Elucidation of natural product biosynthetic pathways provides important insights about the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here we show full reconstitution in vitro of an unusual multi-step oxidative cascade for post-assembly line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and an FAD-dependent oxidase (TamL), which act co-dependently through repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a heretofore unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C-10 oxidation of TirE.

The novel dienoyl tetramic acids tirandamycin C (1) and tirandamycin D (2) with activity against vancomycin-resistant Enterococcus faecalis (VRE) were isolated from the marine environmental isolate Streptomyces sp. 307-9, which also produces the previously identified compounds tirandamycin A (3) and B (4). Spectroscopic analysis of 1 and 2 indicated structural similarity to 3 and 4, with differences only in the pattern of pendant oxygenation on the bicyclic ketal system. The isolation of these putative biosynthetic intermediates was enabled by their sequestration on an adsorbent resin during early stationary-phase fermentation.Tetramic acids comprise a growing set of natural products containing a 2,4-pyrrolidinedione ring system formed from the condensation of an amino acid to a polyketide-derived acyl chain ( Figure 1). 1 These compounds continue to generate significant interest due to their broad structural diversity and breadth of biological activities, with representative examples including the HIV-1 integrase inhibitor equisetin, 2 the mycotoxin cyclopiazonic acid, 3 and the first discovered tetramic acid antibiotic streptolydigin. 4,5 Structurally similar to streptolydigin, the antibiotic tirandamycin A (3) was first isolated from the culture broth of the terrestrial bacterium Streptomyces tirandis in 1971, 6 and again in 1976 from Streptomyces flaveolus along with tirandamycin B (4). 7 These compounds exhibited antimicrobial activity against Grampositive bacteria, and in vitro activity against bacterial RNA polymerase. 8,9 The related compounds tirandalydigin, 10 BU-2313, 11 and nocamycin II 12 have since been characterized, all of which share a bicyclic ketal system and dienoyl tetramic acid moiety characteristic of streptolydigin and tirandamycin. Interest in the biosynthesis of these compounds prompted feeding studies in the streptolydigin system, the results of which support a hybrid polyketidenonribosomal peptide origin. 13 The biogenesis of these compounds would be further informed by the identification of pathway intermediates, but to date these efforts have been quite limited. We report here the use of established resin capture methods to isolate and identify the two new natural products tirandamycin C (1) and tirandamycin D (2) presumed to be key biosynthetic intermediates in the pathway to 3 and 4.During a screen to discover new natural products from marine-derived actinomycetes with activity against vancomycin-resistant Enterococcus faecalis (VRE), we isolated from marine sediments a bacterium of Streptomyces-like morphology designated as strain Streptomyces sp. 307-9. Analysis of extracts from shake-flask fermentation identified the major anti-VRE * To whom correspondence should be addressed: . davidhs@umich.edu † These authors contributed equally to this work. Supporting Information Available: 1 H, COSY, HSQC, HMBC spectra for 1-3, NOESY of 1, 1 H and 13 C of 4; maps of 2D NMR correlations; anti-VRE bioassay data, representative HPLC chromatograms of tirandamycin metabolite profiles; ...

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyltetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307-9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid-polyketide synthase-nonribosomal peptide synthetase gene cluster with a colinear domain organization entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any postassembly line oxidative modifications.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

More pieces for the puzzle. The β‐methylaspartate‐α‐decarboxylase (CrpG) from the cryptophycin biosynthetic pathway was cloned, over‐expressed, and purified. We found that CrpG decarboxylates (2S,3R)‐3‐methylaspartic acid to form 3‐amino‐2(R)‐methylpropionic acid, which is subsequently incorporated into Unit C of cryptophycins (see scheme).