Characterization of the polyoxin biosynthetic gene cluster from Streptomyces cacaoi and engineered production of polyoxin H

Abstract

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics.

FIGURE 1.

Chemical structures of polyoxins (A) and nikkomycin Z (B). Different polyoxin components were indicated as polyoxin-A, -F, -H, and -K, respectively.

FIGURE 2.

Restriction map (A) and genetic organization of the polyoxin biosynthetic gene cluster (B). ∼46-kb sequenced region from S. cacaoi var. asoensis covered by nine overlapping cosmids is indicated by open bars, and the region used as probes by solid squares. See Table 1 for the proposed functions of the pol genes.

FIGURE 3.

Heterologous production of polyoxin H in S. lividans TK24. A, bioassay of the metabolites produced by the recombinant. T. cutaneum was used as indicator strain, and a volume of 35 μl of processed fermentation broths were used for each assay. Recombinants (S. lividans TK24 carrying construct m5A7) spotted individually in spots 2 and 4 were compared with wild-type S. cacaoi var. asoensis as a positive control (spot 1) and S. lividans TK24/pSET152 as a negative control (spot 3). B, HPLC analysis of the metabolites produced by the engineered recombinant. Polyoxin authentic standards (ST), and samples from wild-type (WT) of S. cacaoi var. asoensis, S. lividans TK24/m5A7 (TK24/m5A7), and S. lividans TK24/pSET152 (TK24/pSET152) were compared. C, MS/MS fragmentation of the polyoxin H (601.4) authentic standard (1) into 411.1 (1a), 456.1 (1b), 474.0 (1c), and 540.0(1d). D, identical MS/MS fragmentation pattern of the polyoxin component produced by recombinant S. lividans TK24/m5A7 as polyoxin H (C).

FIGURE 4.

Insertional inactivation into polA and mutant complementation by polA. A, schematic representation for the construction of CY1 mutant. P, PvuII; B, BamHI; B, PCR confirmation of the CY1 mutants as having a 1.4-kb aac(3)IV fragment inserted into polA, as the 2.2-kb PCR product generated from mutants is ∼1.4 kb larger than that (762 bp) from the wild-type strain. C, bioassay of polA mutant CY1 (spot 1) and its complemented strain by polA(spot 3). Mutant CY1 containing empty vector pJTU695 (spot 2) was used as a negative control, and wild-type S. cacaoi (spot 5) as positive control. Mutant CY1 containing nikO (spot 4) was included to show the similar complementation effect of nikO with polA. D, HPLC analysis of the metabolites produced by the mutant CY1 and its complemented strain by polA. Polyoxins authentic standard (ST) and samples from wild-type (WT) of S. cacaoi var. asoensis, mutant CY1 (CY1), mutant CY1 complemented by polA (CY1/polA) and nikO (CY1/nikO), and mutant CY1 containing empty vector pJTU695 (CY1/pJTU695) as negative control were compared.

FIGURE 5.

Characterization of PolA as an enolpyruvyltransferase. A, SDS-PAGE analysis of PolA and NikO proteins. Purified His₆-tagged NikO (lane 1), PolA (lane 2), soluble proteins of cell-free extract from E. coli BL21 (DE3)/pLysE/polA before (lane 4) and after (lane 3) induction with IPTG, and total proteins of cell-free extract of E. coli BL21 (DE3)/pLysE/polA after induction with IPTG (lane 5), were aligned with molecular mass markers (M). B, conversion mechanism of UMP to 3′-EUMP catalyzed by PolA. C, HPLC analysis of the products catalyzed by PolA (III) and NikO (II), respectively. UMP (I) is included as a negative control. D, MS/MS fragmentation of the products 3′-EUMP catalyzed by PolA in negative mode.

FIGURE 6.

Targeted inactivation of polO. A, schematic representation for the construction of mutant CY14. B, PCR confirmation of the CY14 mutants, as the aac(3)IV cassette was recombined into polO the mutants give an ∼1.5-kb PCR product, although the wild-type strain shows 0.45-kb PCR product. C, bioassay of polO mutant CY14 (spots 2–4) with the wild-type S. cacaoi (spot 1). D, HPLC analysis of the metabolites produced by mutant CY14 (CY14) together with the wild-type (WT) S. cacaoi and authentic polyoxin standard (ST).

FIGURE 7.

Characterization of PolO as an O-carbamoyltransferase. A, SDS-PAGE analysis of PolO proteins. Purified His₆-tagged PolO (lane 1), soluble proteins of cell-free extract from E. coli BL21 (DE3)/pLysE/polO before (lane 4) and after (lane 2) induction with IPTG, and total proteins of cell-free extract of E. coli BL21 (DE3)/pLysE/polO after induction with IPTG (lane 3) were aligned with molecular weight markers (M). B, conversion mechanism of AHV to ACV catalyzed by PolO. C, TLC analysis of the products catalyzed by PolO (lane 3) AHV standard (lane 1) and reaction mix with heat-inactivated (90 °C, 10 min) PolO added was included as negative controls (lane 2).

FIGURE 8.

Proposed pathway for polyoxin biosynthesis. The dashed line indicates the protein(s) for the proposed catalytic reaction(s) was/were not identified. R₁ = CH₃,CH₃OH, or COOH; R = H, CH₃,CH₃OH, or COOH.? indicates the highly hypothetic protein functions deduced from bioinformatic analysis only.