The Identification Of Genes Involved In Hexanoate Synthesis And The Development Of Genetic Manipulation Tools For Clostridium Kluyveri

Clostridium kluyveri is an important hexanoate-producing microorganism.For a long time,key enzyme encoding genes for the hexanoate synthesis pathway in Clostridium kluyveri were not identified and due to the lack of genetic manipulation tools of Clostridium kluyveri,result of performing the metabolic engineering studies on Clostridium kluyveri became very hard.In this paper,we identified hexanoate-producing key enzyme encoding genes of C.kluyveri and analyzed the thiolase function for the green biosynthesis provides highly efficient genetic elements.The thiolase was the key enzyme encoding genes for the production of hexanoate and the synthesis of six carbon skeletons.For genetic manipulation tool of Clostridium kluyveri was established which achieved the reformation of the metabolic engineer of the hexanoate synthesis pathway of Clostridium kluyveri.Topics include:?1?When ethanol and acetate were used as carbon sources,Clostridium kluyveri produced butyrate,hexanoate and octanoate.Hexanoate was the major product.However,when ethanol and butyrate were used as carbon sources,Clostridium kluyveri produced hexanoate and trace amounts of octanoate,but butyrate exhibited a net consumption.The fatty acid synthetase of C.kluyveri have the ability to catalyze the synthesis of butyrate and hexanoate,and the in vivo catalytic function of the enzyme was regulated by the type of initial carbon source.Transcription analysis of hexanoate-producing key genes of Clostridium kluyveri showed that each gene showed different expression profiles during fermentation.The thiolase-encoding gene and the 3-hydroxybutyryl-Co A dehydrogenase-encoding gene had the highest levels of transcription.?2?Clostridium kluyveri has three thiolase-encoding genes?thlA1,CKL₃696;thlA2,CKL₃697;thlA3,CKL₃698?.The transcriptome and reverse transcription-quantitative PCR were used to analyze the expression profiles of thiolase-encoding genes during fermentation.Transcription analysis showed that thlA1 gene was constitutively expressed,thlA2 gene was up-regulated and thlA3 gene was down-regulated before the depletion of acetate.We also found that the expression levels of three thiolase-encoding genes were dramatically down-regulated in stationary phase of cell growth.It can be seen that the expression of the three thiolase-encoding genes strictly depended on the cell growth.The kinetic parameters show that the three thiolases from C.kluyveri had similar affinity for acetoacetyl-CoA.However,the catalytic efficiency(k_cat/K_m)of ThlA1 for four-carbon substrates was lower than Thl A2 and ThlA3.?3?In this study,Clostridium kluyveri DSM555 was used to successfully construct a genetic manipulation tool?gene overexpression system?through resistance selection,optimization transformation conditions,replicon selection and promoter selection.Heterologous expression in Clostridium kluyveri was achieved by using this genetic manipulation tool for the first time.In order to test the effectiveness of the expression system,this study overexpressed aldehyde/alcohol dehydrogenasealdol-encoding gene?adhE2?derived from Clostridium acetobutylicum in Clostridium kluyveri.The adhE2 overexpression strain produced butanol 25 mg·L^-1 and hexanol 155 mg·L^-1.The concentration of butanol and hexanol produced by the overexpression of adhE2 were 2.5-fold and 8.2-fold higher than that of the wild-type strain.Based on the above results,the study showed that Clostridium kluyveri has three catalytically activty thiolases that are actively expressed during fermentation.In this parper,we successfully established the genetic manipulation tools of Clostridium kluyveri,which will promote the research on the hexanoate synthesis mechanism of Clostridium kluyveri and its metabolic engineering.