Identification by Genome Mining of a Type I Polyketide Gene Cluster from Streptomyces argillaceus Involved in the Biosynthesis of Pyridine and Piperidine Alkaloids Argimycins P

Abstract

Genome mining of the mithramycin producer Streptomyces argillaceus ATCC 12956 revealed 31 gene clusters for the biosynthesis of secondary metabolites, and allowed to predict the encoded products for 11 of these clusters. Cluster 18 (renamed cluster arp) corresponded to a type I polyketide gene cluster related to the previously described coelimycin P1 and streptazone gene clusters. The arp cluster consists of fourteen genes, including genes coding for putative regulatory proteins (a SARP-like transcriptional activator and a TetR-like transcriptional repressor), genes coding for structural proteins (three PKSs, one aminotransferase, two dehydrogenases, two cyclases, one imine reductase, a type II thioesterase, and a flavin reductase), and one gene coding for a hypothetical protein. Identification of encoded compounds by this cluster was achieved by combining several strategies: (i) inactivation of the type I PKS gene arpPIII; (ii) inactivation of the putative TetR-transcriptional repressor arpRII; (iii) cultivation of strains in different production media; and (iv) using engineered strains with higher intracellular concentration of malonyl-CoA. This has allowed identifying six new alkaloid compounds named argimycins P, which were purified and structurally characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Some argimycins P showed a piperidine ring with a polyene side chain (argimycin PIX); others contain also a fused five-membered ring (argimycins PIV-PVI). Argimycins PI-PII showed a pyridine ring instead, and an additional N-acetylcysteinyl moiety. These compounds seem to play a negative role in growth and colony differentiation in S. argillaceus, and some of them show weak antibiotic activity. A pathway for the biosynthesis of argimycins P is proposed, based on the analysis of proposed enzyme functions and on the structure of compounds encoded by the arp cluster.

Keywords: Streptomyces; alkaloid; cryptic; growth; piperidine; pyridine; thioester reductase; type I polyketide synthase.

FIGURE 1

Genetic organization of cluster 18. Genes are shown to scale. Argimycins P biosynthesis genes (arp) are shown in gray. Other genes are shown in white and are numbered. Triangles indicate those genes that have been inactivated. Bars indicated DNA regions that have been deleted. Black lines represent overlapping cosmids isolated from this chromosomal DNA region.

FIGURE 2

UPLC chromatograms of S. argillaceus wild type and mutant strains in arp genes. Butanol extracts of S. argillaceus wild type strain (WT), S. argillaceus MARPPIII, S. argillaceus MARPRII, and S. argillaceus MARPPIII (pEM4ATCPKS). Chromatograms are shown at 400 nm (A), 272 nm (B) and 230 nm (C). Peaks corresponding to the different argimycins P are indicated as follows: argimycins PI and PII (I and II); nigrifactin (N); argimycin PIV (IV); argimycin PV (V); argimycin PVI (VI); and argimycin PIX (IX).

FIGURE 3

Chemical structures of argimycins P (relative configuration shown for argimycins PI, PII, PIV, PV, and PVI).

FIGURE 4

Morphology of S. argillaceus strains: argimycins P producers (wild type, WT; MARPRII); non-producer mutants (MARPPIII; DARPO-HII); and complemented mutant (MARPPIII + pEM4TCPKS).

FIGURE 5

Proposed biosynthesis pathway for argimycins P.

FIGURE 6

UPLC chromatograms at 350 nm of butanol extracts of S. argillaceus DARPO-HII and S. argillaceus DARPO-HII (pIAGOorf8). N, nigrifactin.

FIGURE 7

UPLC chromatograms of butanol extracts of Streptomyces NRRL S-1022. N, nigrifactin; IV, argimycin PIV; V, argimycin PV; VI, argimycin PVI.