Directed Accumulation of Anticancer Depsipeptides by Characterization of Neoantimycins Biosynthetic Pathway and an NADPH-Dependent Reductase

Neoantimycins (NATs) are members of antimycin-types of depsipeptides with outstanding anticancer activities. We isolated NAT-A (1) and -F (2) from the fermentation extract of Streptomyces conglobatus. The NAT biosynthetic gene cluster ( nat BGC) was identified by genome sequencing and bioinformatics analysis. nat BGC includes two nonribosomal peptide synthetase (NRPS) and one polyketide synthase (PKS) gene, and a gene cassette (10 genes), of which the encoded enzymes share high homology to the ones responsible for 3-formamidosalicylate (3-FAS) biosynthesis in the antimycin biosynthetic pathway. Heterologous expression of the partial nat BGC without the 3-FAS gene cassette in the antimycin producer, Streptomyces albus J1074, results in the production of 1 and 2, suggesting that the nat BGC indeed directs NATs biosynthesis. Targeted in-frame deletion of the reductase gene ( natE) abolished the production of 1 and 2 but accumulated two NAT derivatives, the known NAT-H (3) and a new NAT-I (4). Biochemical verification demonstrated that the recombinant NatE indeed catalyzes an NADPH-dependent reaction of 3 or 4 to 1 or 2, respectively. Compound 3 presented significantly stronger activities against eight cancer cell lines than the ones using cisplatin, the clinical chemotherapy medicine. In particular, 3 displayed 559- and 57-fold higher activity toward human melanoma and cervix epidermoid carcinoma cells, respectively, compared with cisplatin. The new derivative, 4, was 1.5- to 10.9-fold more active than cisplatin toward five cancer cell lines. The evaluation of NATs biosynthesis depicted here will pave the way to generate new NAT derivatives through rational pathway engineering.