Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions
Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions
复制标题
Cupriavidus necator H16 的代谢工程可改善限氧条件下的化能自养生长和 PHB 生产
DOI：
10.1016/j.ymben.2020.04.009
复制
发表时间：
2020
影响因子：
8.4
通讯作者：
Han Yejun
中科院分区：
文献类型：
--
作者：
Tang Ruohao;Weng Caihong;Peng Xiaowei;Han Yejun
氧限制条件促进了钩虫H16聚羟基丁酸酯（PHB）的积累，但限制了其生长。在使用二氧化碳、氢气和氧气作为底物的自养培养中，混合物中低于6.9%（v/v）的氧气浓度被认为是安全条件。在C环境中，在氧限制条件下，细胞有望在快速生长的同时大量积累PHB。necatorH16.本研究基于转录组学分析构建了一株在限氧条件下既能快速生长又能大量积累聚羟基丁酸的代谢工程菌。在比较转录组学分析中，对C. necatorH 16在自养培养中在氧限制条件下（3%，v/v）下调。此外，还对PHB生物合成途径中的关键中间产物（丙酮酸和乙酰辅酶A）代谢相关基因进行了分析。其中大部分下调，除了分别编码L-乳酸脱氢酶、异柠檬酸裂解酶和乙酸激酶的基因ldh、iclA和ackA 2在氧限制条件（3%，v/v）下上调。玻璃体血红蛋白（VHb）具有促进有氧代谢和能量产生的能力。促进细菌生长，提高产能能力。necatorH 16中，将VHb基因导入C. necatorH 16与优化的启动子PphaC 1-j5。此外，VHb定位于周质空间的细菌的膜结合氢化酶（MBH）的信号肽的牵引。通过优化不同基因的敲除，发现敲除fldh 2基因可以提高PHB的产量，减少副产物。最后，将vg和ldh基因敲除，在C. necatorH16.在限氧（3%，v/v）条件下，与对照（Reh（p2））相比，菌株P101（p2 M-pj-v）的细胞干重（DCW）、PHB含量和PHB产量分别增加了31.0%、30.9%和71.5%。从转录组学和代谢工程的角度，该工作为实现细胞在C中的快速生长和大量积累PHB提供了新的思路。necator在氧限制和自养条件下。
The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) inC.necator H16, while the growth of which is restricted. Under autotrophic culture using carbon dioxide, hydrogen, and oxygen as substrates, the oxygen concentration below 6.9% (v/v) in the mixture is considered as a safe condition. It also expected to achieve cell rapid growth and large accumulation of PHB simultaneously under the oxygen-limiting condition inC. necatorH16. In this study, a metabolically engineered strain capable of both rapid growth and large accumulation of PHB under oxygen-limiting conditions was constructed based on the transcriptomic analysis. In the comparative transcriptomic analysis, the genes related to energy-generating ofC. necatorH16 at autotrophic culture were downregulated under oxygen-limiting conditions (3%, v/v). Besides, the genes related to the key intermediates (pyruvate and acetyl-CoA) metabolism in PHB biosynthetic pathway were analyzed. Most of which were downregulated, except the genesldh,iclA, andackA2respectively encoding L-lactate dehydrogenase, isocitrate lyase, and acetate kinase were upregulated under oxygen-limiting conditions (3%, v/v). The Vitreoscilla hemoglobin (VHb) has the ability to promote aerobic metabolism and energy generation. To promote the bacterium growth and improve the energy generation inC. necatorH16 under oxygen-limiting conditions, the VHb gene was introduced intoC. necatorH16 with the optimized promoterPphaC1-j5. Moreover, VHb was localized to the periplasmic space of the bacterium by the traction of membrane-bound hydrogenase (MBH) signal peptide. By optimizing the knockout of different genes, it was found that knockout ofldhcan improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression ofvgbandldhknockout inC. necatorH16. Compared with the control (Reh (p2)) under oxygen-limiting conditions (3%, v/v), the dry cell weight (DCW), PHB content, and PHB production of Reh01 (p2M-pj-v) increased by 31.0%, 30.9%, and 71.5%, respectively. From the perspectives of transcriptome and metabolic engineering, the work provides new ideas to achieve rapid cell growth and large PHB accumulation inC. necatorunder oxygen-limiting and autotrophic conditions.