阻断集胞藻6803 PHB合成途径提高胞内NADPH含量

Increasing reductant NADPH content via metabolic engineering of PHB synthesis pathway in Synechocystis sp. PCC 6803

摘要:

蓝藻是探索利用太阳能生产化学品的重要微生物，但产量低限制了蓝藻化学品的工业应用。提高宿主还原力水平是提高微生物合成化学品产量的重要手段。为提高集胞藻细胞内NADPH含量，利用同源重组方法，获得敲除聚羟基丁酸酯PHB合酶编码基因phaC和phaE的集胞藻Synechocystis sp. PCC 6803突变体S.DphaC&E。PCR结果证明突变体S.DphaC&E基因组中phaC和phaE已完全被氯霉素抗性基因取代。生长曲线结果显示S.DphaC&E的生长与野生型无明显差异，说明敲除phaC和phaE对

Abstract:

Cyanobacteria have become attractive hosts for renewable chemicals production. The low productivity, however, prevents it from industrial application. Reductant NAD(P)H availability is a chief hurdle for the production of reductive metabolites in microbes. To increase NADPH content in Synechocystis sp. PCC 6803, PHB synthase encoding gene phaC and phaE in Synechocystis was inactivated by replacing phaC&E genes with chloromycetin resistance cassette via homologous recombination. PCR analysis showed that mutant S.DphaC&E with complete genome segregation was generated. The comparison between growth curves of S.wt and S.DphaC&E indicated the knockout of phaC & phaE genes did not affect obviously the cell growth. Gas chromatography analysis showed that the accumulation of PHB in wild type was about 2.3% of the dry cell weight, whereas no PHB was detected in the mutant S.DphaC&E. The data indicated that inactivation of PHB synthase gene phaC and phaE interrupted the synthesis of PHB. Further comparative study of wild type and mutant demonstrated that NADPH content in S.DphaC&E was obviously increased. On the third day, the NADPH content in S.DphaC&E was up to 1.85 fold higher than that in wild type. These results indicated that deleting PHB synthase gene phaC and phaE not only can block the synthesis of PHB, but also can save NADPH to contribute reductant sink in cyanobacteria. Hence, the engineered cyanobacterial strain S.DphaC&E, in which carbon flux was redirected and NADPH was increased, will be a potential host strain for chemicals production in cyanobacteria.