Nongenetic Reprogramming of a Fungal Highly Reducing Polyketide Synthase

Yakasai, Ahmed A.; Davison, Jack; Wasil, Zahida; Halo, Laura M.; Butts, Craig P.; Lazarus, Colin M.; Bailey, Andy M.; Simpson, Thomas J.; Cox, Russell J.

doi:10.1021/ja204200x

Cited by 50 publications

(42 citation statements)

References 35 publications

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“…In contrast, genetic analyses of the biosynthetic pathway of the 2-pyridone tenellin revealed that this compound does not appear to contribute to B. bassiana virulence (40,41). This system also has been exploited for novel compound discovery, with expression of the tenellin nonribosomal peptide synthase in Aspergillus oryzae leading to the production of several new compounds and the use of chemical epigenetic modifiers in B. bassiana altering the selectivity of tenellin polyketide synthase to produce different products (42,43).…”

Section: Discussionmentioning

confidence: 99%

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Fan

Liu

Keyhani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

124

119

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The regulatory network and biological functions of the fungal secondary metabolite oosporein have remained obscure. Beauveria bassiana has evolved the ability to parasitize insects and outcompete microbial challengers for assimilation of host nutrients. A novel zinc finger transcription factor, BbSmr1 (B. bassiana secondary metabolite regulator 1), was identified in a screen for oosporein overproduction. Deletion of Bbsmr1 resulted in up-regulation of the oosporein biosynthetic gene cluster (OpS genes) and constitutive oosporein production. Oosporein production was abolished in double mutants of Bbsmr1 and a second transcription factor, OpS3, within the oosporein gene cluster (ΔBbsmr1ΔOpS3), indicating that BbSmr1 acts as a negative regulator of OpS3 expression. Real-time quantitative PCR and a GFP promoter fusion construct of OpS1, the oosporein polyketide synthase, indicated that OpS1 is expressed mainly in insect cadavers at 24-48 h after death. Bacterial colony analysis in B. bassiana-infected insect hosts revealed increasing counts until host death, with a dramatic decrease (∼90%) after death that correlated with oosporein production. In vitro studies verified the inhibitory activity of oosporein against bacteria derived from insect cadavers. These results suggest that oosporein acts as an antimicrobial compound to limit microbial competition on B. bassiana-killed hosts, allowing the fungus to maximally use host nutrients to grow and sporulate on infected cadavers.Beauveria bassiana | oosporein | biological role | transcription factor | fungal-bacterial competition

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Section: Discussionmentioning

confidence: 99%

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Fan

Liu

Keyhani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

124

119

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“…There is no obvious remnants of an inactive enoylreductase (ER°) domain or a methyltransferase (CMeT) domain, as seen in several iterative fungal PKS-NRPS with the similar organization, such as PKS for tenellin, lovastain, and compactin. [24-27] . Although this type of iterative PKS-NRPS is commonly seen in fungi, it is not common in bacteria until recent years.…”

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confidence: 99%

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Chen

Ding

et al. 2014

Angew Chem Int Ed

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Antifungal HSAF (heat‐stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single‐module polyketide synthase–nonribosomal peptide synthetase (PKS‐NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two “clean” strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl‐coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene–PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS‐NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide–peptide natural products.

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“…Fungal iPKSs may be classified into three subgroups (5). Highly reducing iPKSs (hrPKSs) generate complex linear or nonaromatic cyclic products by reducing the nascent β-ketoacyl intermediates to the β-alcohol, the alkene, or the alkane after each condensation step using their ketoreductase, dehydratase, and enoyl reductase domains to execute a cryptic biosynthetic program (2,(6)(7)(8). Partially reducing iPKSs omit enoyl reduction to generate simple cyclic structures (5).…”

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confidence: 99%

Rational reprogramming of fungal polyketide first-ring cyclization

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

115

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Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

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Nongenetic Reprogramming of a Fungal Highly Reducing Polyketide Synthase

Cited by 50 publications

References 35 publications

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Rational reprogramming of fungal polyketide first-ring cyclization

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