Engineering and metabolic characteristics of a Clostridium tyrobutyricum strain

Abstract:

Clostridium tyrobutyricum is suitable for simultaneous saccharification and fermentation of lignocellulosic. It can produce butyric acid, acetic acid as its main fermentation products from a wide variety of carbohydrates such as glucose, xylose, cellobiose and arabinose. In order to decrease acetic acid content and increase butyric acid content in C. tyrobutyricum, we replaced genes on the acetic acid fermentation pathway with genes on the butyric acid fermentation pathway. Three genes were selected. They were acetyl-CoA acetylrtansfers gene (thl) which is the key enzyme gene associated with acetic acid fermentation pathway from Clostridium acetobutylicum, erythromycin gene (em) from plasmid pIMP1 and phosphotransacetylase gene (pta) which is the key enzyme gene associated with butyric acid fermentation pathway from C. tyrobutyricum. We fused these genes with pUC19 to construct nonreplicative integrated plasmids pUC19-EPT. Then we transformed pUC19-EPT into C. tyrobutyricum through electroporation. The recombinant transformants grown on plates containing erythromycin were validated by PCR. A mutant whose pta gene was displaced by thl gene on the chromosome was selected. In the fermentation from glucose, the mutant’s yield of butyric acid is 0.47, increased by 34% compared with wild type; and the yield of acetic acid is 0.05, decreased by 29% compared with wild type.

Key words:Clostridium tyrobutyricum, butyric acid, acetyl coenzyme A acetyltransferase, phosphotransacetylase, metabolic engineering