Rational Domain Swaps Decipher Programming in Fungal Highly Reducing Polyketide Synthases and Resurrect an Extinct Metabolite

Fisch, Katja M.; Bakeer, Walid; Yakasai, Ahmed A.; Song, Zhiyong; Wasil, Zahida; Bailey, Andy M.; Lazarus, Colin M.; Simpson, Thomas J.; Cox, Russell J.

doi:10.1021/ja206914q

Cited by 125 publications

(116 citation statements)

References 34 publications

Supporting

Mentioning

113

Contrasting

Order By: Relevance

“…Transformation of A. oryzae and T. stipitatus, knockout of T. stipitatus genes, and heterologous expression in A. oryzae were conducted using literature procedures (17,43). Strains were fermented in standard production media and extracted using EtOAc and extracts were analyzed by LCMS using a Waters 2767 HPLC linked to a Waters ZQ mass spectrometer.…”

Section: Methodsmentioning

confidence: 99%

Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis

Davison

Fahad

Cai

et al. 2012

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

Self Cite

158

167

View full text Add to dashboard Cite

A gene cluster encoding the biosynthesis of the fungal tropolone stipitatic acid was discovered in Talaromyces stipitatus ( Penicillium stipitatum ) and investigated by targeted gene knockout. A minimum of three genes are required to form the tropolone nucleus: tropA encodes a nonreducing polyketide synthase which releases 3-methylorcinaldehyde; tropB encodes a FAD-dependent monooxygenase which dearomatizes 3-methylorcinaldehyde via hydroxylation at C-3; and tropC encodes a non-heme Fe(II)-dependent dioxygenase which catalyzes the oxidative ring expansion to the tropolone nucleus via hydroxylation of the 3-methyl group. The tropA gene was characterized by heterologous expression in Aspergillus oryzae , whereas tropB and tropC were successfully expressed in Escherichia coli and the purified TropB and TropC proteins converted 3-methylorcinaldehyde to a tropolone in vitro. Finally, knockout of the tropD gene, encoding a cytochrome P450 monooxygenase, indicated its place as the next gene in the pathway, probably responsible for hydroxylation of the 6-methyl group. Comparison of the T. stipitatus tropolone biosynthetic cluster with other known gene clusters allows clarification of important steps during the biosynthesis of other fungal compounds including the xenovulenes, citrinin, sepedonin, sclerotiorin, and asperfuranone.

show abstract

Section: Methodsmentioning

confidence: 99%

Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis

Davison

Fahad

Cai

et al. 2012

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

Self Cite

158

167

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

Rational reprogramming of fungal polyketide first-ring cyclization

Zhou

et al. 2013

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

115

View full text Add to dashboard Cite

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.

show abstract

“…8 This similarity even extends to the position of a CMeT domain which is inactive in vFAS, but which acts during the first and second rounds of chain processing by SQTKS. 9 vFAS produces fully saturated linear 16-18 carbon chains, whereas SQTKS produces the dimethylated and unsaturated 8-carbon chain 3. SQTKS thus displays a complex programme in which the activities of the individual catalytic domains can be varied (Scheme 1).…”

Section: R-2-methyl-3-hydroxybutyryl Snac 2r3r-8 Was a Substrate Cmentioning

confidence: 99%