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Greenhouse gas emissions and dwindling fossil fuel reserves have pushed developed countries like the United States to explore renewable fuel sources. “Biofuels” are an attractive sustainable energy technology because they can be produced from plant biomass, which includes wood, grasses, and agricultural waste. However, the bioenergy industry faces problems converting this inexpensive plant matter into high value fuels. Biomass is tough to break down and requires costly pretreatment processes before it can be converted to fuel. Pretreatment produces toxic byproducts, including furfural and 5-hydroxymethyl furfural (HMF), which will kill cell cultures and inhibit the conversion of biomass to usable sugars.

To solve this problem, we intend to engineer bacteria with a recently discovered metabolic pathway that consumes furfural and HMF. Koopman et. al. identified the six enzyme pathway from Cupriavidus basilensis and showed that it functions in Pseudomonas putida. In C. basilensis or P. putida, HMF can be used as the sole carbon source. Engineering bacteria with this pathway would allow them to survive and produce biofuels but also use the toxic HMF as an energy source.

However, this pathway does not function in Esherichia coli. Based on our recent experiments, the pathway also does not function in Pseudomonas fluorescens, a microbial relative of P. putida.

We want to determine the genomic differences that allow the pathway to function in one organism versus another. We intend to do this using a novel approach, combinatorial dCas9 gene knockdown. The final objective of this research is to engineer the HMF pathway in E. coli and bring us one step closer to sustainable biofuels produced by bacteria

Abstract

Codon level optimization of genes allows for fine tuning of expression due to differences in translational efficiency between degenerate codons. It is theorized that through this technique expression ceilings due to translation becoming the rate limiting step in protein synthesis can be lifted. Traditional methods in E. coli rely on the preference for certain codons across the entire genome, yet this is not the only possible approach. In this project, novel criteria for codon optimization were employed to design and create variants of a reporter gene that was then characterized in vivo. Results show that the new criteria for codon optimization, for example the statistical correlation between a degenerate codon and its presence in highly translated parts of the genome, are feasible for use in future projects. This leads to new theories about the mechanics of translation, and will allow researchers to optimize genes at the codon level with greater fidelity.