Pathogenic fungi are a predominant cause of postharvest losses, accounting for an estimated 30% of the total harvested produce (Wang et al., 2022; Zhang et al., 2021). A key contributor is Botrytis cinerea, which causes gray mold in diverse horticultural crops and results in massive economic losses (Petrasch et al., 2019). Beyond such quality deterioration, many postharvest fungi pose a more critical health risk due to their production of mycotoxins, which are potent toxins hazardous to humans (Sanzani et al., 2016). The control of postharvest fungal pathogens primarily depends on synthetic fungicides (Hosseini et al., 2023). However, their use poses significant threats to ecosystem stability and human health, causing issues such as environmental pollution and toxic residue accumulation, while simultaneously accelerating the evolution of resistance (Alikord et al., 2022; Nicolopoulou-Stamati et al., 2016). These challenges have intensified the search for sustainable and eco-friendly alternatives, among which bioactive metabolites from beneficial microorganisms stand out as a particularly promising solution (Reveglia et al., 2024). Notably, Bacillus spp. have been extensively studied due to their rapid growth, generally recognized as safe (GRAS) status for specific strains and applications, and remarkable capacity to synthesize a vast arsenal of antimicrobial compounds (Abdel-Nasser et al., 2024). The spectrum of bioactive secondary metabolites produced by Bacillus includes lipopeptides, proteolytic antibiotics, bacteriocins, and polyketides, which exhibit diverse activities against phytopathogens (Abriouel et al., 2011; Xiao et al., 2022).

Lipopeptides, such as surfactin, fengycin, and iturin, are among the most potent antimicrobial compounds synthesized by Bacillus strains (Mnif and Ghribi, 2015). These amphiphilic molecules exhibit broad-spectrum activity through membrane disruption and biofilm inhibition (Zhao et al., 2017), enabling their widespread use as biocontrol agents against plant pathogens (Sani et al., 2024). As a particularly effective producer, Bacillus velezensis has been shown to continuously synthesize complex lipopeptide mixtures (Fazle Rabbee and Baek, 2020). However, the large-scale commercial application of microbial antimicrobials, such as lipopeptides (e.g., surfactin and fengycin), is primarily constrained by high fermentation costs and low production yields (Jin et al., 2025; Sun et al., 2025). The production efficiency of these metabolites is highly dependent on culture conditions (Pan et al., 2019; Romero-Rodríguez et al., 2018). In particular, the initial pH, nitrogen source, and carbon source of the fermentation medium have a significant impact on the synthesis of the target antimicrobial substances (Wang et al., 2023). Therefore, optimizing fermentation conditions is essential to enhance metabolite yield and reduce production costs (Wang et al., 2018). One of the key strategies to overcome this bottleneck lies in medium optimization using low-cost, agro-industrial by-products, which not only reduces production expenses but also aligns with circular economy principles (Narendra Kumar et al., 2017; Sun et al., 2025). Nevertheless, many optimization studies remain phenomenological, focusing on process parameters without elucidating the underlying metabolic mechanisms. This knowledge gap impedes the rational design of high-efficiency bioprocesses.

To bridge this gap and advance a more rational design, this study introduces a novel fermentation strategy employing a first-time combination of soluble starch and soybean meal as synergistic, low-cost substrates. We hypothesize that soluble starch, as a slow-release carbon source, can avoid carbon catabolite repression and ensure a sustained carbon supply. At the same time, soybean meal provides a complex mixture of nitrogen and potential precursors. This specific synergistic strategy has not been previously investigated for lipopeptide biosynthesis in Bacillus strains. The integration of metabolomics provides a powerful tool to bridge this gap, enabling the systematic profiling of metabolic changes and offering insights into the regulatory networks that govern product synthesis (Hu et al., 2024a; Yang et al., 2023). Building on our previous findings of the antifungal efficacy of lipopeptides (mainly surfactin and fengycin) produced by B. velezensis YTQ3 (Yang et al., 2026), this study aimed to: (1) systematically optimize fermentation conditions and medium composition through single-factor experiments and response surface methodology (RSM); (2) quantitatively profile the enhanced lipopeptide production using UHPLC-ESI-MS; and (3) most importantly, employ metabolomics and RT-qPCR analysis to unravel the underlying metabolic regulation and transcriptional responses responsible for the yield improvement. The findings not only provide a scalable high-density fermentation process but also establish a mechanistic framework for the targeted regulation of lipopeptide biosynthesis in B. velezensis, laying a solid foundation for developing efficient postharvest biocontrol agents.