Date of Award
Doctor of Philosophy (PhD)
The global energy crisis and increasing environmental concerns have spurred the sustainable production of biofuels and other fossil-fuel-derived chemicals. One approach is to engineer microbial metabolic pathways to convert renewable feedstocks (e.g. lignocellulosic biomass) to compounds that have structures similar to those of petroleum-derived fuels and chemicals. The microbial fatty acid (FA) biosynthetic pathway is attractive because FAs are common precursors that can be converted to several types of chemicals, including alkanes, alkenes, alcohols, and esters. These chemicals have numerous applications as fuels, fragrances, emollients, plasticizers, thickeners, and detergents. Most work has used Escherichia coli as the microbial host due to its exceptionally high rate of fatty acid biosynthesis.
To broaden the use of FA-derived compounds, it is important to diversify the structure of biosynthesized FAs. Unfortunately, wild-type E. coli synthesize only straight long-chain FAs (mostly palmitic acid), which limits their range of application. On the other hand, chemicals derived from non-natural fatty acids (NNFAs) have wider applications. For example, branched-chain FA-derived hydrocarbons have better combustion properties than their straight-chain counterparts when used as fuels, and ω-hydroxy acids and di-acids are monomers for polymers. My dissertation work explored engineering E. coli metabolic pathways to produce NNFAs and their derivatives. I started with the production branched-chain fatty acids (BCFAs), and showed that high percentage BCFA (or derivatives) production can be achieved via my engineered strains. The composition of BCFAs was controlled to produce even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Next, I partitioned the complete pathway into three modules: a precursor formation module, an acyl-CoA activation and malonyl-ACP consumption module, and a final product synthesis module. I engineered and tuned each module separately and combined the optimal modules to produce a high percentage of branched-chain fatty alcohols, alkanes, and esters. Finally, I established a new cell-free framework as a proof-of-concept for in vitro production of fatty acid derivatives, providing a platform for rapid screening and prototyping of fatty acid synthetic pathways to produce bifunctional fatty acid derivatives.
The findings of my work have provided a deeper understanding of the potential of the fatty acid synthetic pathway for NNFA production, and the strain-engineering strategies used in this dissertation will open new opportunities for efficient production of chemicals derived from NNFAs.
Michael C. Jewett, Yinjie Tang, Tae Seok Moon, Himadri Pakrasi,