The Overexpression of Phasin and Regulator Genes Promoting the Synthesis of Polyhydroxybutyrate in Cupriavidus necator H16 under Nonstress Conditions

Cupriavidus necator H16 is an ideal strain for polyhydroxybutyrate (PHB) production from CO₂. Low-oxygen stress can induce PHB synthesis in C. necator H16 while reducing bacterial growth under chemoautotrophic culture. The optimum growth and PHB synthesis of C. necator H16 cannot be achieved simultaneously, which restricts PHB production. The present study was initiated to address the issue through comparative transcriptome and gene function analysis. First, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen stress and nonstress conditions was studied. Three types of genes were discovered to have differential levels of transcription: those involving PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen stress conditions, acetoacetyl-coenzyme A (CoA) reductase gene phaB2, PHB synthase gene phaC2, phasins genes phaP1 and phaP2, and regulator genes uspA and rpoN were upregulated 3.0-, 2.5-, 1.8-, 2.7-, 3.5-, and 1.6-fold, respectively. Second, the functions of upregulated genes and their applications in PHB synthesis were further studied. It was found that the overexpression of phaP1, phaP2, uspA, and rpoN can induce PHB synthesis under nonstress conditions, while phaB2 and phaC2 have no significant effect. Under the optimum conditions, the PHB percentage content in C. necator H16 was increased by 37.2%, 28.4%, 15.8%, and 41.0%, respectively, with overexpression of phaP1, phaP2, uspA, and rpoN, and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5%, respectively, under nonstress chemoautotrophic conditions. Similar promotion by phaP1, phaP2, uspA, and rpoN was observed in heterotrophically cultured C. necator H16. The PHB percentage content and PHB production were increased by 54.4% and 103.1%, respectively, with the overexpression of rpoN under nonstress heterotrophic conditions. IMPORTANCE Microbial fixation of CO₂ is an effective way to reduce greenhouse gases. Some microbes, such as C. necator H16, usually accumulate PHB when they grow under stress. Low-oxygen stress can induce PHB synthesis when C. necator H16 is autotrophically cultured with CO₂, H₂, and O₂, while under stress, growth is restricted, and total PHB yield is reduced. Achieving the optimal bacterial growth and PHB synthesis at the same time is an ideal condition for transforming CO₂ into PHB by C. necator H16. The present study was initiated to clarify the molecular basis of low-oxygen stress promoting PHB accumulation and to realize the optimal PHB production by C. necator H16. Genes upregulated under nonstress conditions were identified through comparative transcriptome analysis and overexpression of phasin, and regulator genes were demonstrated to promote PHB synthesis in C. necator H16.