Isolation And Identification Of The Ficellomycin Biosynthetic Gene Cluster

Ficellomycin is a dipeptide-like antibiotic isolated from the culture broth of Streptomyces ficellus (NRRL8067), displaying high in vitro activity against Penicillium oxcicum and Staphylococcus aureus. This agent can selectively impair semiconservative DNA replication and cause the accumulation of deficient okazaki fragments, which lack the capability to be further integrated into larger DNA fragments while has no effect on normal cell metabolism. As for the special action mode of ficellomycin, it has large potential for developing novel antibiotics especially for some antitumor drugs with low toxicity and fewer side-effects. However, there are no reports on the biosynthesis of ficellomycin and its related genes?In this study, the PCR primers were designed according to the non-ribosomal peptide synthase (NRPS) conserved region (Adenylation domain), which was necessary for the ficellomycin biosynthesis process. The constructed genomic library of S. ficellus NRRL8067 was screened by PCR. Five different nrps gene clusters (nrps1-5) were identified by sequencing and analysis of positive clones. The gene disruption and complementation experiments confirmed that the nrps1 was crucial for ficellomycin biosynthesis. Therefore, the flanking regions of nrps1 were cloned and sequenced by chromosome walking, and finally a 65.9 kb contiguous DNA fragment was obtained. The results of gene disruption and LC-MS analysis of fermentation products revealed that the biosynthetic gene of the ficellomycin was located within 30 kb of contiguous DNA, which contains 26 ORFs (fic11-fic36). Most of these genes corresponded to a functional section of ficellomycin biosynthesis. The nrps1 gene (fic31) was associated with dipeptide compound assembly, the ornithine-oxo-acid transaminase gene (fic16) transketolase gene (fic23, fic24)and N-acetyl-ornithine/N-acetyl-lysine deacetylase gene (fic30) were associated with the synthesis of the functional group 1-azabicyclo [3.1.0] hexane-2-ylidene core (AHYC). The glutamince-scyllo-inositol aminotransferase gene (fic25) and scyllo-inosamine-4-phosphate gene (fic36) were related to the syinthesis of guanidine groups. The ABC transporter permease gene (fic11) and AtrA protein gene (fic12) may function encoding a self-resistant protein to carry the synthesized ficellomycin out of the cells. However, there is a lack of a NRPS encoding genes responsible for valine activation and assembly in the gene cluster,which may be located in other regions of the genome.In this study, the biosynthetic gene cluster and biosynthesis of ficellomycin were elucidated, which provided a theoretical basis for further improving the fermentation yield by means of genetic engineering. It would be of great assistance to acquire a series of corresponding derivates with higher biological activity. The present research would be possible to inspire attempts to apply this knowledge for combinatorial biosynthesis.