A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis

Wang, Tao; Yurkovich, Marie E.; Wen, Su; Lebe, Karen E.; Samborskyy, Markiyan; Yang, Anna; Liu, Y.; Ju, Young Seok; Deng, Zixin; Tosin, Manuela; Sun, Yuhui; Leadlay, Peter F.

doi:10.1039/c5sc03059e

Cited by 35 publications

(55 citation statements)

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“…[23] Leadlay and co-workers previously reported the gene inactivation of stuD1 and tlmD1 , two homologs of the CYP450-encoding gene tlmF , in two separate TLM-producing strains, that led to the elimination of TLM production without detecting any additional products. [18] Thus, our findings not only support the essential role of the CYP450 TlmF in the formation of the 5-membered thiolactone ring, but the identification of compound 5 further implicates that the insertion of sulfur into TLM is independent of TlmF.…”

supporting

confidence: 58%

“…Simultaneously, others identified similar BGCs in four different strains. [18] These findings revealed that 1 and its analogues are constructed via an unconventional PKS-NRPS hybrid assembly line, indicating an unusual biosynthetic logic at play. Here we report the exploration of the biosynthesis of the tlm BGC from S. pacifica CNS-863 through genetic and analytical strategies.…”

mentioning

confidence: 99%

“…While the mechanism for the desulfuration reaction has not yet been established, Leadlay and co-workers suggest that the sulfur atom is incorporated by the action of a cysteine desulfurase along with a sulfur transferase via primary metabolic reactions. [18] Following the generation of 6 , we envision that the competing routes, a or b , proceed divergently in the presence or absence of the CYP450 TlmF (Figure 3). In the presence of the TlmF P450, formation of the 5-membered TLM 1 may proceed via a number of oxidative routes, including an epoxide intermediate as shown via route a .…”

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confidence: 99%

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Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five‐Membered Thiolactone Ring Formation

Tang

Moore

2017

ChemBioChem

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Thiolactomycin (TLM) belongs to a class of rare and unique thiotetronate antibiotics inhibiting bacterial fatty acid synthesis. Although this group of natural product antibiotics was first discovered over 30 years, the study of TLM biosynthesis remains in its infancy. We recently discovered the biosynthetic gene cluster (BGC) for TLM from the marine bacterium Salinispora pacifica CNS-863. Here, we report the investigation of TLM biosynthetic logic through mutagenesis and comparative metabolic analyses. Our results reveal that only four genes (tlmF, tlmG, tlmH, and tlmI) are required for the construction of the characteristic γ-thiolactone skeleton of this class of antibiotics. We further showed that the cytochrome P450 TlmF does not directly participate in the sulfur insertion and C-S bond formation chemistry but rather in the construction of the 5-membered thiolactone ring, as upon its deletion, we observed the alternative production of the 6-membered δ-thiolactomycin. Our findings pave the way for future biochemical investigation of the biosynthesis of this structurally unique group of thiotetronic acid natural products.

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confidence: 99%

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Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five‐Membered Thiolactone Ring Formation

Tang

Moore

2017

ChemBioChem

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“…188,189 This ring, found at the C-terminus of the molecule, contains a sulfur atom that originates from the side chain of cysteine that is selected and activated by the nal module of the PKS/NRPS. Following this, the exact mechanism by which the sulfur is inserted into the molecule is unclear, although the role of the P450 is believed to centre on the epoxidation of the C4-C5 double bond (Fig.…”

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confidence: 99%

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

et al. 2018

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The cytochromes P450 (P450s) are a superfamily of heme-containing monooxygenases that perform diverse catalytic roles in many species, including bacteria. The P450 superfamily is widely known for the hydroxylation of unactivated C-H bonds, but the diversity of reactions that P450s can perform vastly exceeds this undoubtedly impressive chemical transformation. Within bacteria, P450s play important roles in many biosynthetic and biodegradative processes that span a wide range of secondary metabolite pathways and present diverse chemical transformations. In this review, we aim to provide an overview of the range of chemical transformations that P450 enzymes can catalyse within bacterial secondary metabolism, with the intention to provide an important resource to aid in understanding of the potential roles of P450 enzymes within newly identified bacterial biosynthetic pathways. 1.Introduction to P450s 2.Chemistry of P450 enzymes 3.Bacterial biosynthesis pathways involving P450s 3.1Terpene metabolism 3.1. Battersby (1925Battersby ( -2018 to the molecular understanding of biosynthesis over the course of their careers.

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“…However, an increasing number of PKS systems are now known in which the main product apparently requires iterative use of a module to accomplish two or even three successive rounds of chain extension. First noted in the stigmatellin PKS from Stigmatella aurantiaca [20], further examples have been uncovered in the PKSs for aureothin [21–22], borrelidin [23–24], lankacidin [25–26], neoaureothin [27], etnangien [28], crocacin [29], ebelactone [30] and thiolactomycin [31–32]. Given the close mechanistic analogy between fatty acid synthases and an iterative PKS module, it is instructive that a normally colinear extension module of the pikromycin PKS, when studied as a stand-alone protein in vitro, catalyses two rounds of polyketide chain extension [19].…”

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confidence: 99%

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

Hong¹,

Sun²,

Zhou³

et al. 2016

Beilstein J. Org. Chem.

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SummaryThe 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.

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A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis

Abstract: A novel mechanism is proposed for ring formation in the biosynthetic pathway to thiotetronate antibiotics thiolactomycin and Tü 3010.

Cited by 35 publications

References 58 publications

Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five‐Membered Thiolactone Ring Formation

Minimization of the Thiolactomycin Biosynthetic Pathway Reveals that the Cytochrome P450 Enzyme TlmF Is Required for Five‐Membered Thiolactone Ring Formation

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

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

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