Analysis of Intact and Dissected Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase in Vitro and in Saccharomyces cerevisiae

The widely found fungal iterative PKS-NRPS hybrid megasynthetases are highly programmed biosynthetic machines involved in the synthesis of 3-acyltetramic acids and related natural products. In vitro analysis of iterative PKS-NRPS has been hampered by the difficulties associated with obtaining pure a...

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Bibliographic Details
Published inJournal of the American Chemical Society Vol. 132; no. 39; pp. 13604 - 13607
Main Authors Xu, Wei, Cai, Xiaolu, Jung, Michael E., Tang, Yi
Format Journal Article
LanguageEnglish
Published WASHINGTON American Chemical Society 06.10.2010
Amer Chemical Soc
Subjects
Aspergillus nidulans - enzymology
Biological Factors - biosynthesis
Biological Factors - chemistry
Chemistry
Chemistry, Multidisciplinary
Molecular Structure
Peptide Synthases
Physical Sciences
Polyketide Synthases - chemistry
Polyketide Synthases - genetics
Polyketide Synthases - metabolism
Protein Engineering
Saccharomyces cerevisiae - genetics
Science & Technology
Stereoisomerism
CYCLOPIAZONIC ACID
ASPERGILLUS-NIDULANS
BIOSYNTHESIS
ESCHERICHIA-COLI
GENES
CLUSTER
IDENTIFICATION
EXPRESSION
RECONSTITUTION
PKS-NRPS
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Summary:The widely found fungal iterative PKS-NRPS hybrid megasynthetases are highly programmed biosynthetic machines involved in the synthesis of 3-acyltetramic acids and related natural products. In vitro analysis of iterative PKS-NRPS has been hampered by the difficulties associated with obtaining pure and functional forms of these large enzymes (>400 kDa). We successfully expressed Aspergillus nidulans aspyridone synthetase (ApdA) from an engineered Saccharomyces cerevisiae strain. The complete functions of ApdA and its enoylreductase partner ApdC are reconstituted in vitro and in S. cerevisiae with the production of preaspyridone 7. The programming rules of both the PKS and NRPS modules were then examined in vitro. The key interaction between the PKS and the NRPS was dissected and reconstituted in trans by using stand-alone modules. Analogs of 7 were synthesized through heterologous combinations of PKS and NRPS modules from different sources. Our results represent one of the largest, multidomain enzyme reconstituted to date and offer new opportunities for engineered biosynthesis of fungal natural products.
Bibliography:NIH RePORTER
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ISSN:0002-7863
1520-5126
DOI:10.1021/ja107084d