Engineering And Heterologous Expression Of The Nikkomycin Biosynthetic Gene Cluster

Nikkomycins are a group of peptidyl nucleoside antibiotics. They consist of peptidyl and nucleoside moieties which are synthesized separately and then linked together by peptide bond. With structural similarity to UDP-N-acetylglucosamine, nikkomycins act as competitive inhibitors of fungal chitin synthases to prevent cell wall biosynthesis in pathogenic fungi, and it has the value of practical application as the novel agricultural antibiotics.By PCR-targeting method, we replaced the promoters of sanG and sanF in pNIK, which contains nikkomycin biosynthesis gene cluster produced by S.ansochromogenes, with the hrdB promoter to generate pNIKm. We transferred pNIK and pNIKm into S.coelicolor M1146 by intergeneric conjugation. We then evaluated expression of the gene cluster in the heterologous host by RT-PCR. Furthermore, we also observed the antifugal activity and nikkomycin production by bioassay against Alternaria longipes and HPLC analysis.In this paper, the main results were as follows:1. The plasmid pNIKm was constructed successfully. And nikkomycin were expressed in heterologous host successfully. Then we obtained M1146-NIK and M1146-NIKm.2. M1146-NIK and M1146-NIKm exhibited antifungal activity against Alternaria longipes.3. M1146-NIK and M1146-NIKm can produce a trace amount of nikkomycin X, nikkkomycin Z and pseudo-Z.4. There was a substantial accumulation of uridine in M1146-NIK, whereas substantial accumulations of uridine, ribofuranosyl-4-formyl-4-imidazolone and pyridylhomothreonine were observed in M1146-NIKm.We successfully expressed the nikkomycin biosynthetic gene cluster in the heterologous host and identified nikkomycins and some of its key biosynthetic intermediates. This study will provide the basis for enzymatic reaction of the condensation between the two nikkomycin moieties and for the generation of hybrid antibiotics by combinatorial biosynthesis.