Beyond the canonical PHA synthase: insights into transcriptional expression and functions of phaC paralogs in Haloferax mediterranei

The halophilic archaeon Haloferax mediterranei is a promising candidate for polyhydroxyalkanoate production, offering several advantages due to its extremophilic physiology. While its primary polyhydroxyalkanoate 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 polyhydroxyalkanoate synthases among Halobacteriales and underscoring the distinctiveness of H. mediterranei. We further analyzed 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 polyhydroxyalkanoate monomer composition. RT-qPCR 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 polyhydroxyalkanoate metabolism. These findings offer new insights into the functional complexity of polyhydroxyalkanoate biosynthesis in H. mediterranei and lay the groundwork for future metabolic engineering aimed at optimizing biopolymer production.