Construction Of Producing Butanol Clostridium Saccharobutylicum Engineering Bacteria Based On Fermentation Of Poplar

With the development of Global economy,energy consumption is increasing day by day.In the current energy structure,80%of the energy consumption come from fossil fuels,which not only cause fossil energy depletion,but also lead to the deterioration of the environment.Therefore,the search for alternative green energy has become the focus of national energy development.Among the green energy sources,bioenergy is the best environment-friendly energy compared to natural gas,Hydrogen energy and syngas.Biobutanol,as a new generation of renewable energy,has a higher combustion value and octane number,and can be used directly for unmodified motorized engines.Clostridium saccharobutylicum BAA-117 was the main strain producing butanol,and it was enable to ferment poplar hydrolyzate to produce butanol(7.51 g/L).However,Due to the poplar hydrolyzate solution contains acidic,phenolic and other compounds inhibitors,butanol yeild of target clostridium has not been high.6-phosphate fructokinase(PFK)and pyruvate kinase(PYK)as the upstream stage of butanol metabolism,provide the reducing force NADH for the synthesis of butanol.Therefore,this experiment hopes to increase the yield of butanol by heterologous expression of pfkA and pyk genes.In this study,the pyruvate kinase gene(pyk),the 6-phosphofiructokinase gene(pfkA)and pfkA-pyk gene were amplified by PCR using Lactococcus total DNA as template,and then ligated to pMD18-T vector to construct T-pfkA,T-pyk cloning vector.extract the recombinant T vector,double digestes recombinant T vector and pfkA-pyk gene,Digested fragments was ligated to the expression vector pSOS94 after the fragments were verified correct.Then,the recombinant plasmids pSOS94-pyk,pSOS94-pyk,pSOS94-pfkA-pyk were transformed into E.coli R2275(containing plasmid pAN1)to obtain methylated plasmid,and extract the methylated pSOS94-pfkA,pSOS94-pyk and pSOS94-pfkA-pyk and then were electrotransformed into Clostridium saccharobutylicum BAA-117.Finally successfully constructed three Engineering bacteria--Clostridium saccharobutylicum BAA-117(pSOS94-pJkA),Clostridium saccharobutylicum BAA-117(pSOS94-pyk)and Clostridium saccharobutylicum BAA-117(pSOS94-pfkA-pyk).The engineering bacteria were subjected to poplar fermentation test under the induction of 50?g/L erythromycin to produce the butanol.The bacteria were collected and subjected to a series of cell disintegration treatments.Then,SDS protein gel electrophoresis and ABE yeild were analyzed by gas chromatography.The results of protein gel electrophoresis showed that the three strains expressed corresponding exogenous genes.Butanol production of Clostridium saccharobutylicum BAA-117(pSOS94-pfkA),Clostridium saccharobutylicum BAA-117(pSOS94-pyk)and Clostridium saccharobutylicum BAA-117(pSOS94-pfkA-pyk)was 7.68g/L,8.13 g/L and 8.72g/L,respectively.improved 4%,10%,18%than Wild type strain,While the acetone production is relatively decreased,the relative increase in ethanol production.In the experiment,the heterologous genes(pfkA,pyk and pfkA-pyk)were overexpressed firstly in the EMP pathway of Clostridium saccharobutylicum BAA-117.In the process of poplar fermentation,the constructed engineering bacteria could improve the yield of butanol and reduce the formation of acetone,which provides a new method and theoretical basis for further improving the yield of butanol.