Metabolic Engineering Of Escherichia Coli For Ectoine Production

Ectoine is a high-value stabilizer and protective agent with various applications in enzyme industry,cosmetics,and biomedicine.It can be synthesized naturally by some moderate halophilic microorganisms,which however needs stimulation from high concentration of salt.In this study,we constructed a heterologous synthesis pathway of ectoine in Escherichia coli and implemented some rational metabolic engineering strategies,resulting in an efficient strain producing ectoine under the control of fermentation temperature.（1）After codon optimization,the ectoine synthesis gene cluster ect ABC originally from Halomonas elongata was heterologously expressed in E.coli MG1655 by the high copy plasmid p FT28.The expression of the gene cluster ect ABC was controlled by a thermosensitive circuit c I^ts-p _R-p _L,rather than chemical inducer addition or high concentration salt shock.During shake flask fermentation,the constructed strain MG1655/p FT28-ect ABC synthesized ectoine when the temperature rose to 42℃,of which the extracellular ectoine titer was 1.95 g·L^-1 in the end.（2）Improving the accumulation of precursors can enhance ectoine production in E.coli.In order to reduce the consumption of phosphoenolpyruvate,the gene crr,encoding the glucose-specific enzyme II domain A of glucose phosphotransferase system,was knocked out from the genome of MG1655,resulting in the improvement of ectoine titer by 171%.In order to reinforce glyoxylate bypass and increase the accumulation of oxaloacetate,the glyoxylate shunt transcriptional repressor gene icl R was deleted,making the ectoine titer 72%higher.（3）Weakening the L-threonine competitive synthesis pathway and simultaneously compensating the aspartokinase can promote the synthesis of ectoine.The gene thr A encodes the bifunctional aspartokinase/homoserine dehydrogenase.Its deletion can weaken the synthesis of L-threonine and cause the enzymatic activity deficiency of the aspartatokinase,which inhibits the growth of cells.The two feedback-insensitive mutants,which were Eclys C*（C1055T）encoding E.coli aspartokinase III and Cglys C*（G1A,C932T）encoding C.glutamicum aspartokinase were overexpressed respectively.After compensating the aspartokinase,the cells restored the normal growth,and the ectoine titers improved to 11.85g·L^-1and 9.67 g·L^-1,respectively.（4）The overexpression of ppc encoding phosphoenolpyruvate carboxylase was modulated to further enhance ectoine synthesis in E.coli.When too much PEP is converted to oxaloacetate,there will be the flux imbalance between oxaloacetate and acetyl-co A,which are both converted from PEP.This is not beneficial for the synthesis of ectoine,because equimolar oxaloacetate and acetyl-co A are required as precursors for ectoine synthesis.The ectoine titer improved by5.8%when ppc was moderately overexpressed.（5）The gene asp DH encoding a novel aspartate dehydrogenase from Pseudomonas aeruginosa PAO1（Pae Asp DH）was heterologously overexpressed to enhance the synthesis of aspartic acid without competing for glutamate,with the ectoine titer improving by 8.39%.（6）The temperature rising time and initial glucose concentration of medium during shake flask fermentation were optimized.When rising the temperature at 3 h after the fermentation started and the initial glucose concentration was 40 g·L^-1,the ectoine titer of the final engineered strain reached 17.89 g·L^-1.The ectoine production of the final strain MWZ003/p FT28-ect ABC-Eclys C*-asp DH-ppc3 was also investigated in the scale of fed-batch fermentation.After 36 h fermentation,the ectoine titer reached 30.37 g·L^-1 with the production efficiency at 0.844 g·L^-1·h^-1,and the yield was 0.132 g·g^-1 glucose.