Enhanced Production of Virginiamycin with the Maintained Optimal Ratio of T Its Components by a Mutant Streptomyces Virginiae IB 25–8 Strain ⁎ Vyacheslav A
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Biocatalysis and Agricultural Biotechnology 17 (2019) 408–415 Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab Enhanced production of virginiamycin with the maintained optimal ratio of T its components by a mutant Streptomyces virginiae IB 25–8 strain ⁎ Vyacheslav A. Savushkina, Vakhtang V. Dzhavakhiyab,c, , Elena V. Glagolevab,c, Veronika V. Savelyevab,c, Evgeniya D. Voskresenskayab,c, Alexander I. Ovchinnikovb,c, Vladislav I. Glagolevb,c, Nikita V. Novakb,c, Yana O. Grebenevab a Russian State Agrarian University – Moscow Timiryazev Agricultural Academy, Timiryazevskaya ul., 49, Moscow 127550, Russia b Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, Pr. 60-letiya Oktyabrya, 7/1, Moscow 117312, Russia c INGBIO small innovative enterprise, Pr. 60-letiya Oktyabrya, 7/1, Moscow 117312 Russia ARTICLE INFO ABSTRACT Keywords: Virginiamycin, a widely used veterinary antibiotic produced by Streptomyces virginiae, represents a natural mix of Virginiamycin two macrocyclic peptidolactones; its main components are the M1 and S1 factors, which act synergistically when Streptomyces virginiae present in the optimum ratio of 75:25. Because of a large number of genes involved into the virginiamycin Mutagenesis biosynthesis, the development of high-yield strains able to synthesize M1 and S1 at a synergistic ratio with the Fermentation total productivity exceeding 4 g/L still remains a relevant problem. Using a multi-step random UV mutagenesis Synthetic resins with the simultaneous control of the M1:S1 ratio in a final product, a new highly productive and genetically stable S. virginiae strain IB 25–8 was obtained. After the medium improvement combined with the addition of a Diaion™ HP21 resin absorbing up to 98.5% of the total virginiamycin, the productivity of the strain has reached 5.21 ± 0.16 g/L, while the M1:S1 ratio remained to be synergistic (75:25). The effect of various medium components, their concentration, and resin types on the M1:S1 ratio has been shown. The obtained strain is promising for the industrial use due to its high productivity and the optimal M1:S1 ratio. A pre-fermentation addition of the selected DIAION™ HP21 resin to fermentation medium increases the total yield and simplifies the further isolation and purification of virginiamycin. 1. Introduction virginiae simultaneously produces both M1 and S1 in a ratio providing their maximum synergistic activity (Di Giambattista et al., 1989). The In the modern world, maintenance of a high meat and milk pro- maximum synergism is observed when M1 an S1 present in the op- duction rate is provided by active use of veterinary antibiotics, which timum ratio of 75:25; in this case, the total antimicrobial activity of production volume makes almost 2/3 of the global antibiotic produc- virginiamycin increases in 3.5–4 times (Mast and Wohlleben, 2014). tion and is estimated as about $4 billion (Gelband et al., 2015; van This rare feature provides a lack of any significant resistance of mi- Boeckel et al., 2014). The most popular veterinary antibiotics belong to croorganisms to virginiamycin even after its long-term use. the tetracycline, cephalosporin, aminoglycoside, destomycin, and Virginiamycin has a bacteriostatic and bactericidal activity towards streptogramin groups. The last one is of special importance due to the majority of gram-positive and some gram-negative bacteria in- biological properties of streptogramins, which are nontoxic, non- cluding Clostridium perfringens, Staphylococcus spp., Microccocus spp., mutagenic, biodegradable, and are characterized by the lack of accu- Campylobacter spp., Listeria spp., clamydia (C. trachomatis, C. pneumo- mulation in animal tissues and a narrow antimicrobial spectrum con- niae), and mycoplasma (M. pneumoniae)(Grozina et al., 2014). In ad- cerning Staphylococcus bacteria (Cocito, 1979). dition to the antimicrobial activity, virginiamycin also acts as a growth Virginiamycin, a streptogramin antibiotic produced by Streptomyces stimulator, since it optimizes the absorption and metabolism of nu- virginiae, represents a natural mix of macrocyclic peptidolactones M and trients, improves the state of a small intestine epithelium, and inhibits S. The main components of the antibiotic are the M1 and S1 factors. the synthesis of harmful toxins and metabolites by gut organisms One of the unique features of a virginiamycin biosynthesis is that S. (Feighner and Dashkevicz, 1987; Hoyzman et al., 2011; Cervantes et al., ⁎ Corresponding author at: Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, Pr. 60-letiya Oktyabrya, 7/1, Moscow 117312, Russia. E-mail address: [email protected] (V.V. Dzhavakhiya). https://doi.org/10.1016/j.bcab.2018.10.021 Received 16 July 2018; Accepted 5 October 2018 Available online 19 December 2018 1878-8181/ © 2018 Elsevier Ltd. All rights reserved. V.A. Savushkin et al. Biocatalysis and Agricultural Biotechnology 17 (2019) 408–415 2002). Today virginiamycin is widely used in ethanol fuel production Schott funnel (pore size 100 µm) to remove large mycelium conglom- (Arshad et al., 2011) and as an antibiotic feed additive for cattle and erates. A spore concentration was calculated using a Goryaev-Thoma poultry providing better survival of young animals and birds, lower counting chamber and adjusted to 1.5–2·106 spores/mL. The final sus- sickness rate, increased body weight gain, and improved nutrition ef- pension was placed under a 12.5-W UV lamp (250–280 nm, ficiency (Ives et al., 2002; Singh et al., 2008; Shojadoost et al., 2013). Mineralight, USA) at a distance of 40 cm. The radiation intensity during Virginiamycin is registered and actively used in 32 countries in- treatment was 0.25 mW/cm2. After UV treatment, 0.1 mL of the sus- cluding USA, Russia, Brazil, and China. In the first half of 2014, the pension was inoculated onto Petri plate containing the agar medium, import of virginiamycin-based preparations into Russia made 8.32% of spread on the whole agar surface, and incubated for 6–7 days at 28 °C. the total import of antibacterial veterinary preparations (Vetanalytic, To determine the optimum exposure time, the survival rate and 2015) and 40.6% of the total import of macrolide antibiotics mutagenesis efficiency were assessed within the exposure rangeof (Lavrenova, 2016). To 2020, a predicted increase in the consumption of 5–40 min. A survival rate of colonies was determined as the percentage this antibiotic in North America, Europe, and Asia will make 6% of colonies grown in the treated variants comparing to the untreated (Market Research Reports, 2016). control. Mutagenesis efficiency was determined by the percentage of To provide a sufficient production of virginiamycin, efficient tech- morphologically changed colonies grown after the UV treatment. nologies of its industrial production, isolation, and purification are re- After UV treatment, 10–20 mutant colonies, which morphology quired along with overproducing strains. As a rule, productivity of wild significantly differed from initial one, were selected for the further strains is unsufficient for industrial microbiology; for example, pro- work. Selected colonies were reinoculated on agar medium, grown for 7 ductivity of the original ATCC 13161 strain, which was first described days, then fermented (see 2.3), and preliminary analyzed for their as a virginiamycin-producing microorganism, is only 30 U/mL or productivity by a thin-layer chromatography; the best mutants were ~48 µg/mL (de Somer and van Dijck, 1955). In general, according to additionally examined for their productivity and M1:S1 ratio by HPLC. various publications, productivity of developed industrial S. virginiae Mutant strains characterized by a high virginiamycin production and strains under laboratory conditions still remains rather low varying better M1:S1 ratio were selected and used for the further mutagenesis/ from ~150–180 U/mL (~0.28 g/L) to 1200 U/mL (~ 1.9 g/L) (Biot, selection cycles. 1984; Prikrylova et al., 1987; Zvenigorodskii et al., 2001; Zhang et al., 2011). A final virginiamycin yield can be additionally increased bythe 2.3. Fermentation conditions medium improvement and optimization of fermentation conditions, but even in this case productivity of existing industrial strains is reported Spore suspensions were prepared from 7-day mutant cultures grown within the range of 3–4 g/L (Biot, 1984; Zhang et al., 2011; Han et al., on agar medium and adjusted to the concentration of 1·106 CFU/mL. 2013), except for one patent describing a method to increase the vir- The suspension (0.1 mL) was inoculated into 50-mL Erlenmeyer flasks giniamycin output to > 5 g/L (Yong, 2015). Note that none of the containing 10 mL of vegetation medium. Seed cultures were grown for mentioned authors reported about the control of the M1:S1 ratio during 48 h on Innova 44 incubation shakers (New Brunswick, Germany) at optimization of fermentation conditions and in the final product, 28 °C and 250 rpm (5-cm orbit) and then were reinoculated into 50-mL though it is an important characteristics of the virginiamycin efficiency. flasks containing 10 mL of fermentation medium. Fermentation was The purpose of this work was the development of a new over- carried out for 96 h under the same conditions. producing S. virginiae strain and the improvement of medium and fer- mentation conditions to achieve a final virginiamycin output exceeding 2.4. Fermentation medium