Metabolic Engineering of the Thermophilic Fungus Myceliophthora thermophila for l-glutamate Production from Lignocellulosic Biomass.

l-Glutamic acid is conventionally produced by bacteria from starch-derived sugar, while its production in eukaryotic hosts remains rarely reported. The thermophilic ascomycete fungus Myceliophthora thermophila has been developed as a platform for producing value-added chemicals from lignocellulosic biomass. In this study, we explored the feasibility of engineering M. thermophila for l-glutamate production from biomass-derived carbon sources. Genome-scale metabolic modeling indicated that M. thermophila has the stoichiometric capacity to synthesize l-glutamate from glucose and ammonia. Experimental fermentation confirmed extracellular l-glutamate accumulation, and medium optimization revealed that nitrogen source selection and extracellular pH homeostasis strongly affected l-glutamate accumulation. Overexpression of the native NADPH-dependent glutamate dehydrogenase gene gdh1 markedly increased glutamate production. Additional engineering strategies targeting membrane-associated metabolism, glutamate transport, mitochondrial precursor transport, and competing α-ketoglutarate-consuming pathways provided insights into the regulation of glutamate metabolism in filamentous fungi. The engineered strain produced 3.41 g/L l-glutamate from glucose. Moreover, 1.50 g/L and 1.49 g/L l-glutamate were obtained directly from cellulose and raw corncob, respectively. This study establishes M. thermophila as a proof-of-concept thermophilic fungal platform for l-glutamate production from lignocellulosic carbon sources and provides a framework for engineering amino acid biosynthesis in cellulolytic fungi.