α-Substituted 3-hydroxy acid production from glucose in Escherichia coli

Polyhydroxyalkanoates (PHAs) are renewably-derived, microbial polyesters composed of hydroxy acids (HAs). Demand for sustainable plastics alternatives, combined with the unfavorable thermal properties exhibited by some PHAs, motivates the discovery of novel PHA-based materials. Incorporation of α-substituted HAs yields thermostable PHAs; however, the reverse β-oxidation (rBOX) pathway, the canonical pathway for HA production, is unable to produce these monomers because it utilizes thiolases with narrow substrate specificity. Here, we present a thiolase-independent pathway to two α-substituted HAs, 3-hydroxyisobutyric acid (3HIB) and 3-hydroxy-2-methylbutyric acid (3H2MB). This pathway involves the conversion of glucose to various branched acyl-CoAs and ultimately to 3HIB or 3H2MB. As proof of concept, we engineered Escherichia coli for the specific production of 3HIB and 3H2MB from glucose at titers as high as 66 ± 5 mg/L and 290 ± 40 mg/L, respectively. Optimizing this pathway for 3H2MB production via a novel byproduct recycle increased titer by 60%. This work illustrates the utility of novel pathway design HA production leading to PHAs with industrially relevant properties.