Cloning of butyrogenic genes from Clostridium difficile and metabolic pathway reconstitution in Escherichia coli

In this study, a novel strategy for systematic applications in synthetic biology aiming the transfer of metabolic capacities from a donor to an acceptor organism was elucidated. As a model system, it was chosen the butyrate fermentation of Clostridium difficile, which has not been studied in details before. In order to succeed, individual enzymes, thiolase (EC 2.3.1.9), -hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157), crotonase (EC 4.2.1.17), butyryl-CoA dehydrogenase with its two electron transferring flavoprotein subunits (EC 1.3.99.2), phosphate butyryltransferase (EC 2.3.1.19) and butyrate kinase (EC 2.7.2.7), were initially produced in E. coli in order to demonstrate their functional production, followed by an initial characterization in vitro. Substrate specificity, stability and kinetic parameters were established in order to address their capability for the synthesis of various metabolic intermediates in volatile fatty acid biosynthetic pathways. In this context, novel enzyme tests for phosphate butyryl-transferase and butyrate kinase were developed and a facile method for testing of bi-forking butyryl-CoA dehydrogenase in the physiological reaction was established. Using the latter assay, evidence was obtained for a bi-forking butyryl-CoA dehydrogenase in C. difficile, which could allow energy conservation aided by reduced ferredoxin, an ability lacking in the most intensively studied pathway of C. acetobutylicum. The impact of such enzymes, and of the second rate limiting one (3-hydroxybutyryl-CoA dehydrogenase), in butan-1-ol-forming recombinant strains is discussed. Successively, the enzyme encoding genes were assembled in a synthetic operon in order to create an artificial metabolic pathway module, which was expressed from a single plasmid and enabled the formation of the predicted product, butyrate. Two different butyrate metabolic pathway modules, pASG.wt_BUTD and pASG.wt_BUTAC, were constructed, which contained the butyrate fermentation genes (thia1, hbd, crt2, pbt and buk) from C. difficile, but differed in the origin of the butyryl-CoA dehydrogenase/ETF complex genes. These genes were either from C. difficile (pASG.wt_BUTD) or C. acetobutylicum (pASG.wt_BUTAC). The recombinant butyrogenic pathways were analyzed in vivo by a combination of enzymes activity measurements in cell free extracts and analysis of the fermentation process: substrate uptake, biomass-, product- and by-product- formations were established in order to understand the mass flows in the process. Only the recombinant strains were capable to produce butyrate with a maximum concentration of 3.1 and 3.7 mM, respectively. While growth characteristics, substrate consumption and product yields were rather similar in the recombinant strains, the butyryl-CoA dehydrogenase of different origin and sub-class caused remarkable different kinetics of butyrate formation, which are discussed with regard to their potential applications in solventogenis.