Metabolomic analysis of the effect of glutamate on the fengycin-overproducing Bacillus subtilis ATCC 21332 with an enhanced fatty acid synthesis pathway (2023)

Fengycin is a cyclolipopeptide antibiotic synthesized by a non-ribosomal peptide synthase (NRPS) [1], [2]. It consists of a lipophilic β-hydroxy fatty acid chain and a hydrophilic cyclic peptide of 10 amino acid residues, resulting in a unique amphiphilic cyclolipopeptide structure that gives fengycin a variety of biological activities and functions [3], [4]. Due to its extensive antifungal activity and its harmlessness to the environment and health, fengycin has wide applications as a biological control agent in agriculture [5], [6], [7]. Fengycin has antitumor, antimicrobial, anticoagulant, and other effects, which have led to increased interest in potential clinical applications [8], [9], [10]. Fengycin works by damaging the cell membrane, which is a complex cell organelle that is difficult to change, resulting in a reduced likelihood of the formation of resistant strains[11],[12],[13]. Above all, fengycin is characterized by a wide margin of safety and must be developed as part of the next generation of antibacterial drugs [14]. In addition, fengycin has strong surface activity, indicating potential applications in the food, cosmetic and pharmaceutical industries [15], [16]. However, the slow growth and difficult genetic modification of the production strain led to low yields and high production costs of fengycin, which precluded its large-scale utilization[17], [18].

There are three viable methods to overcome strain limitations for the synthesis of fengycin. First, wild strains with high titers of fengycin can be screened and domesticated. Naturally occurring strains have excellent genetic stability that meets the production requirements of the fermentation industry [19], [20]. However, the screening process is complicated, labor intensive and may not guarantee success. In addition, adaptive laboratory evolution was also used to obtain mutant strains with increased fengycin titers [ 21 ]. However, the mutagenesis process is untargeted and requires lengthy and time-consuming screening. Third, genetically engineered strains with a high titer of fengycin can be constructed by metabolic engineering and synthetic biology methods based on the existing fengycin production strains [22], [23]. The process is manageable and convenient, but the genetically engineered strains are often unstable, which is a major obstacle to industrial fermentation. In addition to these strain modification approaches, the production cost of fengycin can be reduced by using an inexpensive fermentation substrate such as lignocellulose, crude glycerol or kitchen waste [24], [25], [26].

The three key steps in the fengycin pathway are the synthesis of side chain fatty acids, activation and loading of the fatty acids followed by elongation and cyclization of the peptide chain [27]. As direct precursors for fengycin synthesis, the availability of fatty acids and amino acids directly determines the fengycin titer, and adding them externally to the culture medium or increasing the supply using genetic engineering can significantly improve product synthesis [28], [29], [30]. Compared to the amino acid synthesis pathway, the fatty acid synthesis pathway is more concentrated in fengycin metabolic networks, making it more suitable for systematic and combined transformation. Therefore, most of the studies have focused on increasing the expression of the fatty acid synthesis pathway in strains to improve fengycin titers [31]. Fatty acids not only affect the synthesis of fengycin, but also have a significant impact on its biological activity [32], [33]. The fatty acid supply can be increased either by exogenous fatty acid supply or by up-regulation of the intracellular fatty acid metabolism [34], [35]. Dinge et al. increased fengycin synthesis ofB. amyloliquefaciensPc3 by adding exogenous fatty acids, which upregulated the transcription levelbadGene related to the synthesis of fengycin [29]. Tan et al. overexpressed genes associated with the malonyl-CoA pathway, a rate-limiting pathway for fatty acid synthesis iBacillus subtilis168, which increased the production of fengycin [36]. Jin et al. enhanced fatty acyl-CoA release and promoted fengycin synthesis by modifying the fatty acid pathway iBacillus subtilis168[17]. Similarly, Wu et al. 20.8-fold increase in surfactin production by enhancing the branched-chain fatty acid synthesis pathwayB. subtilis168 to increase the intake of fatty acids [37]. Hu et al. engineered fatty acid precursor delivery route fromB. subtilis168, leading to increased surfactin production and an increased proportion of C14 isomers [38]. Lu et al. performed proteomic analysis ofB. amyloliquefaciensX030 at different growth stages and it was concluded that the strain is highly efficient in the synthesis of bacillomycin, which was highly dependent on the increase in fatty acid synthesis during the exponential growth phase [39].

The compatibility between the functional modules of the fatty acid synthesis pathway and chassis cells is also worth investigating [40]. The titers of lipopeptides produced by NRPS are often limited by metabolic imbalances [41]. Thus, both overexpression and underexpression of fatty acid pathway genes can have negative effects on lipopeptide synthesis. Too low expression levels result in insufficient precursors for fengycin synthesis, limiting the final titer. Conversely, excessive expression levels divert resources to unnecessary intermediate metabolites and enzymes, placing excessive metabolic pressure on chassis cells and leading to slow growth, reducing fengycin yield and productivity [42], [43].

In this study, the fatty acid synthesis pathway forB. subtilisATCC 21332 was methodologically improved, including by overexpressing important genes from the fatty acid synthesis pathwayB. subtilisATCC 21332 andB. amyloliquefaciensFZB42, which greatly increased the titer of fengycin. In addition, we also expressed discomfortE coliMG1655 encodes enzymes that activate and release fatty acids. We investigated the different effects of homologous and heterologous expression of fatty acid synthesis genes on the synthesis of fengycin inB. subtilisATCC 21332. We constructed the recombinant strain BSA034 that produced 442.51 mg/l fengycin with an improved fatty acid synthesis pathway. The expression level of fengycin biosynthase in BSA034 was analyzed by RT-qPCR, and the possible reason for the increase in fengycin titers was revealed. We also identified the glutamate as the limiting precursor amino acid and it was added exogenously to the fermentation medium to improve the fengycin titer to 657.55 mg/L. The metabolic regulatory target of exogenous glutamate in BSA034 was analyzed by metabolomics, and the mechanism by which exogenous glutamate promotes the synthesis of fengycin was revealed.