C-Nucleoside Formation in the Biosynthesis of the Antifungal Malayamycin A.

Malayamycin A is an unusual bicyclic C-nucleoside, with interesting antiviral, antifungal, and anticancer bioactivity. We report here the discovery and characterization of the biosynthetic pathway to malayamycin by using genome mining of near-identical clusters both from the known producer Streptomyces malaysiensis and from Streptomyces chromofuscus. The key precursor 5'-pseudouridine monophosphate (5'-Ψ-MP) is supplied chiefly through the action of MalD, a TruD-like pseudouridine synthase. In vitro assays showed that MalO is an enoylpyruvyltransferase acting almost exclusively on 5'-Ψ-MP rather than 5'-UMP, while in contrast the counterpart enzyme NikO in the nikkomycin pathway readily accepts either substrate. As a result, deletion of malD in S. chromofuscus coupled with introduction of the gene for NikO led to production of non-natural N-malayamycin, as well as malayamycin A. Conversely, cloning malO into the nikkomycin producer Streptomyces tendae in place of nikO diverted biosynthesis toward C-nucleoside formation.BBSRC Syngenta plc U

The polyketide backbone of thiolactomycin is assembled by an unusual iterative polyketide synthase

Following the in vivo investigation of thiotetronate assembly in Lentzea sp. and in S. thiolactonus NRRL 15439 (Havemann et al., Chem. Commun., 2017, DOI: 10.1039/c6cc09933e), the minimal set of genes required for thiolactomycin production was determined through heterologous expression and the mechanism for polyketide assembly was established in vitro through incubation of recombinant TlmB with its substrates in the presence of either nonhydrolysable or hydrolysable chemical probes. The results presented here constitute unequivocal evidence of enzymatic processing by an unusual iterative polyketide synthase

Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes.

Clethramycin from Streptomyces malaysiensis DSM4137, and mediomycins (produced together with clethramycin from Streptomyces mediocidicus), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific O-sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene (slf) encoding a candidate sulfotransferase has been located in both gene clusters. Deletion of this gene in DSM4137 led to accumulation of desulfoclethramycin only, instead of a mixture of desulfoclethramycin and clethramycin. The mediomycin gene cluster does not encode an amidinohydrolase, but when three candidate amidinohydrolase genes from elsewhere in the S. mediocidicus genome were individually expressed in Escherichia coli and assayed, only one of them (medi4948), located 670 kbp away from the mediomycin gene cluster on the chromosome, catalysed the removal of the amidino group from desulfoclethramycin. Subsequent cloning of medi4948 into DSM4137 caused mediomycins A and B to accumulate at the expense of clethramycin and desulfoclethramycin, respectively, a rare case where an essential biosynthetic gene is not co-located with other pathway genes. Clearly, both desulfoclethramycin and clethramycin are substrates for this amidinohydrolase. Also, purified recombinant sulfotransferase from DSM4137, in the presence of 3'-phosphoadenosine-5'-phosphosulfate as donor, efficiently converted mediomycin B to mediomycin A in vitro. Thus, in the final steps of mediomycin A biosynthesis deamidination and sulfotransfer can take place in either order

Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase.

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm

Broadening substrate specificity of a chain-extending ketosynthase through a single active-site mutation.

An in vitro model system based on a ketosynthase domain of the erythromycin polyketide synthase was used to probe the apparent substrate tolerance of ketosynthase domains of the mycolactone polyketide synthase. A specific residue change was identified that led to an emphatic increase in turnover of a range of substrates.BBSRC (BB/J007250/1)This is the final version of the article. It first appeared from Royal Society of Chemistry] via https://doi.org/10.1039/C6CC03501A

Uncovering the origin of Z-configured double bonds in polyketides: intermediate E-double bond formation during borrelidin biosynthesis

Formation of Z-configured double bonds in reduced polyketides is uncommon and their origins have not been extensively studied. To investigate the origin of the Z-configured double bond in the macrolide borrelidin, the recombinant dehydratase domains BorDH2 and B0rDH3 were assayed with a synthetic analogue of the predicted tetraketide substrate. The configuration of the dehydrated products was determined to be E in both cases by comparison to synthetic standards. Detailed NMR spectroscopic analysis of the biosynthetic intermediate pre-borrelidin confirmed the E,E-configuration of the fulllength polyketide synthase product. In contrast to a previously-proposed hypothesis, our results show that in this case the Z-configured double bond is not formed via dehydration from a 3 L-configured precursor, but rather as the result of a later isomerization process.Marie Curie programme of the European UnionEmmy Noether programme of the Deutsche ForschungsgemeinschaftDAA

Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.

Conglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assembly line. The cloned cluster directs conglobatin biosynthesis in a heterologous host strain. Using a model substrate to mimic the conglobatin monomer, we also show that the conglobatin cyclase/thioesterase acts iteratively, ligating two monomers head-to-tail then re-binding the dimer product and cyclizing it. Incubation of two different monomers with the cyclase produces hybrid dimers and trimers, providing the first evidence that conglobatin analogs may in future become accessible through engineering of the polyketide synthase.We gratefully acknowledge BBSRC (project grant BB/J007250/1 to P.F.L.), the European Commission (Marie Curie Fellowship to Y.Z.), and the University of Cambridge (Herchel Smith Research Fellowship to A.C.M.), and Ms. Asha Boodhun (Department of Chemistry, University of Cambridge) for help in HR-MS analysis. L.C.D. acknowledges the support of Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Proc. 2012/04616-3 and 2012/02230-0). P.F.L. is an International Research Awardee of the Alexander von Humboldt Foundation.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.chembiol.2015.05.01