| As a bulk chemical,butanol is widely used in chemical,pharmaceutical and energy fields With the exhaustion of fossil energy and the deterioration of global environmental problems,butanol as the second-generation biofuel has drawn much attention for its physical and chemical properties which are superior to ethanol and similarity to gasoline.Butanol can be produced by chemical synthesis and microbial fermentation and now chemical synthesis is the predominant way due to its high efficiency and economy.However,butanol production by microbial fermentation has drawn an increasing attention for its advantages of sustainable development and environmentally friendly over chemical synthesis which leads to exhaustion of fossil energy and the serious environmental pollution.Nowadays,the market competitiveness of bio-butanol is weak due to its low butanol titer and productivity and high raw material cost.Therefore,to enhance the market competitiveness of bio-butanol,the titer and productivity of butanol should be improved and the raw material cost should be reduced In this study,Clostridium acetobutylicum ATCC 824 was modified by genetic engineering to improve the butanol production and the lignocellulosic biomass utilizationOverexpressing the VB1 biosynthesis related genes thiC,thiG and thiE in C.acetobutylicum ATCC 824 could significantly enhance carbon source metabolism and energy production,which eventually led to the improvement of the performance of ABE fermentation In the case of fermentation in P2 medium without VB1,when glucose was used as the substrate,carbon source consumption and butanol production increased by 11.6±2.8%and 13.0±0.1%,respectively;When xylose was used as the substrate,carbon source consumption and butanol production increased by 11.6±0.4%and 12.7±1.2%,respectively;When mixed sugar was used as the substrate,xylose consumption and butanol production increased by 45.8±1.9%and 20.4±0.3%respectivelyThe cofactor NADH plays a key role in the butanol production of C.acetobutylicum and insufficient NADH supply will restrict the biosynthesis of butanol.Therefore,increasing the intracellular NADH supply may be an effective way to improve the butanol production of C.acetobutylicum ATCC 824.When aspartate(a precursor of NADH)was added exogenously during ABE fermentation,the results showed that the butanol titer increased significantly Further studies found that the expression levels of NAD+de novo synthesis related genes nadA,nadB and nadC were significantly up-regulated,and the intracellular concentration of total NAD(H)was significantly increased.Based on these results,we investigated the effect of overexpression of NAD+de novo synthesis related genes on the butanol production of C.acetobutylicum ATCC 824,and the results showed that the butanol titer was increased by overexpressing of nadA,nadB or nadC.Among them,the engineered strain C.acetobutylicum 824(nadC)had the highest butanol titer,13.5±0.4%higher than that of the wild type strain.In order to further improve the butanol production,the VB1 biosynthesis related genes thiC,thiG,thiE and NAD(H)de novo synthesis related genes nadC were co-expressed for strengthening supply of NADH as well as to enhance the carbon source metabolism and energy production As a result,the butanol titer of the engineered strain C.acetobutylicum 824(thiCGE-nadC)reached 13.96±0.11 g/L,7.2±0.4%and 18.1±0.1%higher than that of the engineered strains C acetobutylicum 824(thiCGE)and C.acetobutylicum 824(nadC)respectively,and 34.1±0.1%higher than that of the wild type strainThe effect of the heat shock proteins from extremophile on the cell performance of C.acetobutylicum ATCC 824 was studied.Results showed that the tolerances to butanol,low pH and lignocellulose-derived inhibitors were significantly improved by heterogenous expressing of the heat shock proteins GroESL and DnaK from the extremely radioresistant bacterium Deinococcus wulumuqiensis R12 in C.acetobutylicum ATCC 824.Meanwhile,it was also found that the performance of heat shock protein from the extremely radioresistant bacterium D.wulumuqiensis R12 was superior to that from C.acetobutylicum ATCC 824 and the engineered strain C.acetobutylicum 824(dnaK R12),which was overexpressed the DnaK from D.wulumuqiensis R12 in C.acetobutylicum ATCC 824,had the best cell performance When the undetoxified soybean straw,corn straw and rice straw hydrolysate were used as the substrates for ABE fermentation,the butanol titer of the engineered strain C.acetobutylicum ATCC 824(dnaK R12)reached 5.82±0.07,3.32±0.15 and 4.38±0.30 g/L,respectively,and 29.4±2.5%,104.8±17.1%and 115.7±10.9%higher than that of the wild type strainThe effect of proline on the tolerance of C.acetobutylicum ATCC 824 to lignocellulose-derived inhibitors was investigated.The tolerance of C.acetobutylicum ATCC 824 to formic acid,ferulic acid,syringaldehyde and p-coumaric acid were significantly improved by exogenous proline addition.Further studies showed that,the intracellular proline synthesis ability,ROS scavenging ability,and the tolerances to formic acid,ferulic acid,syringaldehyde and p-coumaric acid were significantly improved by co-expressing of proline biosynthesis related genes proA,proB and proC.When fermented using undetoxified soybean straw,rice straw and corn straw hydrolysate as the substrates,the butanol titer of engineered strain C.acetobutylicum 824(proABC)was 8.0±0.07,7.80±0.20 and 6.30±0.10 g/L,respectively,and 1-,2.4-and 3.4-fold higher than that of the wild type strainAll the results show that the strategies used in this study successfully improved the butanol production and the lignocellulosic biomass utilization of C.acetobutylicum,it provides a valuable reference for molecular breeding of other biofuel producing microorganism... |
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