Engineering Escherichia coli for Conversion of Glucose to Medium-Chain ω-Hydroxy Fatty Acids and α,ω-Dicarboxylic Acids

Abstract

In search of sustainable approaches to plastics production, many efforts have been made to engineer microbial conversions of renewable feedstock to short-chain (C2-C8) bifunctional polymer precursors (e.g., succinic acid, cadaverine, 1,4-butanediol). Less attention has been given to medium-chain (C12-C14) monomers such as ω-hydroxy fatty acids (ω-OHFAs) and α,ω-dicarboxylic acids (α,ω-DCAs), which are precursors to high performance polyesters and polyamides. Here we engineer a complete microbial conversion of glucose to C12 and C14 ω-OHFAs and α,ω-DCAs, with precise control of product chain length. Using an expanded bioinformatics approach, we screen a wide range of enzymes across phyla to identify combinations that yield complete conversion of intermediates to product α,ω-DCAs. Finally, through optimization of culture conditions, we enhance production titer of C12 α,ω-DCA to nearly 600 mg/L. Our results indicate potential for this microbial factory to enable commercially relevant, renewable production of C12 α,ω-DCA-a valuable precursor to the high-performance plastic, nylon-6,12.

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…In that study, both thioesterase and fatty acid hydroxylase P450 BM3 were involved in the production of free fatty acids and in their conversion into oxygenated monomers. Bowen et al (2015) also engineered E. coli for sugar-based C12 and C14 ω-hydroxy FAs and α,ω-DCAs production [6]. This was achieved by expressing high-specificity acyl ACP thioesterases (TEs) to detach free fatty acids from the fatty acid synthase (FAS II).…”

“…For example, inactivation of FAA1 and FAA4 genes in S. cerevisiae was found to mitigate the feedback inhibition of acyl-CoA on fatty acid synthesis and undermine the β-oxidation upregulating-effect of fatty acid overproduction [86]. Overexpression of thioesterases has also been employed to release the free fatty acids of specific chain lengths [6].…”

Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

“…AlkJ was applied before to catalyse the conversion of alcohols to aldehydes in E. coli 78,113,117 . This is the first report of its functionality on 9-hydroxy ethyl nonanoate.…”

“…Bowen et al have also used AlkJ in combination with a set of aldehyde dehydrogenases in E. coli. They applied these enzymes for the production of α,ω-DCAs, from ω-hydroxy fatty acids that were produced de novo from glucose 113 . AlkJ was clearly the best performer among the tested alcohol dehydrogenases.…”

“…This results in irreversible alcohol oxidation 78 , which is desirable for DCA production. Recently, AlkJ was also identified as the best among 5 alcohol dehydrogenases for C12 and C14 DCA production with E. coli 113 . The ubiquinol generated by AlkJ can be oxidized in the electron transport chain, which yields ATP.…”