Redox engineering of thermophilic fungus Myceliophthora thermophila enhances production of L-malic acid by consolidated bioprocessing

Consolidated bioprocessing (CBP) is a promising strategy for low-cost malic acid production from plant biomass. In this study, we report a comprehensive redox engineering strategy to redirect reducing equivalents towards the reductive tricarboxylic acid (rTCA) pathway for enhanced malic acid production directly from cellulose in the thermophilic fungus Myceliophthora thermophila JG207. Firstly, deletion of key NADH-consuming pathways, including glycerol-3-phosphate dehydrogenase (gpdh) and mitochondrial external NADH dehydrogenases (nde1 and nde2), resulted in a significant increase in the NADH/NAD⁺ ratio and a corresponding 17% increase in malic acid titre. Subsequent removal of alternative oxidase (AOX) further heightened the NADH pool, although it induced oxidative stress and slowed growth. To alleviate redox imbalance, we overexpressed the native transhydrogenase SthA, which effectively converted NADPH to NADH and improved malate production by an additional 6%. Fine-tuning NAD⁺ kinase (UTR1) expression further optimised cofactor homoeostasis, bringing about the highest malate titre of 89.2 g/L, a 38.9% improvement over the starting strain JG207. Finally, overexpression of lytic polysaccharide monooxygenase gene (lpmo) and cellobiose dehydrogenase gene (cdh) enhanced cellulose degradation, shortening the fermentation cycle by two days on Avicel and crushed corncob. This work not only advances the fundamental understanding of redox metabolism in filamentous fungi but also provides a proof-of-concept for sustainable organic acid production from inexpensive lignocellulosic feedstocks.