pH-driven metabolic reprogramming in Bacillus velezensis 83 regulates metabolite synthesis and sporulation: A transcriptional approach for bioprocess development

Abstract

Bacillus velezensis 83 (Bv83) spores are the active ingredient in Fungifree AB, an agricultural biocontrol agent whose industrial production is negatively affected by poly-γ-glutamic acid (γ-PGA) biopolymer synthesis at pH 6.8. At pH 5, γ-PGA production and sporulation are suppressed, but the latter is restored through pH-shift to 6.8 after glucose depletion. This work presents a comprehensive Bv83 spore bioprocess development through physiological and transcriptional analyses. At pH 5, Bv83 present a strategic response to pH stress by reducing its growth rate and redirecting energy towards survival rather than differentiation (downregulating lipopeptide and γ-PGA production genes) and maintaining SigB-mediated stress response rather than sporulation. Transcriptome analysis revealed downregulation of comX and phrC genes at pH 5, indicating sporulation limitation from insufficient signaling molecule production. Indeed, our results confirmed the essential role of the competence and sporulation factor (CSF) in differentiation of elongated cells into forespores. Furthermore, spent medium addition from high-cell-density cultures induced complete sporulation at pH 5, suggesting critical metabolite concentrations (besides CSF) are required. A pH-shift strategy during fed-batch cultivation suppressed γ-PGA synthesis, leading to enhanced mixing and oxygen transfer. Moreover, this strategy led to a 2.6-fold increase in spore productivity (7.86 ×10¹⁰ spores L⁻¹ h⁻¹) compared to a batch at pH 6.8, reducing operational costs. This research has identified pH-regulated metabolic networks, establishing a foundation for designing efficient, cost-effective industrial-scale B. velezensis fermentation strategies that comply with regulatory requirements for biocontrol applications.