Genetic and Process Engineering for the Simultaneous Saccharification and Biocatalytic Conversion of Lignocellulose for Itaconic Acid Production by Myceliophthora thermophila

Itaconic acid (IA), one of the top twelve renewable platform chemicals, is a key precursor for polymer synthesis. Here, we engineered Myceliophthora thermophila for efficient consolidated biocatalytic IA production from lignocellulose by introducing the heterologous IA pathway (cis-aconitic acid decarboxylase (CAD), mitochondrial tricarboxylic transporter (MTT), major facilitator superfamily transporter (MFS) from Aspergillus terreus), and boosting CAD expression and precursor supply. A critical issue was temperature mismatch: optimal fungal growth vs. CAD activity. Transcriptomics analysis identified reduced expression of glycolytic rate-limiting enzymes (fructose-bisphosphate aldolase, FBA; phosphofructokinase, PFK) at 40 °C. Overexpressing these enzymes in strain IA32 generated strain IA41 (with 3.1-fold and 2.8-fold higher expression of pfk and fba, respectively), which accelerated glucose consumption by 53.2% and increased IA yield by 55.1% A two-stage temperature-shift strategy (45 °C for growth/saccharification, 40 °C for CAD activity) was developed. The engineered strain achieved 3.93 g/L IA in shake flasks and 10.51 g/L in corncob fed-batch fermentation—the highest reported titer for consolidated lignocellulose-to-IA processes. This establishes M. thermophila as a robust platform for cost-effective IA production from lignocellulose.