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

Abstract

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.

Biosynthesis of depsipeptides with a 3-hydroxybenzoate moiety and selective anticancer activities involves a chorismatase

“…We previously established a heterologous system to produce NAT-A (5) and -F (6) in S. albus J1074 by incorporation of a phiC31 integrative plasmid, pRJ4, containing natA-E genes ( Fig. 4b and d) (2). The metabolite profile of pRJ4/J1074 reveals no production of 1-4, suggesting that no functional 3-hydroxybenzoate synthase homolog exists in J1074.…”

“…The nat-hyg5 gene was amplified from the genomic DNA of RJ2 with the primers Hyg5-S and Hyg5-A (Table S1) and was cloned under the strong constitutive promoter ermE*p in ppYJ10, which contains a Streptomyces replication element derived from pIJ101 (27). Through conjugation, the resultant plasmid pRJ71 was used to transform the strain pRJ4/J1074, which produces compounds 5 and 6 with genes natA-E cloned in the plasmid pRJ4 (2). The resultant strain pRJ71+pRJ4/J1074, which is resistant to both thiostrepton and apramycin, was identified from among the exconjugants by colony PCR with the primers C-ppYJ10-S and C-ppYJ10-A (Table S1).…”

“…The majority of NATs show excellent anticancer activities, e.g. NAT-A, -F and -H have IC 50 0.1 -0.8 µM against human ovarian cancer cells (2), and IC 50 0.2 -0.6 µM against human colon cancer cells (3), respectively. In molecular pharmacological studies, the NAT analogs prunustatin A, JBIR-04, and JBIR-05 have been shown to down-regulate GRP-78, a molecular chaperone contributing to chemotherapy resistance (4,5).…”

“…The macrolide skeleton of NATs is generated from a NRPS-PKS multienzyme assembly line by loading the starter unit of 3-FAS and the elongation units of one L-threonine, three α-keto acids, and one malonic acid (2, 9, and 10). The offloaded 15-membered macrocyclic rings are converted into the final NATs via C1 hydroxylation conducted by a NAD(P)H-dependent ketoreductase (2).…”

Leveraging synthetic biology for producing bioactive polyketides and non-ribosomal peptides in bacterial heterologous hosts

“…134 The BGCs producing neoantimycins from Streptomyces conglobatus and Streptomyces orinoci were recently characterized through heterologous expression of the clusters in S. albus. 135,136 Heterologous expression of the BGC from S. conglobatus yielded a novel derivative of neoantimycin that was up to 10 times more active toward cancer cell lines than cisplatin, a common anti-cancer drug. Cas9-assisted genome editing was used to introduce part of the neoantimycin BGC from S. orinoci into a related antimycin BGC on the chromosome of S. albus to generate a chimeric pathway that produces several neoantimycin derivatives at levels comparable to S. orinoci.…”