiles.However,this method has high demands on equipment and is cost prohibitive.In this study,we introduced ectoine synthesis pathway to E.coli to construct an ectoine engineering bacteria that can accumulate ectoine without high osmolarity and improved its production by metabolic engineering.First,ectABC gene cluster from Halomonas elongata was introduced to E.coli W3110 MG1655 and K27 by pTrc99a.Fed-batch cultures revealed that the ectABC gene cluster was functionally expressed without high salt concentration and ectoine was produced and secreted.The extracellular ectoine accumulation reached 4.88±0.07 g/L,4.14±0.02 g/L and 2.97±0.08 g/L respectively.When the codon usage was adjusted to that preferred by E.coli ectoine production reduced 84.6%unexpected.Then,thrA and lysA were deleted to weaken L-threonine and L-lysine branch.Results showed that ectoine accumulation increased 49.6%and 109.1%respectively.The results indicated that carbon flux towards ectoine haven been improved a lot as a result of sufficient supply of L-aspartate-p-semialdehyde.But when lysA and thrA was deleted together ectoine production reduced 38.9%.Furthermore,a feedback resistant lysC gene from Corynebacterium glutamicum and a feedback resistant lysC from E.coli were introduced by pSTV28,Fed-batch cultures revealed that the extracellular ectoine increased 9.2%and 3.1%respectively.Last,the promoter of ppc was replaced by a trc promoter together with iclR deletion.The final ectoine engineering strain was able to produce ectoine with a yield of 13.60±0.12 g·L-1 by fed-batch culture in low salt concentration.The final ectoine engineering strain was able to produce ectoine with a production of 25.1 g/L by fed-batch culture in low salt concentration with glucose as carbon source while the production yield and productivity reached 0.8 g/g DCW and 0.84 g·L-1·day-1 respectively and glucose conversion was 10.7%.The production we got is the highest in all the reported ectoine fermentation with different strains.