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A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae.

ABSTRACT: Resorcylic acid lactones represent a unique class of fungal polyketides and display a wide range of biological activities, such as nanomolar inhibitors of Hsp90 and MAP kinase. The biosynthesis of these compounds is proposed to involve two fungal polyketide synthases (PKS) that function collaboratively to yield a 14-membered macrolactone with a resorcylate core. We report here the reconstitution of Gibberella zeae PKS13, which is the nonreducing PKS associated with zearalenone biosynthesis. Using a small molecule mimic of the natural hexaketide starter unit, we reconstituted the entire repertoire of PKS13 activities in vitro, including starter-unit selection, iterative condensation, regioselective C2-C7 cyclization, and macrolactone formation. PKS13 synthesized both natural 14-membered and previously uncharacterized 16-membered resorcylic acid lactones, indicating relaxed control in both iterative elongation and macrocyclization. PKS13 exhibited broad starter-unit specificities toward fatty acyl-CoAs ranging in sizes between C6 and C16 and displayed the highest activity toward decanoyl-CoA. PKS13 was shown to be active in Escherichia coli and synthesized numerous alkyl pyrones and alkyl resorcylic esters without exogenously supplied precursors. We demonstrated that PKS13 can interact with E. coli fatty acid biosynthetic machinery and can be primed with fatty-acyl ACPp at low-micromolar concentrations. PKS13 synthesized new polyketides in E. coli when the culture was supplemented with synthetic precursors, showcasing its utility in precursor-directed biosynthesis. PKS13 is therefore a highly versatile polyketide macrolactone synthase that is useful in the engineered biosynthesis of polyketides, including resorcylic acid lactones that are not found in nature.

SUBMITTER: Zhou H

PROVIDER: S-EPMC2359803 | biostudies-other | 2008 Apr

REPOSITORIES: biostudies-other

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A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae.

Zhou Hui H Zhan Jixun J Watanabe Kenji K Xie Xinkai X Tang Yi Y

Proceedings of the National Academy of Sciences of the United States of America 20080421 17

Resorcylic acid lactones represent a unique class of fungal polyketides and display a wide range of biological activities, such as nanomolar inhibitors of Hsp90 and MAP kinase. The biosynthesis of these compounds is proposed to involve two fungal polyketide synthases (PKS) that function collaboratively to yield a 14-membered macrolactone with a resorcylate core. We report here the reconstitution of Gibberella zeae PKS13, which is the nonreducing PKS associated with zearalenone biosynthesis. Usin ...[more]

PMID: 18427109

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Project description:BackgroundFor filamentous fungi, the basic growth unit of hyphae usually makes it sensitive to shear stress which is generated from mechanical force and dynamic fluid in bioreactor, and it severely decreases microbial productions. The conventional strategies against shear-sensitive conundrum in fungal fermentation usually focus on adapting agitation, impeller type and bioreactor configuration, which brings high cost and tough work in industry. This study aims to genetically shape shear resistant morphology of shear-sensitive filamentous fungus Aspergillus glaucus to make it adapt to bioreactor so as to establish an efficient fermentation process.ResultsHyphal morphology shaping by modifying polarized growth genes of A. glaucus was applied to reduce its shear-sensitivity and enhance aspergiolide A production. Degenerate PCR and genome walking were used to obtain polarized growth genes AgkipA and AgteaR, followed by construction of gene-deficient mutants by homologous integration of double crossover. Deletion of both genes caused meandering hyphae, for which, ΔAgkipA led to small but intense curves comparing with ΔAgteaR by morphology analysis. The germination of a second germ tube from conidiospore of the mutants became random while colony growth and development almost maintained the same. Morphology of ΔAgkipA and ΔAgteaR mutants turned to be compact pellet and loose clump in liquid culture, respectively. The curved hyphae of both mutants showed no remarkably resistant to glass bead grinding comparing with the wild type strain. However, they generated greatly different broth rheology which further caused growth and metabolism variations in bioreactor fermentations. By forming pellets, the ΔAgkipA mutant created a tank environment with low-viscosity, low shear stress and high dissolved oxygen tension, leading to high production of aspergiolide A (121.7 ± 2.3 mg/L), which was 82.2% higher than the wild type.ConclusionsA new strategy for shaping fungal morphology by modifying polarized growth genes was applied in submerged fermentation in bioreactor. This work provides useful information of shaping fungal morphology for submerged fermentation by genetically modification, which could be valuable for morphology improvement of industrial filamentous fungi.

Project description:Pseudocercospora fijiensis, causal agent of black Sigatoka of banana, produces polyketide synthase (PKS) pathways shown to be important in disease development by related Dothideomycete fungi. Genome analysis of the P. fijiensis PKS8-1 gene identified it as part of a gene cluster including genes encoding two transcription factors, a regulatory protein, a glyoxylase/beta-lactamase-like protein, an MFS transporter, a cytochrome P450, two aldo/keto reductases, a dehydrogenase, and a decarboxylase. Genome analysis of the related pathogens Pseudocercospora musae, Pseudocercospora eumusae, and Pseudocercospora pini-densiflorae, identified orthologous clusters containing a nearly identical combination of genes. Phylogenetic analysis of PKS8-1 identified homology to PKS proteins in the monodictyphenone and cladofulvin pathways in Aspergillus nidulans and Cladosporium fulvum, respectively. Analysis of clustered genes showed that the PKS8-1 cluster shares genes for enzymes involved in the production of the emodin intermediate in the monodictyphenone and cladofulvin pathways, but differs in many genes, suggesting production of a different metabolic product. Time course analysis of gene expression in infected banana showed up-regulation of PKS8-1 and four of eight clustered genes as early as 2 weeks post-inoculation and remaining high through 9 weeks. Overexpression of the pathway through constitutive expression of an aflR-like transcription factor gene in the cluster resulted in increased expression in culture of PKS8-1 as well as the four clustered genes that are up-regulated in infected plants. No differences were seen in timing or severity of disease symptoms with the overexpression strains relative to controls, however gene expression analysis showed no difference in expression in planta by an overexpression strain relative to controls. Thus constitutive expression of the aflR-like gene is not sufficient to upregulate the pathway above normal expression in planta. Pathway expression during all phases of disease development and conservation of the pathway in related Pseudocercospora species support a role for this pathway in disease.

Dataset Information

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae.

Publications

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae.

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