Engineering thiamine pyrophosphate metabolism improves consolidated bioprocessing for cellulosic ethanol with inorganic nitrogen in Myceliophthora thermophila

Ammonium sulfate supported higher ethanol production than nitrate, but still resulted in a lower titer than yeast extract. Under ammonium sulfate, transcriptomic and metabolomic analyses revealed altered amino acid metabolism together with reduced carbohydrate metabolic and cellulose catabolic functions, while supplementation with selected amino acids provided only limited improvement.

Supplementation with 5 mg/L thiamine pyrophosphate (TPP) increased ethanol production under ammonium sulfate from 14.3 g/L to 23 g/L, reaching a level comparable to that obtained with yeast extract. Moreover, α-ketoglutarate was identified as a responsive metabolic node associated with the altered fermentation state. Stepwise strengthening of the endogenous TPP biosynthetic pathway progressively improved ammonium-based ethanol fermentation, and additional overexpression of pyruvate decarboxylase further enhanced ethanol production.