Molecular analysis of starch utilization in solventogenic Clostridium

Abstract

Solventogenic clostridia were commonly used in the 1900’s for the production of acetone, butanol and ethanol for various industries through ABE fermentation prior to being phased out in the late 1950’s and 60’s due to competition from cheaper oil based products. With the recent increase in interest in green technologies and the move away from oil, these solventogenic fermentations have again become industrially relevant. Particular interest has been shown in how clostridia break down a variety of industrial feedstocks, from cellulose to starch. Prior analysis of a protein secreted by C. saccharoperbutylacetonicum when grown in the presence of starch, revealed that it matched closely to a family of proteins called cyclodextrin glycosyltransferases (Cgt) responsible for breaking down starch into small ring-shaped polymers called cyclodextrins, a possibly novel method of starch metabolism for clostridial species. Further in-silico analysis identified a further 7 genes, encoding for possible starch degrading enzymes, transporter proteins and a putative operon regulator. The main aims of this project was to measure the expression of the genes of the starch operon when C. saccharoperbutylacetonicum was grown on different carbon sources. Expression levels were measured by qPCR and showed that the presence of starch had a significant effect on the expression of the genes of the starch operon. Further gene expression analysis carried out in a mutant strain of C. saccharoperbutylacetonicum which had a truncated version of the putative operon regulator lead to unregulated expression, no matter the carbon source suggesting that the putative regulator does indeed act on the expression of the operon. Further to the gene expression analysis, cloning and in-silico analysis was carried out to identify the function of the proteins expressed from the genes of the starch operon. Cloning efforts were focused on the three putative starch degrading proteins encoded by cgt, gdp, and aac, but efforts to clone any of the three genes into E. coli were unsuccessful. The in-silico analysis added further evidence to the role of several of the proteins, and found a possible function for the protein previously annotated as AAC, as part of a cyclodextrin degrading family of proteins called cyclodextrinase’s.