Insights into transcriptional expression and putative functions of multiple polyhydroxyalkanoate synthase paralogs in Haloferax mediterranei

The halophilic archaeon Haloferax mediterranei is a promising candidate for polyhydroxyalkanoate (PHA) production, offering several advantages due to its extremophilic physiology. While its primary PHA synthase, a class III enzyme composed of PhaCHme and PhaEHme subunits, has been well characterized, the genome encodes three additional phaC paralogs (phaC1, phaC2, and phaC3), which were previously labeled as cryptic and remain poorly understood. In this study, we systematically investigated these paralogs by employing a targeted bioinformatics pipeline, revealing notable diversity in PHA synthases among Halobacteriales and underscoring the distinctiveness of H. mediterranei. We further analysed the native transcriptional expression profiles of all phaC paralogs under three physiologically relevant conditions: growth-limiting and growth-permissive conditions, as well as valeric acid supplementation to alter PHA monomer composition. Quantitative RT-PCR analysis demonstrated that all three paralogs are transcriptionally active and differentially expressed, refuting earlier assumptions of their cryptic nature. Expression patterns were found not to correlate to polymer composition but to be dependent on growth phase, suggesting a potential physiological role for each paralog in native PHA metabolism. These findings offer new insights into the functional complexity of PHA biosynthesis in H. mediterranei and lay the groundwork for future metabolic engineering aimed at optimizing biopolymer production.