Engineering Of Butanol-production Pathway From Clostridium Saccharobutylicum In Escherichia Coli And Its Fermentation Optimization

With the increasing problems on energy and climate,new energy becomes urgent needs of the whole society.Due to its higher energy density and the advantages of easier blending with gasoline,butanol has become one of the most important new energies.Clostridium saccharobutylicum has been proved to be efficient in butanol fermentation from various feedstocks.Whereas,lack of genetic manipulation system has severely hindered the engineering of C.saccharobutylicum for more extensive applications.Escherichia coli has been a recognized model strain with simple genetic background and mature gene manipulation system.In the former study,the butanol synthesis pathway from C.saccharobutylicum has been constructed in E.coli and knockout the lactate dehydrogenase gene.Based on the butanol synthesis pathway of C.saccharobutylicum,combination technologies of genomics and metabolic engineering,various strategies were implemented in engineered E.coli to improve its butanol production in this study.The rate-limiting enzyme acetoacetyl-CoA thiolase encoding gene thlA was replaced by the acetyl-CoA acetyltransferase gene?atoB?from E.coli,and the butanol titer was increased to 1.8-fold(120.6 mg·L^-1).Comparison of butanol titer between these two enzymes revealed that acetyl-CoA degradation is the limiting step in butanol production,and atoB has better catalytic activity.In order to reduce the consumption of carbon source and key intermediates,some competitive branch pathways were knockout,such as aldehyde/alcohol dehydrogenase gene?adhE1?,fumarate reductase gene?frdBC?and so on.Then,the butanol titer was successfully improved for 3.8-fold(254 mg·L^-1).To reduce the burden of plasmids,the genes atoB and adhE2 from pRSFDuet-atoB-adhE2?pAA?were integrated into the genome of E.coli.However,the results showed that the butanol production in chromosomal integrated strain was reduced by 17%(236 mg·L^-1).It was presumed that the expression level of target genes in the chromosome was lower than that on the high-copy plasmids,and therefore the relevant flux was reduced.To increase NADH and acetyl-CoA,glyceraldehyde 3-phosphate dehydrogenase?gapA?,glucose dehydrogenase?gdh?and acetyl-Co A synthetase?acs?were overexpressed,and butanol production can improve 5.5%,12.7% and 18% respectively.Under the optimum fermentation conditions,the final butanol titer reached 584 mg·L^-1 after 120 h.This result demonstrates butanol production by recombinant E.coli could be successfully promoted through metabolic engineering and fermentation optimization.