EXPANDING METABOLISM FOR BIOPRODUCTION OF BIOLFUELS

PROJECT ABSTRACT

The University of Oklahoma iGEM team has chosen to focus on the anaerobic and solvent-producing bacterium, Clostridia acetobutylicum ATCC 824. Our goal is to engineer metabolic pathways for the purpose of expanding the range of alcohol production. C. acetobutylicum is a native producer of alcohols and provides an optimal model organism for the expression of alcohol cassettes, as it is tolerant to toxic substrates and metabolites.

Ultimately, we are interested in designing and building a biological system for the greater purpose of producing biofuels. Additionally, we are involved in educating the public about the applications of genetic engineering.

METHODOLOGY

1. Plasmid Construction of Shuttle Vector

A plasmid was constructed containing an origin of replication for Clostridia. The promoter P_ptb-, absent of a ribosome binding site, was added to increase the efficiency of expression levels. The reasoning behind this specific promoter is because E. coli promoters are not know for functioning well in gram positive species. Additionally, an mlsR antibiotic resistance gene was incorporated into the pSB1C3 plasmid. mlsR was chosen because it provided a resistance not already found in C. acetobutylicum or the backbones.

2. Confirmation of Shuttle Vector In Vivo Methylation

C. acetobutylicum ATCC 824 excises foreign DNA by the restriction endonuclease Cac824I. This is a major barrier for electrotransformation due to Cac8241 restriction sites present in the plasmid. Escherichia coli DH5α contains the methylating plasmid pAN1, which is hypothesized to methylate the vector plasmid for protection.

Figure 1. Gel confirmation of methylated shuttle vector after transformation into E. coli strain DH5α containing the methylating plasmid pAN1. From left to right: lane two contains the pAN1 plasmid, lane three contains an unmethylated shuttle vector, lanes four through six contain the pAN1 plasmid (top band) and methylated shuttle vector (bottom band). Lanes 1 and 6 contain reference ladders.

3. Future Endeavors: Expression of Alcohol Cassettes in C. acetobutylicum and E. coli

The future endeavor of the Oklahoma iGEM team will be to express alcohol constructs into C. acetobutylicum. While electrotransformation of the shuttle vector into C. acetobutylicum is still in progress, plasmids containing alcohol cassettes are being constructed containing beta-ketothiolase and trans-2-enoyl reductase, which will yield hexanol in addition to the original pathway. The metabolic pathway of hexanol is shown below. Accordingly, the natural products of the genus Clostridia’s solvent producing capabilities is shown below.