National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing, 100049, China.
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China.
Metab Eng. 2020 Sep;61:11-23. doi: 10.1016/j.ymben.2020.04.009. Epub 2020 Apr 26.
The oxygen-limiting condition promotes the accumulation of ployhydroxybutyrate (PHB) in C. 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 in C. necator H16. 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 of C. necator H16 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 genes ldh, iclA, and ackA2 respectively 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 in C. necator H16 under oxygen-limiting conditions, the VHb gene was introduced into C. necator H16 with the optimized promoter P. 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 of ldh can improve PHB production and reduce the by-products. Finally, a recombinant strain Reh01 (p2M-pj-v) was constructed by heterologous expression of vgb and ldh knockout in C. necator H16. 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 in C. necator under oxygen-limiting and autotrophic conditions.
在限制供氧的条件下，C. necator H16 会积累大量的聚羟基丁酸酯（PHB），而其生长则受到限制。在以二氧化碳、氢气和氧气为底物的自养培养中，混合物中低于 6.9%（v/v）的氧浓度被认为是安全的条件。人们还期望在 C. necator H16 的限氧条件下同时实现细胞的快速生长和大量 PHB 的积累。在这项研究中，基于转录组分析，构建了一株能够在限氧条件下快速生长和大量积累 PHB 的代谢工程菌株。在自养培养条件下，C. necator H16 的转录组比较分析显示，与能量生成相关的基因在限氧条件下（3%，v/v）下调。此外，还分析了 PHB 生物合成途径中关键中间产物（丙酮酸和乙酰辅酶 A）代谢相关的基因。大多数基因下调，只有编码 L-乳酸脱氢酶、异柠檬酸裂解酶和乙酰激酶的 ldh、iclA 和 ackA2 基因在限氧条件下（3%，v/v）上调。Vitreoscilla 血红蛋白（VHb）具有促进有氧代谢和能量产生的能力。为了在 C. necator H16 限氧条件下促进细菌生长和提高能量生成，将 VHb 基因通过优化的启动子 P 引入 C. necator H16。此外，通过膜结合氢化酶（MBH）信号肽的牵引，将 VHb 定位到细菌的周质空间。通过优化不同基因的敲除，发现敲除 ldh 可以提高 PHB 的产量并减少副产物。最后，通过在 C. necator H16 中异源表达 vgb 和敲除 ldh，构建了重组菌株 Reh01（p2M-pj-v）。与限氧条件（3%，v/v）下的对照（Reh（p2））相比，Reh01（p2M-pj-v）的干重（DCW）、PHB 含量和 PHB 产量分别提高了 31.0%、30.9%和 71.5%。从转录组和代谢工程的角度来看，这项工作为在限氧和自养条件下实现 C. necator 细胞的快速生长和大量 PHB 积累提供了新的思路。
