Heterologous polyhydroxyalkanoate synthase expression enables poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) production from fatty acids in Rhodobacter capsulatus.

Abstract

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) is a biodegradable copolymer whose mechanical properties can be tuned by the 3-hydroxyhexanoate (3HHx) fraction. However, current industrial production largely relies on established hosts and plant oil-based feedstocks. Here, we developed Rhodobacter capsulatus SB1003 as a new PHBH-producing platform by focusing on the two key determinants of copolymer formation: polyhydroxyalkanoate (PHA) synthase substrate specificity and intracellular monomer supply. A PHA synthase with broad-substrate specificity was integrated into the native phaC locus to generate a heterologous phaC strain. All PHA production experiments were performed under anaerobic photoheterotrophic conditions in 8-mL screw-cap tubes containing 7.6 mL of medium and illuminated with continuous white light. During butyrate cultivation under these conditions, the engineered strain accumulated polymer up to 41.5% of cell dry weight and incorporated detectable 3HHx, whereas the wild type showed no 3HHx incorporation. To increase 3HHx-CoA availability from butyrate, we introduced C4-to-C6 precursor-supply modules involving β-ketothiolase (BktB)/β-ketoacyl-CoA reductase (PhaB) and crotonyl-CoA carboxylase/reductase (Ccr)/ethylmalonyl-CoA decarboxylase (Emd), but these modifications led to only marginal improvements in the 3HHx fraction. In contrast, supplying C6 or longer fatty acids under the same conditions markedly increased 3HHx incorporation; cultivation on hexanoate yielded PHBH containing 32.4 mol% 3HHx. Collectively, this study demonstrates PHBH biosynthesis in R. capsulatus and indicates that limited 3HHx-CoA supply rather than polymerization capacity is the primary bottleneck, providing a foundation for further pathway and host optimization toward flexible PHBH production from diverse substrates.