The over-expression of phasin and regulator genes promoting the synthesis of polyhydroxybutyrate in Cupriavidus necator H16 under non-stress 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. Firstly, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen-stress and non-stress conditions was studied. Three types of transcription different genes were discovered: PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen-stress condition, acetoacetyl-CoA reductase gene phaB2, PHB synthase gene phaC2, phasins genes phaP1 and phaP2, regulators genes uspA and rpoN were up-regulated 3.0, 2.5, 1.8, 2.7, 3.5, 1.6 folds, respectively. Secondly, the functions of up-regulated genes and their applications in PHB synthesis were further studied. It was found that the over-expression of phaP1, phaP2, uspA, and rpoN can induce PHB synthesis under non-stress condition, while phaB2 and phaC2 have no significant effect. Under the optimum condition, PHB percentage content in C. necator H16 was respectively increased by 37.2%, 28.4%, 15.8%, and 41.0% with the over-expression of phaP1, phaP2, uspA, and rpoN, and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5% under non-stress 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 respectively increased by 54.4% and 103.1% with the over-expression of rpoN under non-stress 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 up-regulated under non-stress conditions were identified through comparative transcriptome analysis and over-expression of phasin and regulator genes were demonstrated to promote PHB synthesis in C. necator H16.