Metabolic Engineering Of Clostridium Tyrobutyricum For Enhanced Butyric Acid Production And Lignocellulosic Utilization

Butyric acid is a C₄-carboxylic acid which is widely used as food addtives,industrial chemicals,animal feed supplements,pharmaceuticals and perfume.At present,butyric acid is mainly and cheaply produced by chemical synthesis from petroleum cracking.Recently,with diminishing fossil resources,concerns about environmental pollution by the consumption of petrochemicals,and consumer preferences toward natural ingredients for food,animal feed supplements and pharmaceuticals,butyric acid obtained from renewable source by microbial fermentation has attracted much attention as an alternative to petroleum-based products.However,butyric acid from biological fermentation was hampered by many factors,such as its low yield and productivity,high cost of substrate and downstream process.To overcome these difficulties faced in industrial application of butyric acid production by microbial fermentation,the objective of this study was to enhance butyric acid concentration,butyrate/acetate ratio and lignocellulosic hydrolysate utilization of Clostridium tyrobutyricum ATCC 25755 through metabolic engineering strategies.Overexpressing the genes of 6-phosphofructokinase（pfkA）and pyruvate kinase（pykA）in Clostridium tyrobutyricum ATCC 25755 could significantly improve intracellular NADH and ATP levels,which has been shown to enhance the butyric acid tolerance and productivity.Fed-batch fermentation results showed that the final concentration of butyric acid and productivity of ATCC 25755/pfkA+pykA reached 48.19 g/L and 0.50 g/L·h,respectively,increased by 38.00%and 38.89%as compared with the wild-type strain.Furthermore,ATCC25755/pfkA+pykA still had a higher glucose tolerance.When the glucose concentration reached 120 g/L,the butyric acid productivity of ATCC 25755/pfkA+pykA was up to 0.82g/L·h.Enhancing the acetyl-Co A to butyrate flux by co-overexpression of butyryl-CoA/acetate CoA transferase（cat1）and crotonase（crt）could significantly reduce acetic acid production of C.tyrobutyricum ATCC 25755.Fed-batch fermentation results showed that the acetic acid produced by ATCC 25755/cat1+crt was reduced to 2.38 g/L,and the butyrate/acetate ratio was increased by 124.04%as compared with the wild-type strain.However,overexpression of cat1 and crt had no significant effect on butyric acid production.Hence,the recombination strain of ATCC 25755/ppcc（co-overexpression of pfkA,pykA,cat1 and crt）was constructed to enhance both butyric acid production and butyrate/acetate ratio.The fed-batch fermentation results of ATCC 25755/ppcc indicated that the butyric acid concentration and butyrate/acetate ratio reached 47.63 g/L and 13.72 g/g,respectively,increased by 36.44%and 95.16%as compared with the wild-type strain.To test whether butyric acid stress can induce transcriptional changes of the Class I heat shock protein genes（HSGs）in C.tyrobutyricum ATCC 25755,quantitative real-time PCRs were carried out.The results indicated that the Class I HSGs grpE,dnaK,dnaJ,groEL,groES,and htpG were significantly upregulated under butyric acid stress,which indicated that Class I HSPs may have an important role in the process of dealing with butyric acid stress in C.tyrobutyricum.And then,the effects of overexpressing Class I HSGs on butyric acid tolerance and production were detected by butyric acid tolerance test and fed-batch fermentation.Finally,the butyric acid production and inhibition constant of C.tyrobutyricum ATCC25755/groESL reached 44.13 g/L and 3.52 g/L,respectively,increased by 28.21%and 101.14%as compared with the wild-type strain,which indicated that ATCC 25755/groESL showed both enhanced butyric acid production and tolerance.To overcome inhibitory effects of lignocellulosic hydrolysate-derived inhibitors on cell growth and butyric acid fermentation,the feasibility of enhancing the resistance of C.tyrobutyricum ATCC 25755 to lignocellulosic inhibitory compounds by overexpression of groESL was tested.The results showed that the butyric acid concentration of wild-type reached 17.62 g/L,17.22 g/L and 16.71 g/L with hydrolysates of corncob（CCH）,soybean hull（SHH）and soybean straw（SSH）as carbon source,respectively.However,the butyric acid concentrations of wild-type were only 14.23 g/L,12.84 g/L and 11.22 g/L with hydrolysates of rice straw（RSH）,corn straw（CSH）,and wheat straw（WSH）,respectively,indicating their high toxicity for C.tyrobutyricum.As expected,the butyric acid concentrations of ATCC 25755/groESL increased by 18.90%,25.62%and 18.89%as compared with wild-type,implying that ATCC 25755/groESL had a higher tolerance to lignocellulosic hydrolysate-derived inhibitors than the wild-type strain.And then,the toxicity tests of single inhibitor confirmed that overexpression of groESL could enhance the tolerance of C.tyrobutyricum to phenolic compounds,furan derivatives and dilute acids,especially for phenolic compounds.The batch fermentation results indicated that compared to the wild-type strain,the butyric acid produced by ATCC 25755/groESL reached 29.62 g/L（CSH）and 30.12g/L（RSH）,increased by 26.53%and 19.57%.And more importantly,the butyric acid productivity reached 0.31 g/L·h（vs.0.20-0.21 g/L·h for the wild-type strain）due to the shortened lag phase.In addition,the tolerance of C.tyrobutyricum ATCC 25755 to furfural was significantly enhanced by expressing the short-chain dehydrogenase/reductase（sdr）gene from Clostridium beijerinckii NCIMB 8052.The results of furfural tolerance test indicated that the growth of wild-type was completely inhibited with 2.5 g/L furfural,while the tolerance of ATCC25755/sdr to furfural increased to above 3.0 g/L due to its higher recovery efficiency for furfural,which was confirmed by the subsequent enzyme activity assays of furfural-related reductase.The results of fermentation in serum bottles showed that the butyric acid concentration and productivity of ATCC 25755/sdr reached 20.84 g/L and 0.15 g/L·h,increased by 10.15%and 36.36%as compared with the wild-type strain,respectively.The focus of this study was to enhance the butyric acid fermentability of C.tyrobutyricum by metabolic engineering,which has done some research on the enhancement of butyric acid production and selectivity,butyric acid tolerance,and lignocellulosic hydrolysates utilization by C.tyrobutyricum.And these strategies can provide some new ideas for the development of butyric acid fermentation by microorganisms.