Identification and Predictions Regarding the Biosynthesis Pathway of Polyene Macrolides Produced by Streptomyces roseoflavus Men-myco-93-63

Abstract

A group of polyene macrolides mainly composed of two constituents was isolated from the fermentation broth of Streptomyces roseoflavus Men-myco-93-63, which was isolated from soil where potato scabs were repressed naturally. One of these macrolides was roflamycoin, which was first reported in 1968, and the other was a novel compound named Men-myco-A, which had one methylene unit more than roflamycoin. Together, they were designated RM. This group of antibiotics exhibited broad-spectrum antifungal activities in vitro against 17 plant-pathogenic fungi, with 50% effective concentrations (EC₅₀) of 2.05 to 7.09 μg/ml and 90% effective concentrations (EC₉₀) of 4.32 to 54.45 μg/ml, which indicates their potential use in plant disease control. Furthermore, their biosynthetic gene cluster was identified, and the associated biosynthetic assembly line was proposed based on a module and domain analysis of polyketide synthases (PKSs), supported by findings from gene inactivation experiments.IMPORTANCEStreptomyces roseoflavus Men-myco-93-63 is a biocontrol strain that has been studied in our laboratory for many years and exhibits a good inhibitory effect in many crop diseases. Therefore, the identification of antimicrobial metabolites is necessary and our main objective. In this work, chemical, bioinformatic, and molecular biological methods were combined to identify the structures and biosynthesis of the active metabolites. This work provides a new alternative agent for the biological control of plant diseases and is helpful for improving both the properties and yield of the antibiotics via genetic engineering.

Keywords: Streptomyces roseoflavus Men-myco-93-63; antibiotic; antifungal activity; biosynthetic gene cluster; polyene macrolide.

FIG 1

HPLC chromatogram of crude antibiotic. (a) 3D plot (200 to 800 nm). (b) 2D plot at λ = 363 nm. Compound 1, roflamycoin; compound 2, Men-myco-A.

FIG 2

Chemical structures of the compounds 1 and 2.

FIG 3

Proposed biosynthetic pathway of roflamycoin. The dotted circles indicate inactivity. AT, acyl transferase; ACP, acyl carrier protein; KS, ketosynthase; DH, dehydratase; KR, ketoreductase; ER, enoyl reductase; TE, thioesterase.

FIG 4

Alignment of active-site sequences of AT, DH, and KR domains from RM PKSs. Only the regions containing the proposed active sites are shown. The active-site residues are marked with asterisks. The residue numbers shown in the first row of the AT alignment correspond to the positions in the E. coli FAS acyltransferase (FabD) crystal structure (PDB entry 1MLA). Arg117, which is conserved in all malonate- and methylmalonate-specific AT domains (AT1 to AT17), is the nonpolar amino acid Phe in AT0, which is specific for monocarboxylic substrates. The presence of Gln93 and Ser200 in AT1 and AT17 indicates that their substrate is methylmalonate, whereas Ile93 and Phe200 in AT2 to AT5 and AT7 to AT16 indicate that their substrate is malonate. In AT6, Gln93 indicates that its substrate is methylmalonate, whereas Pro200 indicates that its substrate is malonate; however, considering that position 200 is more selective in terms of amino acid occurrence than position 93, the substrate should be malonate, which also corresponds to the real chemical structure. The unique Ala93 and Ala200 residues in AT0 were not predicted from the key residue comparison.

FIG 5

Phylogenetic analysis of RfmA-F AT domains. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches.

FIG 6

HPLC analysis of the fermentation products from Men-myco-93-63 after the addition of l-valine (left) and l-isoleucine (right) into each 100-ml fermentation.

FIG 7

HPLC analysis of the fermentation products from Menmyco-93-63 and its ΔrfmA, ΔrfmB, ΔrfmD, ΔrfmE, and ΔrfmF gene inactivation mutants.