Activation and characterization of a cryptic gene cluster reveals a cyclization cascade for polycyclic tetramate macrolactams

Abstract

Polycyclic tetramate macrolactams (PTMs) are a growing class of natural products and are derived from a hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway. PTM biosynthetic gene clusters are conserved and widely distributed in bacteria, however, most of them remain silent. Herein we report the activation of a PTM gene cluster in marine-derived Streptomyces pactum SCSIO 02999 by promoter engineering and heterologous expression, leading to the discovery of six new PTMs, pactamides A-F (11-16), with potent cytotoxic activity upon several human cancer cell lines. In vivo gene disruption experiments and in vitro biochemical assays reveal a reductive cyclization cascade for polycycle formation, with reactions sequentially generating the 5, 5/5 and 5/5/6 carbocyclic ring systems, catalysed by the phytoene dehydrogenase PtmB2, the oxidoreductase PtmB1, and the alcohol dehydrogenase PtmC, respectively. Furthermore, PtmC was demonstrated as a bifunctional cyclase for catalyzing the formation of the inner five-membered ring in ikarugamycin. This study suggests the possibility of finding more bioactive PTMs by genome mining and discloses a general mechanism for the formation of 5/5/6-type carbocyclic rings in PTMs.

Fig. 1. Representative PTMs (1–9) and the key precursor (10).

Fig. 2. (A) (A) Genetic organization of the ptm gene cluster in pCSG2404 from a genomic library of S. pactum SCSIO 02999. (B) HPLC analysis of metabolite profile of different strains: (i) S. pactum 2999XM47i, where xiaP is in-frame deleted; (ii) S. lividans TK64/pCSG2801, no promoter inserted; (iii) S. lividans TK64/pSET152; (iv) S. lividans TK64/pCSG2804, where ermE*p is inserted in front of ptmA; (v) S. lividans TK64/pCSG2805, where ermE*p is inserted in front of ptmD; (vi) S. lividans TK64/pCSG2809, where ermE*p is inserted in front of ptmA and ptmC is in-frame deleted; (vii) S. lividans TK64/pCSG2811, where ermE*p is inserted in front of ptmA and ptmB1 is in-frame deleted; (viii) S. lividans TK64/pCSG2814, where ermE*p is inserted in front of ptmA and ptmB2 is in-frame deleted; (ix) S. pactum 02999PTMp1, where ermE*p is inserted in front of ptmA. (i)–(vii) and (ix), detection at 300 nm; (viii), detection at 360 nm. The “#” symbols denote uncharacterized PTM-like products. (C) Chemical structures of patamides A–F (11–16).

Fig. 3. Biochemical characterization of PtmC and the proposed reaction mechanism. (A) HPLC analysis of the enzyme assays. (i) 13 + PtmC + NADPH; (ii) 13 + PtmC + NADH; (iii) standard 11; (iv) 13 + NADPH; (v) 13 + PtmC; (vi) 13 + IkaC + NADPH; (vii) 17 + NADPH; (viii) 17 + PtmC + NADPH; (ix) standard 7. (B) The proposed mechanism of PtmC catalysis. (C) The proposed PtmC mechanism to convert 17 to 7 and 18.

Fig. 4. Proposed biosynthetic pathway for pactamides A–F (11–16).