Engineering Penicillium oxalicum to boost bioethanol production from uncooked starch

Bioethanol production via biorefining uncooked cassava starch using raw-starch-degrading enzymes (RSDEs) is feasible, with filamentous fungi serving as producers of RSDEs. However, the low yield of fungal RSDEs has impeded industrial-scale bioethanol production from raw starch. Herein, we developed a Penicillium oxalicum mutant (GXUR003) through a combination of genetic engineering and random mutagenesis. This mutant achieved the highest reported RSDE production at 510.9 U/mL when using raw cassava flour as the substrate, induced by wheat bran combined with Avicel, representing a 174.5% increase compared to the starting strain. Comparative transcriptome and secretome analyses revealed that in GXUR003, increased transcription of the key regulatory gene amyR boosted expression of the primary raw-starch-degrading glucoamylase gene PoxGA15A and α-amylase gene amy13A, resulting in higher amylase secretion. Moreover, the crude RSDEs from GXUR003 efficiently hydrolyzed raw starch from various sources at a concentration of 150 g/L into glucose. An optimized preparation of crude RSDEs supplemented with commercial α-amylase and glucoamylase achieved a fermentation efficiency of 93.1% during simultaneous saccharification and fermentation using raw cassava flour at a concentration of 28% (w/v). These findings indicate that RSDEs derived from GXUR003 have potential for converting raw starch into bioethanol.