Proteomic and lignin analyses reveal metabolic bottlenecks in corn ethanol production

Corn bioethanol production generates large volumes of animal feed coproducts with nutritional value largely determined by their fiber and protein content. Here, we combined microscopic and biochemical lignin analysis with untargeted proteomics to characterize corn flour (CF) feedstock and the downstream dried distiller’s grains with solubles (DDGS) and corn fermented protein (CFP) fractions from an industrial bioethanol plant. DDGS contained up to 8% lignin on a dry-weight basis, representing an opportunity for its recovery and utilization. Proteomic profiling identified ~4200 maize ( Zea mays ) proteins, and ~300 yeast ( Saccharomyces cerevisiae ) and ~90 bacterial ( Escherichia coli ) proteins. Yeast and bacteria each contributed ~3% of the total protein present in DDGS and CFP, whereas 90% of the maize proteins detected in CF were retained in DDGS and CFP, suggesting mild protein breakdown during processing. We mapped the relative abundance of enzymes involved in lignin and starch biosynthesis in the maize endosperm, and in yeast glycolysis/fermentation, revealing limiting enzymatic steps in each pathway. Seed storage proteins, particularly globulins and oleosins, were enriched in the high-protein CFP fraction, consistent with its higher nutritional value. This study provides the first proteomic analysis of corn ethanol coproducts and identifies targets to enhance their utilization via maize genetics, microbial engineering, or lignin valorization strategies.