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. 2014 Oct;58(10):6185-96.
doi: 10.1128/AAC.03512-14. Epub 2014 Aug 11.

Overproduction of Ristomycin A by activation of a silent gene cluster in Amycolatopsis japonicum MG417-CF17

Marius Spohn  1 , Norbert Kirchner  2 , Andreas Kulik  1 , Angelika Jochim  1 , Felix Wolf  1 , Patrick Muenzer  3 , Oliver Borst  4 , Harald Gross  2 , Wolfgang Wohlleben  5 , Evi Stegmann  6
Affiliations

Overproduction of Ristomycin A by activation of a silent gene cluster in Amycolatopsis japonicum MG417-CF17

Marius Spohn et al. Antimicrob Agents Chemother. 2014 Oct.

Abstract

The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is Amycolatopsis. However, Amycolatopsis japonicum does not produce an antibiotic under standard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina (bbrAba), into A. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of A. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum/pRM4-bbrAba synthesizes ristomycin A.

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Figures

FIG 1
FIG 1
Bioassay after cluster activation by expression of bbrAba and ajrR, respectively. Wild-type A. japonicum (1), A. japonicum/pRM4-bbrAba (2), and A. japonicum/pRM4-ajrR (3) were grown for 5 days in R5 medium, and 20 μl of culture supernatant was assayed for bioactivity against B. subtilis.
FIG 2
FIG 2
HPLC chromatograms of wild-type A. japonicum and A. japonicum/pRM4-bbrAba after growth for 5 days in R5 medium and ristomycin standard. The boxed regions show the corresponding DAD spectra. Rt, retention time; mAU, milliabsorbance units.
FIG 3
FIG 3
Genetic organization of the ristomycin A (ris) cluster identified in A. japonicum. Predicted ORFs are represented by an arrow drawn to scale and are numbered as in Table 1. Gene names are indicated underneath the corresponding ORFs. Predicted functions of genes are listed. TE, thioesterase.
FIG 4
FIG 4
Classification of the glycopeptides. (A) Type I glycopeptides exemplified by vancomycin. These glycopeptides contain aliphatic chains in amino acids 1 and 3. (B) Type II glycopeptides exemplified by actinoidin A. These glycopeptides contain aromatic aliphatic chains in amino acids 1 and 3. (C) Type III glycopeptides exemplified by ristomycin A. These glycopeptides are like type II glycopeptides, and they contain an extra F-O-G ring system. Ring abbreviations: A, Me-l-Dpg; B, D-Hpg; C, L-β-Ht; D, D-Hpg; E, D-β-Ht; F, Me-l-Dpg; G, L-Hpg. (D) Type IV glycopeptides exemplified by teicoplanin. These glycopeptides are like type III glycopeptides plus they have aliphatic side chains on sugar.
FIG 5
FIG 5
Transcriptional pattern of representative ristomycin biosynthesis genes. The gene names are indicated to the right of the gel. Cultures were grown in R5 medium for 25 h. sigB is the major sigma factor of A. japonicum and was used as a housekeeping gene to normalize the RNA. WT, wild type.
FIG 6
FIG 6
HPLC–ESI-MS analysis (positive mode) of A. japonicum glycopeptide and ristomycin A. (Top) Ristomycin A standard (0.1 mg ml−1). (Bottom) A. japonicum/pRM4-bbrAba glycopeptide. [M + 2H]2+ was observed at m/z 1,034.8 and 1,034.9. The relative intensity is shown on the y axes.
FIG 7
FIG 7
Batch fermentation of A. japonicum/pRM4-bbrAba in R5 medium. Biomass (△), ristomycin A (■), and partial O2 pressure (pO2) (●) are depicted. A. japonicum/pRM4-bbrAba reached its maximal biomass value after 44 h of incubation, decreasing afterwards slowly during 2 days of further fermentation. Metabolizing of different sugars in R5 medium (sucrose [α-1,2-glycosidic linked glucose and fructose] and glucose) is reflected by a diauxic shift (between 24 and 44 h) during cultivation, apparent in growth retardation and pO2.
FIG 8
FIG 8
Ristomycin-dependent platelet aggregation. (Left) Representative tracings of aggregometry after stimulation of human platelets with 2.5 mg ml−1 commercial (black line) or A. japonicum ristomycin A (light gray line) as well as 10 μM ADP (dark gray line) and water control. (Right) Results of aggregometry after stimulation of human platelets with 2.5 mg ml−1 commercial or A. japonicum ristomycin A as well as 10 μM ADP. The values are arithmetic means plus standard errors of the means (SEM) (error bars) for four experiments.
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