Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005

Abstract

Lantibiotics are ribosomally synthesized oligopeptide antibiotics that contain lanthionine bridges derived by the posttranslational modification of amino acid residues. Here, we describe the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005, the first, to our knowledge, lantibiotic gene cluster from a high G+C bacterium to be cloned and sequenced. The cin cluster contains many genes not found in lantibiotic clusters from low G+C Gram-positive bacteria, including a Streptomyces antibiotic regulatory protein regulatory gene, and lacks others found in such clusters, such as a LanT-type transporter and a LanP-type protease. Transfer of the cin cluster to Streptomyces lividans resulted in heterologous production of cinnamycin. Furthermore, modification of the cinnamycin structural gene (cinA) led to production of two naturally occurring lantibiotics, duramycin and duramycin B, closely resembling cinnamycin, whereas attempts to make a more widely diverged derivative, duramycin C, failed to generate biologically active material. These results provide a basis for future attempts to construct extensive libraries of cinnamycin variants.

Figure 1

Sequence alignment of the propeptides of cinnamycin, duramycin, duramycin B, and duramycin C, highlighting the positions of posttranslational modifications present in the mature molecules. Lanthionine and methyllanthionine rings are indicated by thio-ether linkages, between cysteines (gray boxes) and dehydrated serines or threonines, respectively (black boxes). The hydroxylated aspartate residue, D₁₅, and the lysinoalanine bridge between K₁₉ and S₆ are indicated by white boxes. Differences between the amino acid sequence of cinnamycin and the closely related family of duramycin variants are ringed.

Figure 2

The cinnamycin gene cluster of S. cinnamoneus cinnamoneus DSM 40005 showing the order and direction of transcription of all of the genes, plus restriction sites used in cloning the cluster. Genes containing a TTA codon are indicated by asterisks. The position and sequence of the potential CinR1 binding site are shown above the map. The individual clones that covered this region of the chromosome were as follows: pIJ10101 and pIJ10102, the central BglII fragment cloned in BamHI cut pBluescriptKSII in opposite orientations; pIJ10103, the BamHI fragment downstream of cinH cloned in BamHI cut pBluescriptIIKS; pIJ10104, the left-most BamHI fragment cloned in BamHI cut pBluescriptIIKS; pIJ10105, the right-most Asp718 fragment cloned into Asp718-cut pBluescriptIIKS; pIJ10108, the small BamHI–Asp718 fragment from pIJ10101 and the insert from pIJ10104 cloned, sequentially, in pOJ436 to give a fragment that corresponds to the left-most portion of the cluster up to the first Asp718 site.

Figure 3

Undersides of plates streaked with various Streptomyces strains overlaid with B. subtilis EC1524. Lantibiotic production is indicated by a zone of inhibition of B. subtilis EC1524 around the Streptomyces culture. The strains are (a) S. cinnamoneus cinnamoneus DSM 40005 (cinnamycin) and S. lividans derivatives carrying the following plasmids; (b) pOJ436; (c) pIJ10108; (d) pIJ10110; (e) pIJ10109 (cinnamycin); (f) pIJ10119 (duramycin B); (g) pIJ10120 (duramycin C); (h) pIJ10121 (duramycin). The lantibiotic predicted to be made by the strain is given in parentheses.

Figure 4

Matrix-assisted laser desorption ionization time-of-flight mass spectra of extracts of media in which the following strains were grown: (a) S. cinnamoneus cinnamoneus DSM 40005 (cinnamycin); (b) S. lividans/pOJ436; (c) S. lividans/pIJ10109 (cinnamycin); (d) S. lividans/pIJ10119 (duramycin B); (e) S. lividans/pIJ10121 (duramycin). The lantibiotic predicted to be made by the strain is given in parentheses. The masses shown are 1 Da in excess of actual masses because of the addition of a proton during ionization. Masses quoted elsewhere have been rounded up and the mass of the extra proton has been subtracted. The larger peaks to the right of the peak for duramycin B in d correspond to masses predicted for the unmodified duramycin B propeptide and its oxidized form.