[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-386] Heterologous polyhydroxyalkanoate synthase expression enables PHBH production in Rhodobacter capsulatus

○Kako Miura¹, Takayuki Shimizu², Kenya Tanaka^1,3,4, Tomohisa Hasunuma^1,3,5,6 (1. Grad. Sch. Sci. Technol. Innov., Kobe Univ. (Japan), 2. Div. Nat. Sci., Nara Women’s Univ. (Japan), 3. EGBRC, Kobe Univ. (Japan), 4. Grad. Sch. Eng. Sci., Univ. Osaka (Japan), 5. CSRS, RIKENS (Japan), 6. Grad. Sch. Eng., Dept. Chem. Sci. Eng., Kobe Univ. (Japan))

Keywords:

purple non-sulfur bacteria,PHBH,PhaC,β-oxidation,photosynthesis

Biodegradable polymers have attracted attention as a potential solution to marine plastic pollution. Among them, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) is a biodegradable copolymer whose mechanical properties can be tuned by varying the molar fraction of 3-hydroxyhexanoate (3HHx). PHBH containing approximately 10 mol% 3HHx exhibits material properties similar to petroleum-derived plastics such as polyethylene and polypropylene and is already produced on an industrial scale. However, the current production process relies on plant oils as feedstocks, raising concerns about competition with food resources and tropical deforestation associated with plant oil production. In addition, the process requires aerobic cultivation, and the energy cost associated with aeration remains a challenge.To address these limitations, this study focused on Rhodobacter capsulatus SB1003 as a novel platform for PHBH production. This bacterium possesses several advantageous characteristics, including the ability to utilize wastewater-derived resources, established genetic manipulation systems, an intrinsic polyhydroxyalkanoate (PHA) biosynthetic pathway, and the capability for anaerobic photosynthetic growth without aeration. Although R. capsulatus can synthesize poly(3-hydroxybutyrate) (PHB), neither previous studies nor the wild-type strain examined in this study have demonstrated PHBH production, suggesting that this organism lacks the inherent ability to synthesize PHBH.To enable PHBH production, we focused on two key factors governing copolymer formation: the substrate specificity of PHA synthase and the intracellular monomer supply pathway. A heterologous phaC gene encoding a PHA synthase with broad substrate specificity was introduced into the native phaC locus to construct a recombinant strain. Under photoheterotrophic cultivation with butyrate as the carbon source, the engineered strain accumulated polymer corresponding to approximately 41% of the cell dry weight and incorporated detectable amounts of 3HHx, whereas no incorporation of 3HHx was observed in the wild-type strain.To increase the supply of 3HHx-CoA from butyrate, a reverse β-oxidation module and an additional crotonyl-CoA carboxylase/reductase module were introduced. However, these modifications resulted in only limited improvement in the 3HHx fraction of the polymer. In contrast, supplementation with long-chain fatty acids (C6 or longer) markedly enhanced 3HHx incorporation. When hexanoate was used as the carbon source, PHBH containing approximately 32 mol% 3HHx was produced.This study demonstrates, for the first time, PHBH biosynthesis in R. capsulatus and reveals that the supply of 3HHx-CoA, rather than the polymerization capacity itself, is the primary bottleneck in copolymer production. These findings provide a foundation for further optimization of metabolic pathways and host strains toward flexible PHBH production from diverse substrates.