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. 2012 Oct 8:11:135.
doi: 10.1186/1475-2859-11-135.

Novel polyoxins generated by heterologously expressing polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes

Jine Li  1 , Lei Li, Chi Feng, Yihua Chen, Huarong Tan
Affiliations

Novel polyoxins generated by heterologously expressing polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes

Jine Li et al. Microb Cell Fact. .

Abstract

Background: Polyoxins are potent inhibitors of chitin synthetases in fungi and insects. The gene cluster responsible for biosynthesis of polyoxins has been cloned and sequenced from Streptomyces cacaoi and tens of polyoxin analogs have been identified already.

Results: The polyoxin biosynthetic gene cluster from Streptomyces cacaoi was heterologously expressed in the sanN inactivated mutant of Streptomyces ansochromogenes as a nikkomycin producer. Besides hybrid antibiotics (polynik A and polyoxin N) and some known polyoxins, two novel polyoxin analogs were accumulated. One of them is polyoxin P that has 5-aminohexuronic acid with N-glycosidically bound thymine as the nucleoside moiety and dehydroxyl-carbamoylpolyoxic acid as the peptidyl moiety. The other analog is polyoxin O that contains 5-aminohexuronic acid bound thymine as the nucleoside moiety, but recruits polyoximic acid as the sole peptidyl moiety. Bioassay against phytopathogenic fungi showed that polyoxin P displayed comparatively strong inhibitory activity, whereas the inhibitory activity of polyoxin O was weak under the same testing conditions.

Conclusion: Two novel polyoxin analogs (polyoxin P and O) were generated by the heterologous expression of polyoxin biosynthetic gene cluster in the sanN inactivated mutant of Streptomyces ansochromogenes. Polyoxin P showed potent antifungal activity,while the activity of polyoxin O was weak. The strategy presented here may be available for other antibiotics producers.

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Figures

Figure 1
Figure 1
Structures of polyoxins, nikkomycins, hybrid antibiotics and their biosynthetic gene clusters.A, structures of polyoxin analogs; B, structures of two representative nikkomycins; C, structures of two hybrid antibiotics; D, gene organization of polyoxin and nikkomycin biosynthetic gene clusters. The red arrows indicate genes related to the nucleoside moiety biosynthesis, and the homolog genes are linked by oblique lines.
Figure 2
Figure 2
HPLC analyses of the antibiotics produced by ΔsanN/pPOL. The corresponding peaks of different compounds are marked by arrows. A, HPLC traces identifying as polyoxin P; B, HPLC traces identifying as polyoxin O. Polyoxin N and thymine polyoxin C had the similar retention time at about 11.8 min, the retention times of polynik A, polyoxin J and P were 12.9 min, 15.2 min and 17.2 min, respectively. The retention times for polyoxin O and H were about 19.5 min and 22 min, respectively.
Figure 3
Figure 3
MS and NMR analyses of polyoxin P and O.A, structure of polyoxin P. The fragmentation pattern of MS/MS is marked in dash lines. The bold lines indicate COSY correlations, and the HMBC correlations are showed by arrows; B, structure of polyoxin O. The fragmentation pattern of MS/MS is marked in dash lines; C, MS and MS/MS spectra of polyoxin P; D, MS and MS/MS spectra of polyoxin O.
Figure 4
Figure 4
Bioassay of polyoxin H, polyoxin P and polyoxin O against fungi.A, Alternaria kikuchiana; B, Aspergillus fumigates; C,Rhizoctonia solani; D, Botrytis cinerea; E, Trichoderma viride. 1, polyoxin H; 2, polyoxin P; 3, polyoxin O.
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