Production of Novel Antibiotics Zeamines through Optimizing Dickeya zeae Fermentation Conditions

Abstract

Dickeya zeae strain EC1 was recently shown to produce a new type of phytotoxins designated as zeamine and zeamine II, which are potent wide-spectrum antibiotics against Gram-positive and Gram-negative bacterial pathogens, suggesting their promising potential as clinical medicines. In this study, the optimized medium composition and culture conditions for biosynthesis of novel antibiotics zeamines have been established by using response surface methodology, largely increasing the yield of zeamines from original about 7.35 µg·mL−1 in minimal medium to about 150 µg·mL−1 in LS5 medium. The study identified the major factors contributing to zeamines production, which include nitrate, sucrose, asparaginate, mineral elements Mg2+ and K+, and optimized amount of phosphate. In addition, the results showed that overexpression of zmsK in D. zeae strain EC1 could further increase zeamines yield to about 180 µg·mL−1 in LS5 medium. The findings from this study could facilitate further characterization and utilization of these two novel antibiotics, and also provide useful clues for understanding the regulatory mechanisms that govern D. zeae virulence.

SlyA regulates phytotoxin production and virulence in Dickeya zeae EC1

“…Our subsequent studies led to the identification of two phytotoxins, i.e. zeamine and zeamine II, with strong antimicrobial activities and a toxic effect against rice seed germination (Cheng et al, 2013;Liao et al, 2014;Wu et al, 2010;Zhou et al, 2011). Zeamine production is encoded by the zms gene cluster, and deletion of zmsA, which encodes a polyketide synthase, abolished zeamine production and the bacterial virulence on rice seeds (Cheng et al, 2013;Zhou et al, 2011Zhou et al, , 2015.…”

Genetic Modulation of c-di-GMP Turnover Affects Multiple Virulence Traits and Bacterial Virulence in Rice Pathogen Dickeya zeae

“…The widths of inhibition zones were measured and converted to zeamines toxin units using the formula described previously[13, 27]. In the optimized zeamines inducing medium LS5[27], deletion of genes encoding c-di-GMP metabolism resulted in minor changes in most cases, except mutants of W909_11190 (Δ11190) and W909_11910 (Δ11910) which showed significantly increased production of zeamines by about 19% and 37% (Fig 5). In contrast, overexpression of the known GGDEF or EAL genes in strain EC1 did not significantly affect the production of zeamines.…”

“…zeae EC1 and its derivatives were grown at 28°C in LB medium as indicated[4]. For testing the antibiotic production, bacteria were grown in the optimized medium LS5 with minor modifications (each liter contains 5.25 g K 2 HPO 4 , 2.25 g KH 2 PO 4 , 10.0 g sucrose, 3.6 g NH 4 NO 3 , 1.0 g KCl and 0.25 g MgSO 4 , pH 7.0)[27]. The cell aggregation inducing medium, SOBG was modified from SOB (each liter contains 20 g of tryptone, 5 g of yeast extract, 2.4 g of MgSO 4 , 0.5 g of NaCl, 0.186 g of KCl) plus 2% glycerol [10].…”

“…The bioassay plates were incubated at 28°C for 24 h before photography. The radius of the inhibition zone was measured and the toxin units were transformed according to the toxin formula: zeamines (units) = 0.5484 e0.886x (R 2 = 0.9957), X is the width in millimeters of the growth inhibition zone surrounding each well[27]. …”

A Sfp-type phosphopantetheinyl transferase ZmsO is essential for zeamines production and the virulence of Dickeya zeae

“…Since zeamines will inhibit the E. coli growth by formation an inhibition zone on bioassay plate (Zhou et al 2011;Cheng et al 2013;Liao et al 2014;Masschelein et al 2013), we can use the same bioassay plate to compare the antimicrobial activity between different mutants by the size of their inhibition zones on plates. To further study the biosynthesis mechanisms of zeamines in D. zeae, a Tn5 insertion mutant library was screened and over 100 candidate mutants with a decreased antimicrobial activity were collected.…”