Development of a highly degenerate primer-based molecular tool for detecting and classifying the four major classes of polyhydroxyalkanoate synthase (phaC) genes in bacteria

Polyhydroxyalkanoate synthase (phaC) gene encodes for PHA synthase enzyme which plays a key role in PHA polymerisation. To screen, unknown bacterial strains for their potential to produce PHAs, the presence of phaC gene is essential. Currently published primer sets targeting phaC gene are inadequate and often work only for well-studied genera (e.g., Pseudomonas, Cupriavidus). Few studies validate them in vitro, even fewer use degenerate primers to address phaC sequence diversity, and many fail to target all four phaC classes. In this study, nine novel highly degenerate primers were designed using the HYDEN (HighlY DEgeNerate) tool. The design included 65 phaC gene sequences from class I, 10 from class II, 19 from class III, 30 from class III/IV, and 6 from class IV, carefully selected as a representative sample size to capture the variations among bacterial strains and phaC sequences. The primer specificity was then assessed in silico with De-MetaST-BLAST against all known phaC sequences in the NCBI database. This was followed by in vitro screening of seven bacterial strains known to express the four major classes of phaC genes and 15 novel marine bacterial strains in which phaC presence were unknown. Seven strains-namely Halomonas alkaliphila DINO, Marinobacter sp. MB2, Halomonas profundus NQ7, Halomonas titanicae MC2, Bacillus pacificus C4, Bacillus pacificus B4 and Bacillus mycoides B12 tested positive. All 15 strains were subjected to nutrient limiting growth conditions to assess PHA production with results confirming molecular screening. This study demonstrates the successful development and validation of a highly degenerate primer-based molecular screening tool capable of detecting and differentiating the four major classes of phaC genes in well-known non-marine and novel marine PHA-storing bacteria.