13 C-MFA helps to identify metabolic bottlenecks for improving malic acid production in Myceliophthora thermophila.

Background: 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. ¹³C-MFA (¹³C metabolic flux analysis) offers a means to analyze cellular metabolic mechanisms and pinpoint critical nodes for improving product synthesis. Here, we employed ¹³C-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.

Results: 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 ¹³C-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 ¹³C-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.

Conclusions: 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.