Filamentous fungi are a rich source of agriculturally and pharmaceutically important natural products. For example, the fungal polyketide statins, such as lovastatin, are among the most widely-prescribed drugs for the prevention and treatment of atherosclerosis by inhibiting cholesterol biosynthesis. Fungal polyketides belonging to the resorcylic acid lactone family exhibit potent antiproliferative activities as selective kinase inhibitors. Iterative fungal polyketide synthases (PKSs) use a unique set of biochemical rules in the synthesis of complex polyketides. These rules dictate polyketide starter unit selection, chain length control, and post-PKS processing. While the biosynthetic origins of bacterial polyketides have been studied extensively and have led to the combinatorial biosynthesis of pharmaceutically important ?unnatural?natural products, the biosynthetic mechanisms of fungal PKSs are not well understood and their potential for combinatorial biosynthesis has not yet been realized. This is largely due to difficulties associated with manipulating these megasynthases in their native or related fungal hosts, and with obtaining intact enzymes for biochemical analysis. The objective of this proposal is to bridge these important knowledge and technical gaps and provide a multi- angled picture of the fungal polyketide biosynthesis employing the workhorse organism Escherichia coli. We have obtained extensive preliminary biochemical data on the expression, reconstitution and engineering of PKS4 from Gibberella fujikuroi (gfPKS4) and PKS13 from Gibberella zeae (gzPKS13) using E. coli as the heterologous host. This proposal will evaluate the following hypotheses: 1) Fungal PKS megasynthases can be functionally reconstituted in a bacterial host, such as E. coli;2) Fungal PKS contains initiation and cyclization domains that can be exploited for combinatorial biosynthesis;3) Fungal and bacterial catalytic components can be catalytically integrated towards the synthesis of novel polyketides. To address these hypotheses in a five-year period, we have defined the following three SPECIFIC AIMS: 1) Biochemical Characterization of gfPKS4 and gzPKS13 Initiation Domains;2) Biochemical Characterization of gfPKS4 and gzPKS13 Cyclization Domains and 3) Catalytic Integration of fungal and bacterial PKSs. Project Narrative We have proposed biochemical and metabolic engineering studies to investigate fungal polyketide synthases. Fungal polyketide synthases are iterative megasynthases that catalyze the biosynthesis of a number of biological active compounds, including those that are anticancer and antihypercholesterolemia. We will use the robust heterologous host Escherichia coli to study the initiation, elongation, termination and cyclization steps of the intact synthases. Knowledge gained from these studies will be valuable in the engineering of these enzymes towards synthesis of novel compounds both in vivo and in vitro.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085128-05
Application #
8269735
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Gerratana, Barbara
Project Start
2008-08-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
5
Fiscal Year
2012
Total Cost
$291,527
Indirect Cost
$72,324
Name
University of California Los Angeles
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Cochrane, Rachel V K; Sanichar, Randy; Lambkin, Gareth R et al. (2016) Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent. Angew Chem Int Ed Engl 55:664-8
Sato, Michio; Winter, Jaclyn M; Kishimoto, Shinji et al. (2016) Combinatorial Generation of Chemical Diversity by Redox Enzymes in Chaetoviridin Biosynthesis. Org Lett 18:1446-9
Bond, Carly; Tang, Yi; Li, Li (2016) Saccharomyces cerevisiae as a tool for mining, studying and engineering fungal polyketide synthases. Fungal Genet Biol 89:52-61
Cacho, Ralph A; Tang, Yi (2016) Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro. Methods Mol Biol 1401:103-19
Gao, Shu-Shan; Duan, Abing; Xu, Wei et al. (2016) Phenalenone Polyketide Cyclization Catalyzed by Fungal Polyketide Synthase and Flavin-Dependent Monooxygenase. J Am Chem Soc 138:4249-59
Winter, Jaclyn M; Cascio, Duilio; Dietrich, David et al. (2015) Biochemical and Structural Basis for Controlling Chemical Modularity in Fungal Polyketide Biosynthesis. J Am Chem Soc 137:9885-93
Mao, Xu-Ming; Zhan, Zha-Jun; Grayson, Matthew N et al. (2015) Efficient Biosynthesis of Fungal Polyketides Containing the Dioxabicyclo-octane Ring System. J Am Chem Soc 137:11904-7
Cochrane, Rachel V K; Gao, Zhizeng; Lambkin, Gareth R et al. (2015) Comparison of 10,11-Dehydrocurvularin Polyketide Synthases from Alternaria cinerariae and Aspergillus terreus Highlights Key Structural Motifs. Chembiochem 16:2479-83
Sato, Michio; Yagishita, Fumitoshi; Mino, Takashi et al. (2015) Involvement of Lipocalin-like CghA in Decalin-Forming Stereoselective Intramolecular [4+2] Cycloaddition. Chembiochem 16:2294-8
Cacho, Ralph A; Thuss, Justin; Xu, Wei et al. (2015) Understanding Programming of Fungal Iterative Polyketide Synthases: The Biochemical Basis for Regioselectivity by the Methyltransferase Domain in the Lovastatin Megasynthase. J Am Chem Soc 137:15688-91

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