Harnessing Algal Peroxisomes for Efficient Poly Hydroxybutyrate Production

Poly hydroxybutyrate (PHB) is considered a biodegradable plastic that is compatible with broad applications. Microalgae, as photosynthetic microorganisms, offer a promising platform for the sustainable production of PHB on an industrial scale. However, cytosolic PHB accumulation commonly caused compromised cell growth, resulting in a low PHB yield. Peroxisomes are promising compartmentalized microfactories, but peroxisomal bioproduct production in microalgae has not been explored. In this study, we reconstructed a complete PHB biosynthesis pathway in the peroxisomes of the model green microalga Chlamydomonas reinhardtii (Chlamydomonas) by the expression of three PHB synthesis genes (phaA, phaB, and phaC) and targeting the gene products to the peroxisome. PHB granules were observed in the peroxisomes of transgenic algal cells under a transmission electron microscope. Furthermore, overexpression of 3-ketoacyl-CoA thiolase (CrKAT1), a key enzyme in fatty acid beta-oxidation, further enhanced PHB production. In the phaA/B/C/CrKAT1 quadruple transgenic lines, the PHB content was up to 21.6 mg/g (dry weight) and was 3600-fold increased over cytosolic production in Chlamydomonas. Furthermore, the PHB-producing strains showed higher cellular dry weight and biomass production without compromising cell growth or altering cell size. Taken together, our data indicate that peroxisomal engineering could be a promising strategy for the efficient production of high-value compounds in microalgae.