Engineering Escherichia coli for production of C12–C14 polyhydroxyalkanoate from glucose

Abstract

Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C12 and C14 fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW – the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from butyrate using engineered Ralstonia eutropha

“…In addition, plant oils are more expensive in comparison to waste organic acids and are often considered controversial due to the conflicting production of biochemicals versus food, similar to the issue of corn being used for biofuel versus food (Flammini 2008). Therefore, using unrelated carbon sources such as glucose, fructose, and gluconate for P(HB-co-HHx) synthesis will be helpful to overcome those problems (Agnew et al 2012;Wang et al 2012). However, to achieve them, many pathways in wild-type strains must be modified to synthesize P(HB-co-HHx) from unrelated carbon sources and the resulting 3HHx incorporation is very low (Fukui et al 2002;Qiu et al 2005).…”

Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha

“…Several previous studies have utilized fatty acid de novo biosynthesis pathway to provide (R)-3HA-CoAs. These were further divided into two groups based on the engineering strategy: channeling from acyl-acyl carrier protein (ACP) intermediates to (R)-3HA-CoAs (Matsumoto et al, 2001;Nomura et al 2005;Wang et al, 2012b); and cleavage of long acyl-ACPs by thioesterases followed by (R)-3HA-CoA formation through β-oxidation of the released acyl moieties (Agnew et al, 2012;Klinke et al, 1999;Rehm and Steinbuchel, 2001). In terms of the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from sugars, Qiu et al (2005) applied the former strategy in Pseudomonas putida, which resulted in the accumulation of P(3HB-co-6.8 mol% 3HHx) with 12.8 wt% of the dry cell mass from gluconate, and the latter strategy in Aeromonas hydrophila which resulted in the accumulation of P(3HB-co-19.2 mol% 3HHx) with 14.7 wt% from glucose.…”

Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum

“…In this study, an engineered R. rubrum strain synthesized PHA MCL at up to 7.1% (wt/wt of CDW), consisting predominantly of 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD), with traces of 3-hydroxyhexanoate (3HH), from an artificial syngas mixture. Expectedly, this amount of synthesized PHA together with the produced biomass is smaller than that reported in foregoing studies focusing on the production of PHA MCL from heterotrophic unrelated carbon sources in recombinant E. coli or various strains of Pseudomonas (4,(39)(40)(41). However, the feedstocks used in these studies differ significantly from the newly applied potential waste gases in the present study.…”