| The exhaustion of fossil energy is an important issue in the world.In order to relieve the energy crisis,developing new energy has become the trend of the times.Among the various ways,and using biotechnology to produce liquid fuel is an important one.There are three main platforms to convert biomass into liquid fuels:thermochemical platform,sugar platform,and carboxylate platform.The carboxylate platform has the highest energy conversion.As one of the important bacteria in the carboxylate platform,Clostridium kluyveri can produce mediate chain fatty acid(MCFAs,C5-C8)and alcohol by elongating chain of short chain fatty acid through reverse β oxidation.The longer chain fatty acid has the higher energy density,which is more suitable for producing biofuels.In this work,the physiological mechanism of chain elongation in producing fatty acid and the pathway to pruduct alcohol of Clostridium kluyveri were studied.Clostridium kluyveri can ferment acetate and ethanol to produce butyrate and caproate,and the metabolic model has been reproted.It is also reported that propionate could replace the acetate to produce odd number chain of organic acid.In order to clarify the physiological mechanism of chain elongation in C.kluyveri,acetate,propionate,and butyrate were used as substrate to do the fermentation experiment,respectively.Then the GC-MS analysis was carried out to detect the fermentation products.The products of acetate fermentation were found to be butyrate,caproate and caprylate.The products of propionate fermentation were valerate and heptylate.The products of butyrate fermentation were caproate and caprylate.The yields of the products were measured by HPLC further.About 2.64 g/L butyrate,8.2 g/L caproate and 0.46 g/L caprylate were got from the fermentation of acetate and ethanol.About 5.1 g/L valerate,1.87 g/L caproate,1 g/L heptylate and 1.1 g/L caprylate were obtained from the fermentation of propionate and ethanol.About 7 g/L caproate and 1.1 g/L caprylate were produced from the fermentation of butyrate and ethanol.All of the results were meeting the reverse β oxidation mechanism and the products of caproate and caprylate were predicted from the oxidation of ethanol.Interestingly,an ORF annotated to encode Rex was found in the upstream of the gene cluster crtl-bcdl-etfABl-hbdl-nfnAB,which is responsible to butyrate synthesis.Rex was reported as a redox-sensing transcriptional regulator,and it can regulate many genes by sensing the change of NADH/NAD+ ratio.Thus it is possible that the Rex can regulate the genes which play important roles in the chain elongation reaction.In order to study the genes regulated by Rex and the function of Rex,the RegPredict Web server tool was used to predict the genes that may be regulated by Rex.The results showed that the genes related to the synthesis of butyrate and butanol,and the oxidation of ethanol was probably regulated by Rex.What’s more,some genes related to the energy metabolism and electron transfer were also found to be possibly regulated by Rex.After heterologous expression and purification,the Rex was used to perform EMSA and ITC experiments to confirm the interaction of the Rex and the promoter of the targeted genes.The result showed that Rex can bind the promoters of some genes related to the synthesis of butyrate,such as crtl,cat3 and thlA3,where the interaction was effected by NADH/NAD+ ratio.All the results indicated that Rex is a global regulator in C.kluyveri.The redox state in vivo was different in different growth periods of C.kluyveri.In order to study the function of Rex further,the transcriptome of C.kluyveri in different growth periods was performed.In addition,in order to find whether the genes related to the chain elongation reaction are same in different substrates the transcriptome sequencing of C.kluyveri grown in the three different substrates were carried out.By comparision of the transcriptomes in different growth periods,some genes were found be possibly regulated by Rex.By comparision of the transcriptomes from different substrates,some genes were found to play different roles in different substrates,such as the expreession level of hbd2 gene was higher in the substract of propionate.What’s more,there may be another way to elongate the chain in C.kluyveri,that is,two carbon dioxide molecules combine into acetate.However,this prediction needs experimental evidence.Based on the results of transcriptome analyses,some genes related to produce fatty acid were selected for further study.There are two kinds of 3-hydroxybutyryl-CoA dehydrogenase(Hbd)in C.kluyveri,Hbdl(NADPH dependent)and Hbd2(NADH dependent).After heterologous expresions,their enzymatic properties were characterized.Hbdl is NADPH dependent and has a high activity for acetoacetyl-CoA,but Hbd2 has no activity of acetoacetyl-CoA.The reason may be that Hbd2 uses different substract.According to the assay of the Hbd activities in C.kluyveri and the transcriptome analyses,Hbd2 was predicted to catalyze odd number carbon chain substracts.In addition,the activities of NfnAB in cell extract of C.kluyveri were also measured.In order to measure the activities of NfnAB,the ferredoxin from C.pasteurianum was heterologously overexpressed,which was used as a substrate for NfnAB assay.The yields of ferredoxin were much higher than the wild type bacterium.After optimizing the expression condition,round 40 mg[2Fe2S]type ferredoxin and 3.7 mg 2[4Fe4S]type ferredoxin was obtained from 1 L culture,respectively.It is reported that C.kluyveri can produce alcohol in some old papers,but the exact pathway to produce alcohol was not clear.Based on the GC-MS analysis,butanol,hexanol and octanol were found in the fermentation broth.About 11.9 μg/L propanol,12.8 μg/L butanol and 8.3μg/L hexanol were got from the fermentation of acetate and ethanol.About 0.98 mg/L propanol,15.9μg/L butanol and 6.7 μg/L pentanol were got from the fermentation of propionate and ethanol.About 95 μg/L propanol,19.8 μg/L butanol and 14.7 μg/L hexanol were got from the fermentation of butyrate and ethanol.Several feasible pathways to produce butanol were postulated,and the several key enzymes in the pathways were expressed and their Km values were measured.Finally,a pathway catalyzed by a difunctional protein Aad was confirmed,which can catalyze the reduction of butyryl-CoA to form butanol.Above all,the physiological mechanism in chain elongation of fatty acid and the pathway to produce alcohol in C.kluyveri were investigated in this study.C.kluyveri can produce C3(propionate)to C8(caprylate)fatty acids,then the C3(propanol)to C8(Octanol)alcohols are further produced.The transcriptome analysis indicated that almost the same genes were used in extending the carbon chain of fatty acids.The redox-sensing transcriptional regulator Rex was likely to regulate the genes related to chain elongation reaction,such as crtl-bcdl-etfAB1-hbd1-nfnAB and cat3.In addition,the pathway to produce alcohol in C.kluyveri was studied and a feasible pathway catalyzed by the Aad was identified.These results prove the basic knowledge for application of C.kluyveri in the production of medium-chain fatty acid and alcohol.It’s the first time to use the transcriptome to analyze the mechanism of chain elongation.These results also provide a new insight into the physiological mechanism of chain elongation in C.kluyveri.However,there are some points need to be improved in this study.For example,construction of rex-disrupted strain was failed,which hinders to clarify the function of the Rex.And some proteins possible involved in the pathway to produce butanol were not successfully expressed,so further study is required... |
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