Science for Life Laboratory, School of Engineering Science in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, P-Box 1031, 171 21, Solna, Sweden.
Science for Life Laboratory, School of Engineering Science in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, P-Box 1031, 171 21, Solna, Sweden.
Metab Eng. 2022 Sep;73:256-269. doi: 10.1016/j.ymben.2022.08.005. Epub 2022 Aug 18.
The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria.
化能自养菌 Cupriavidus necator H16 是一种天然的生物塑料聚合物 PHB 生产菌，其代谢具有多样性，可以利用不同的底物，包括甲酸盐作为唯一的碳源和能源。根据底物的进入点，这种多功能性需要调整热力学景观，以维持生物过程足够高的驱动力。在这里，我们采用了 C. necator H16 的核心代谢模型来分析不同代谢工程策略下甲酸盐的热力学驱动力和 PHB 产量。为此，我们对网络的基本通量模式 (EFMs) 进行了枚举，并通过最大-最小驱动力 (MDF) 分析和驱动力的随机抽样来评估它们的 PHB 产量和热力学。预测异源 ATP:柠檬酸裂解酶反应将增加产生乙酰辅酶 A 的驱动力。预测异源磷酸酮酶反应将增加最大 PHB 产量和驱动力。然后，在选定的条件下，通过实验验证了这些酶可以将 PHB 滴度提高 60%至 300%。EFM 分析还表明，甲酸盐生产 PHB 可能受到柠檬酸裂解酶和乌头酸酶的低驱动力、辅因子平衡以及与低和高 PHB 产量相关的其他反应的限制。工程菌株的蛋白质组学分析证实了乌头酸酶和辅因子平衡的丰度增加。本研究的结果有助于理解代谢适应。此外，所概述的方法将有助于设计其他非模式细菌的代谢工程策略。
