Increased dipicolinic acid production with an enhanced spoVF operon in Bacillus subtilis and medium optimization

Abstract

Dipicolinic acid (DPA) is a multi-functional agent for cosmetics, antimicrobial products, detergents, and functional polymers. The aim of this study was to design a new method for producing DPA from renewable material. The Bacillus subtilis spoVF operon encodes enzymes for DPA synthase and the part of lysine biosynthetic pathway. However, DPA is only synthesized in the sporulation phase, so the productivity of DPA is low level. Here, we report that DPA synthase was expressed in vegetative cells, and DPA was produced in the culture medium by replacement of the spoVFA promoter with other highly expressed promoter in B. subtilis vegetative cells, such as spoVG promoter. DPA levels were increased in the culture medium of genetically modified strains. DPA productivity was significantly improved up to 29.14 g/L in 72 h culture by improving the medium composition using a two-step optimization technique with the Taguchi methodology.

Antifungal Mechanism of Dipicolinic Acid and Its Efficacy for the Biocontrol of Pear Valsa Canker

“…Toya et al (2015) reported that the replacement of the spoVFA promoter and simultaneous deletion of acetoin synthesis genes (alsSD) led to 30 mM DPA after 40 h of fermentation in synthetic medium. In contrast with our results, Takahashi et al (2015) indicated that DPA was not detected in the culture medium when using wild-type B. subtilis 168. Although the detection limit of the analysis method was not described, the lowest DPA concentration reported in that article was 1.26 mM when using recombinant strains.…”

“…Although the detected concentration of DPA in wild-type B. subtilis 168 secretions (0.24 mM) was lower than the minimal inhibitory concentration (5 mM), previous reports have demonstrated that recombinant B. subtilis 168 strains can produce high amounts of DPA (>5 mM), indicating that B. subtilis 168 is an interesting tool for DPA production. In this sense, the replacement of the spoVFA promoter with another highly expressed promoter, spoVG, in B. subtilis vegetative cells, together with improving the medium composition, increased DPA production up to 170 mM (Takahashi et al, 2015). Toya et al (2015) reported that the replacement of the spoVFA promoter and simultaneous deletion of acetoin synthesis genes (alsSD) led to 30 mM DPA after 40 h of fermentation in synthetic medium.…”

Enhanced dipicolinic acid production during the stationary phase in Bacillus subtilis by blocking acetoin synthesis

“…[11][12][13] The resultant strain OA105-DPA was cultured in M(MOPS) medium as described above, but it did not grow in the presence of 100 g L −1 glucose (data not shown), although it did grow when the cells were initially cultured in M(MOPS) with 50 g L −1 glucose. To achieve a final input glucose concentration of 100 g L −1 , additional glucose at 50 g L −1 was supplied after entry into stationary phase (12 h).…”

“…10) Moreover, DPA is expected to be useful for a wide variety of applications, including for use in antimicrobial agents, antioxidants, chelating reagents, and polymer precursors. 11) In B. subtilis, DPA is synthesized via the lysine biosynthetic pathway from pyruvate in glycolysis and from oxaloacetate in the TCA cycle as precursors (Fig. 1).…”

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

“…DPA is found in all endospore formers, constitutes up to 15% dry weight of mature endospores and has been linked to increased heat resistance (Powell, 1953;Church and Halvorson, 1959;Setlow et al, 2006). Synthesis of DPA occurs in the mother cell under the control of σ K in late stages of sporulation, and is mediated by the enzymes DpaA and DpaB of the spoVF operon, through transformation of L-2,3-dihydrodipicolinate, an intermediate of the lysine biosynthesis pathway (Daniel and Errington, 1993;Takahashi et al, 2015). Interestingly, an alternative pathway for DPA synthesis, possibly of ancient origin, has been identified in Clostridia and is performed by the protein EtfA using the product of DpaA activity (Orsburn et al, 2010;Donnelly et al, 2016).…”