Integrated RSM and Genomic Analysis for Optimized Sporulation in Heyndrickxia coagulans

Industrial spore production of the probiotic Heyndrickxia coagulans is hindered by its generally low and highly variable sporulation efficiency across strains. To address this, we selected the representative model strain ATCC 7050 and applied an integrated strategy combining statistical medium optimization with genomic analysis. Key factors (glucose, yeast extract, CaCl2) were screened and optimized using Plackett–Burman and Box–Behnken designs, yielding an optimal formulation that achieved 1.84 × 108 spores/mL in a bioreactor, consistent with the model prediction. Further genomic analysis revealed 112 sporulation-associated genes and identified key homologous genes related to spore resistance and germination. Among them, the successful identification of spoVA, which is implicated in calcium-dipicolinate transport in bacilli, allowed us to hypothesize why calcium ions play a critical role. This work not only enhances the spore yield of a model strain but also provides a framework to tackle the widespread sporulation variability in H. coagulans for industrial applications.