<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>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 <jats:italic>Streptomyces cacaoi</jats:italic> and tens of polyoxin analogs have been identified already. </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>The polyoxin biosynthetic gene cluster from <jats:italic>Streptomyces cacaoi</jats:italic> was heterologously expressed in the <jats:italic>sanN</jats:italic> inactivated mutant of <jats:italic>Streptomyces ansochromogenes</jats:italic> 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 <jats:italic>N</jats:italic>-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. </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Two novel polyoxin analogs (polyoxin P and O) were generated by the heterologous expression of polyoxin biosynthetic gene cluster in the <jats:italic>sanN</jats:italic> inactivated mutant of <jats:italic>Streptomyces ansochromogenes</jats:italic>. 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. </jats:sec>