Identification and analysis of the biosynthetic gene cluster encoding the thiopeptide antibiotic cyclothiazomycin in Streptomyces hygroscopicus 10-22

Abstract

Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides.

FIG. 1.

Structures of thiostrepton, thiocillin, microncoccinate, and cyclothiazomycin (3).

FIG. 2.

Heterologous expression of the cyclothiazomycin biosynthesis gene cluster cloned from S. hygroscopicus 10-22. (A) Organization of the ORFs in the gene cluster. The putative functions of the ORFs are indicated and are summarized in Table 3. The restriction enzymes used for gene deletion are indicated (B, BclI; E, EcoRI; X, XbaI; N, NdeI). (B) Heterologous expression profile of cyclothiazomycin in S. lividans 1326. The black bars represent the genomic fragments in the plasmids (see Table 1) corresponding to the regions in the gene cluster shown in panel A. The inhibition zones for the plasmids shown on the right are inhibition zones on the same bioassay agar plate (see Materials and Methods). (C) MS spectrum of the cyclothiazomycin produced by S. lividans 1326 harboring pJTU4892. Two characteristic peaks at m/z 737.1506 ([M+2]²⁺) and 1473.2877 ([M+1]⁺) with corresponding amplified isotopic peaks were extracted from the LC-ES-QTOF profile for heterologous production.

FIG. 3.

LC-ES-QTOF and MS/MS analysis of methanol extract of an S. hygroscopicus 10-22 culture. (A) LC-ES-QTOF analysis of methanol extract from an S. hygroscopicus 10-22 culture. Two distinct peaks were extracted with m/z 737.1515 ± 5 from total ion current chromatography, indicating the two isomers of cyclothiazomycin. (B) Accurate-mass QTOF spectrum of the active product obtained from S. hygroscopicus 10-22. The two signals at m/z 1473.2933 and 737.1515, corresponding to [M+1]⁺ and [M+2]²⁺, corresponded well to the theoretical value for cyclothiazomycin (m/z 1473.2967) within the error of the instrument (<2 ppm). The relative isotope abundances in the two isotopic graphs could be calculated, which resulted in measured average abundances of 88 and 70 for [M+2]⁺ and [M+3]⁺, respectively. (C) Cyclothiazomycin structure (left panel) and skeleton of the [M+1]⁺ ion indicated by its structural peptide (right panel). The dashed lines indicate the two cyclization sites: the substituted pyridine formed by S₁*, A₁₀*, and S₁₁* (red) and the tertiary thioether formed by S5* and C18* (green). Asterisks indicate the amino acid residues that are posttranslationally modified. (D) MS/MS spectrum of cyclothiazomycin. Several fragments (fragments 2, 3, 4, 5, and 6) generated by double cleavage or multiple cleavage at an acrylamide bond or the tertiary C—S bond were assigned at m/z 784, 1163, 283, 441, and 1231. Differences between the theoretical values and the measurements, which were greater than the instrument errors for MS/MS (<5 ppm), might be caused by low abundances and impurities.

FIG. 3.

LC-ES-QTOF and MS/MS analysis of methanol extract of an S. hygroscopicus 10-22 culture. (A) LC-ES-QTOF analysis of methanol extract from an S. hygroscopicus 10-22 culture. Two distinct peaks were extracted with m/z 737.1515 ± 5 from total ion current chromatography, indicating the two isomers of cyclothiazomycin. (B) Accurate-mass QTOF spectrum of the active product obtained from S. hygroscopicus 10-22. The two signals at m/z 1473.2933 and 737.1515, corresponding to [M+1]⁺ and [M+2]²⁺, corresponded well to the theoretical value for cyclothiazomycin (m/z 1473.2967) within the error of the instrument (<2 ppm). The relative isotope abundances in the two isotopic graphs could be calculated, which resulted in measured average abundances of 88 and 70 for [M+2]⁺ and [M+3]⁺, respectively. (C) Cyclothiazomycin structure (left panel) and skeleton of the [M+1]⁺ ion indicated by its structural peptide (right panel). The dashed lines indicate the two cyclization sites: the substituted pyridine formed by S₁*, A₁₀*, and S₁₁* (red) and the tertiary thioether formed by S5* and C18* (green). Asterisks indicate the amino acid residues that are posttranslationally modified. (D) MS/MS spectrum of cyclothiazomycin. Several fragments (fragments 2, 3, 4, 5, and 6) generated by double cleavage or multiple cleavage at an acrylamide bond or the tertiary C—S bond were assigned at m/z 784, 1163, 283, 441, and 1231. Differences between the theoretical values and the measurements, which were greater than the instrument errors for MS/MS (<5 ppm), might be caused by low abundances and impurities.

FIG. 4.

Proposed pathway for biosynthesis of cyclothiazomycin. Eight proteins, CltB, CltC, CltD, CltE, CltF CltG, CltM, and CltN, are the candidate enzymes involved in posttranslational modification, and one transport protein, CltO, is predicted to transport the final product. Reaction a, dehydration reaction to produce thiazolines (red); reaction b, selective dehydrogenation reaction to produce thiazoles (green); reaction c, dehydration reaction to produce dehydroamino acids (blue); reaction d, simultaneous hetero-Diels-Alder cyclization and LP cleavage to produce a dehydropiperidine intermediate (purple); reaction e, deamination of the amino group generated by LP cleavage to form a trisubstituted pyridine (purple); reaction f, Michael addition reaction to generate the putative thioether intermediate; reaction g, molecular rearrangement suggested to generate a tertiary thioether.

FIG. 5.

Bioassay for cyclothiazomycin production by S. hygroscopicus 10-22 with B. cinerea Persoon. An agar patch containing S. hygroscopicus 10-22 was placed on B. cinerea Persoon growing on medium to determine the zone of inhibition due to cyclothiazomycin production; S. lividans 1326 was used as a negative control (see Materials and Methods).