Multidimensional strategy enables scalable metabolome diversity in microbial fermentations.

Natural products (NPs) are critical sources of new drug leads, but their biosynthesis is highly sensitive to bioprocess parameters and environmental conditions, making NP discovery especially susceptible to challenges in achieving robust scalability and reproducibility of the metabolome across various cultivation systems. Here, we identified key factors that improved the metabolic footprint overlap of Streptomyces griseochromogenes across three cultivation systems, i.e. baffled shake flasks (BSF), 48 flower plates (48 FP), and a stirred tank bioreactor (STR), by 50 %. Using the classical scale-up criterion of constant oxygen availability, the metabolic footprints were found to differ considerably, with only an 18 % overlap in mass feature (MF) number. Efforts to improve overlap by varying ethanol concentration in culture medium and oxygen availability resulted in an 18 % increase. Factor analysis of 80 cultivations including variations in bioprocess conditions, growth, and MF detection identified morphology and total MF number, mainly influenced by the cultivation systems, as key factors in metabolic footprint reproducibility. Molecular network analysis revealed that 48 FP and STR share the highest number of molecular clusters. Our findings reveal that only multidimensional optimization unlocks robust metabolome scalability and reproducibility across cultivation systems, paving the way for the discovery of novel NPs.