Effects Of Intracellular NADH/NAD~+ Ratio Regulation On Butyric Acid Biosynthesis In Clostridium Tyrobutyricum ATCC 25755

Butyric acid is a basic chemical and fine chemical product with a large market capacity and is widely used in the food,chemical and pharmaceutical fields.As a key cofactor in the metabolic network of C.tyrobutyricum,NADH could regulate the metabolic flux and achieve fast and maximum distribution of carbon resource to the target metabolite butyric acid.In this paper,the intracellular NADH/NAD~+ratio was regulated by metabolic engineering to increase the final concentration and selectivity of butyric acid in Clostridium tyrobutyricum,thereby reducing the production cost of bio-based butyric acid.In wild-type C.tyrobutyricum ATCC 25755,the following metabolic engineering strategies were adopted:1)overexpression of glyceraldehyde-3-phosphate dehydrogenase(GAPA)in the glycolytic pathway to increase the supply of intracellular NADH;2)overexpression of NADH-dependent butyryl-Co A dehydrogenase(BCD1)to increase the metabolic rate of NADH in the butyrate biosynthetic pathway;3)co-expression of glyceraldehyde-3-phosphate dehydrogenase(GAPA)and acyl-Co A dehydrogenase(BCD1),and the recombinant strains of C.tyrobutyricum ATCC 25755/gap A,25755/bcd1 and25755/gap A+bcd1 were successfully obtained.In addition,the expression of glyceraldehyde-3-phosphate dehydrogenase(gap A)and butyryl-Co A dehydrogenase(bcd1)genes in C.tyrobutyricum were confirmed by RT-PCR and SDS-PAGE analysis.The results of flask fermentation and the analysis of intracellular NADH/NAD~+ratio showed that the consumption and regeneration of intracellular NADH/NAD+ratio could be adjusted by overexpressing glyceraldehyde-3-phosphate dehydrogenase gene(gap A)and butyryl-Co A dehydrogenase gene(bcd1)individually or simultaneously.As a result,the final concentration,yield and selectivity of butyric acid could be comprehensively improved by regulating intracellular NADH/NAD~+ratio.Compared with batch fermentation with high concentration glucose for the production of butyric acid by C.tyrobutyricum,although the productivity of butyric acid was decreased in fed-batch fermentation,obvious reabsorption of acetic acid and accumulation of butyric acid were observed due to the prolonged fermentation time.Consequently,the concentration of by-product acetate was decreased drastically at the end of the fed-batch fermentation.Among three recombinant strains,ATCC 25755/gap A+bcd1 showed the best fermentation performance.For example,compared with the control,the acetate concentration decreased by56.28%,and the butyrate/acetate ratio increased by 154.09%.Furthermore,the concentration of butyric acid reached 59.53 g/L,which was 11.16%higher than the wild-type strain.To further increase the concentration,productivity and yield of butyric acid,the fermentation was carried out in a fibrous bed bioreactor(FBB)by immobilized C.tyrobutyricum.Among all the strains,the improvement in fermentation performance of25755/gap A+bcd1 was the significant.The concentration,productivity and yield of butyrate reached 63.75 g/L,0.85 g/L·h and 0.36 g/g,respectively,increased by 7.1%,34.9%and 12.5%as compared with the results obtained in free cell fed-batch fermentation.In addition,the concentration of by-product acetate was reduced from 9.14 g/L to 5.25 g/L,and the butyrate/acetate ratio was increased by 97.88%when compared with wild-type strain.By overexpressing the key enzymes involved in NADH synthesis and metabolism in the C.tyrobutyricum ATCC 25755,including glyceraldehyde-3-phosphate dehydrogenase(GAPA)and butyryl-Co A dehydrogenase(BCD1),intracellular NADH/NAD~+ratio was changed,and butyric acid synthesis was strengthened.By comparing the various fermentation modes,butyric acid fermentation in FBB was finally selected because it could achieve efficient acetate reabsorption and butyrate biosynthesis.increasing the economic feasibility and reducing the downstream cost of bio-based butyric acid production.In summary,this study indicates that there is a close relationship between the metabolic network of butyrate biosynthesis and the cofactor regulatory network,and cofactor engineering was an efficient method to regulate the metabolic flux in C.tyrobutyricum.