State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Science, Tianjin, 300308, China.
Microb Cell Fact. 2024 Nov 2;23(1):295. doi: 10.1186/s12934-024-02570-3.
Myceliophthora thermophila has been engineered as a significant cell factory for malic acid production, yet strategies to further enhance production remain unclear and lack rational guidance. C-MFA (C metabolic flux analysis) offers a means to analyze cellular metabolic mechanisms and pinpoint critical nodes for improving product synthesis. Here, we employed C-MFA to investigate the metabolic flux distribution of a high-malic acid-producing strain of M. thermophila and attempted to decipher the crucial bottlenecks in the metabolic pathways.
Compared with the wild-type strain, the high-Malic acid-producing strain M. thermophila JG207 exhibited greater glucose uptake and carbon dioxide evolution rates but lower oxygen uptake rates and biomass yields. Consistent with these phenotypes, the C-MFA results showed that JG207 displayed elevated flux through the EMP pathway and downstream TCA cycle, along with reduced oxidative phosphorylation flux, thereby providing more precursors and NADH for malic acid synthesis. Furthermore, based on the C-MFA results, we conducted oxygen-limited culture and nicotinamide nucleotide transhydrogenase (NNT) gene knockout experiments to increase the cytoplasmic NADH level, both of which were shown to be beneficial for malic acid accumulation.
This work elucidates and validates the key node for achieving high malic acid production in M. thermophila. We propose effective fermentation strategies and genetic modifications for enhancing malic acid production. These findings offer valuable guidance for the rational design of future cell factories aimed at improving malic acid yields.
嗜热毁丝霉已被工程化为生产苹果酸的重要细胞工厂，但进一步提高产量的策略仍不清楚，缺乏合理的指导。C-MFA（C 代谢通量分析）提供了一种分析细胞代谢机制和确定关键节点以提高产物合成的方法。在这里，我们采用 C-MFA 研究了一株高产苹果酸的嗜热毁丝霉菌株的代谢通量分布，并试图揭示代谢途径中的关键瓶颈。
与野生型菌株相比，高产苹果酸的嗜热毁丝霉 JG207 表现出更高的葡萄糖摄取率和二氧化碳释放率，但更低的氧气摄取率和生物量产量。与这些表型一致，C-MFA 结果表明 JG207 通过 EMP 途径和下游 TCA 循环的通量增加，同时氧化磷酸化通量减少，从而为苹果酸合成提供更多的前体和 NADH。此外，根据 C-MFA 结果，我们进行了氧限制培养和烟酰胺核苷酸转氢酶（NNT）基因敲除实验，以增加细胞质 NADH 水平，这两者都有利于苹果酸的积累。
本研究阐明并验证了在嗜热毁丝霉中实现高苹果酸生产的关键节点。我们提出了有效的发酵策略和遗传修饰方法，以提高苹果酸的产量。这些发现为未来旨在提高苹果酸产量的细胞工厂的合理设计提供了有价值的指导。
