pAo: A Consensus Genome-Scale Metabolic Model for Aspergillus oryzae Capturing Intraspecies Diversity

Aspergillus oryzae (koji mold) is a key microorganism in traditional food fermentations including soy sauce, sake, and miso and is important in novel culinary applications and modern biotechnology, such as sustainable meat alternatives and enzyme production. Despite its industrial importance, until recently, the most recent genome-scale metabolic model (GEM) for A. oryzae dated back to 2008 and was limited to a single strain (RIB40). Here, we present pAo, a pan-GEM for A. oryzae, integrating genomic data from 187 strains to capture species-wide metabolic diversity. Our model comprises 2,025 reactions, representing a 52% increase in metabolic coverage over the RIB40-based model and includes previously overlooked pathways, such as cytochrome P450-mediated xenobiotic metabolism and extended amino acid metabolism. Using this pan-GEM, we derived strain-specific GEMs and validated 8 of them through high-throughput phenotypic screening on 285 substrates. Growth experiments on 4 industrially relevant carbon sources revealed substantial interstrain metabolic diversity, although flux balance analysis indicated that this variability originates at the regulatory rather than network-structural level. This resource provides a foundation for informed strain selection for biotechnological applications and future metabolic engineering in A. oryzae.