Characterization of the Biosynthetic Pathway of Fungal Aromatic Polyketides

212 Pages·2012·7.38 MB·English

by  Yanran Li

UCLA
UCLA Electronic Theses and Dissertations
Title
Characterization of the Biosynthetic Pathway of Fungal Aromatic Polyketides
Permalink
https://escholarship.org/uc/item/1kn4q35x
Author
Li, Yanran
Publication Date
2012

Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA
Los Angeles

Characterization of the Biosynthesis of Fungal Aromatic
Polyketides

A dissertation submitted in partial satisfaction of the requirements for the degree Doctor
of Philosophy in Chemical Engineering

By

Yanran Li

2012

ABSTRACT OF THE DISSERTATION

Characterization of the Biosynthetic Pathway of Fungal Aromatic Polyketides

By

Yanran Li
Doctor of Philosophy in Chemical Engineering
University of California, Los Angeles, 2012
Professor Yi Tang, Chair

Nature has endowed microbes and plants with enormous power to synthesize complex
natural products from simple building blocks. Compounds derived from natural sources that
show biological activities are defined as natural products. Over the past century, natural
products have played an indispensible role in the pharmaceutical discovery, and have become an
important element of modern therapy. Aromatic polyketides are an important group of natural
products that exhibit extraordinary medicinal activities. Actinomycetes and filamentous fungi
are the two major producers of aromatic polyketides, especially polycyclic aromatic polyketides;
and they employ distinct enzymatic system to synthesize these complex structures from malonyl-
CoA. Fungi utilize iterative multidomain megasynthase of type I nonreducing polyketide
synthases (NRPKSs), while actinomycetes employ bacterial type II PKSs that comprise a group
of dissociated enzymes. Compared to the well-studied type II PKS, NRPKS has been much less
understood.
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In this study, we first identified the unique product template (PT) domain of NRPKS that
is not shared among the other PKS families as the first ring aldol cyclase. Based on the
understanding of PT domain function, we performed phylogenetic analysis of PT domains,
which classified the NRPKSs into five major groups. Subsequently, we proposed a sequence-
function correlation of PT domain that allowed us to predict the activities of unknown PTs
uncovered from genome sequencing. These target PT domains were assayed with the PKS4 (a
previously identified NRPKS) minimal PKS domains in trans or in cis, and the proposed PT
functions were confirmed through such product based assays. According to the established
sequence-function relationship, we conducted genome mining of A. niger for the biosynthetic
locus of TAN-1612, which contains an interesting tetracyclic naphthacenedione core structure.
Subsequent investigations of the TAN-1612 biosynthetic pathway uncovered a novel α-
hydroxylation-dependent Claisen cyclization cascade that involves a bifunctional dimanganese
metallo-β-lactamase-type thioesterase (MβL-TE). Also, the identification of the TAN-1612
biosynthetic pathway revealed a decaketide synthase AdaA, which enabled us to realize the in
vitro enzymatic total synthesis of several important aromatic decaketide intermediates including
rabelomycin and tetracenomycin D1.

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The dissertation of Yanran Li is approved.

Daniel T. Kamei

James C. Liao

Harold Monbouquette

Yi Tang, Committee Chair

University of California, Los Angeles
2012

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TABLE OF CONTENTS
1 Background and Motivation…………………………………………………….. 1
1.1 Cyclization of Bacterial Aromatic Polyketides…………………………. 4
1.1.1 The First Ring Cyclization………………………………………… 4
1.1.2 Additional Cyclization Steps……………………………………… 11
1.2 Cyclization of Fungal Aromatic Polyketides…………………………… 19
1.2.1 Product Template Domain Mediated Cyclization………………… 20
1.2.2 Thioesterase Domain Mediated Cyclization……………………… 24
2 Results…………………………………………………………………….……… 26
2.1 Identification of PT Domain Function and Engineered Biosynthesis of
Bacteroides Aromatic Polyketides in Escherichia coli ………………... 26
2.1.1 Introduction……………………………………………….……… 26
2.1.2 Results and Discussion…………………………………………… 29
2.1.3 Conclusion………………………………………………………… 42
2.1.4 Materials and Methods……………………………………………. 42
2.2 Classification, Prediction, and Verification of the Regioselectivity of Fungal
Polyketide Synthase Product Template Domains …………………….. 47
2.2.1 Introduction………………………………………………………. 48
2.2.2 Results...………………………………………………………….. 52
2.2.3 Discussion………………………………………………………… 62
2.2.4 Materials and Methods…………………………………………… 65
2.3 Comparative Characterization of Fungal Anthracenone and
Naphthacenedione Biosynthetic Pathways Reveals an α-Hydroxylation-
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Dependent Claisen-Like Cyclization Catalyzed by a Dimanganese
Thioesterase……………………………………………………………… 69
2.3.1 Introduction………………………………………………………. 69
2.3.2 Results……………………………………………………………. 73
2.3.3 Discussion………………………………………………………… 87
2.3.4 Conclusion……………………………………………………….. 94
2.3.5 Materials and Methods…………………………………………… 95
2.4 Enzymatic Total Synthesis of Aromatic Decaketides in vitro………… 100
2.4.1 Introduction……………………………………………….……… 100
2.4.2 Results and Discussion…………………………………………… 101
2.4.3 Conclusions………………………………………………………. 106
2.4.4 Materials and Methods…………………………………………… 107
3 Conclusions……………………………………………………………………… 111
4 Appendix…………………………………………………………………………. 115
4.1 Compound List………………………………………………………….. 115
4.2 Supporting information for chapter 2.1……………………………….. 117
4.3 Supporting information for chapter 2.2……………………………….. 123
4.4 Supporting information for chapter 2.3……………………………….. 139
4.5 Supporting information for chapter 2.4……………………………….. 176
5 References……………………………………………………………………….. 184

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LIST OF FIGURES
Figure 1:Comparison between bacterial and fungal aromatic PKSs…………………….. 2

Figure 2: Biosynthesis of tetracyclines and aureolic acids………………………………. 12
Figure 3: Biosynthesis of tetracenomycins………………………………………………. 15
Figure 4: Biosynthesis of angucyclines………………………………………………….. 16
Figure 5: Biosynthesis of 50 by PKS4…………………………………………………… 22
Figure 6: Biosynthesis of 53 by PksA……………………………………………………. 23
Figure 7: Comparison between fungal and bacterial iterative polyketide synthases…….. 27
Figure 8: Construction of PKS4 minimal PKS domains without the PT domain………… 30
Figure 9: (A) HPLC analysis (280nm) of minimal PKS activities in vitro. (B) LC-MS analysis
and selected ion monitoring of nonaketides produced by KS-MAT+ACP and PKS_WJ in
vitro……………………………………………………………………………………….. 31
Figure 10: UV and mass spectrum of 15 and 51; and two possible cyclization patterns to afford
51…………………………………………………………………………………………. 33
Figure 11: (A) PKS_WJ exhibited comparable kinetic properties as the parent PKS4. (B)
Michaelis-Menten properties of the dissociated KS-MAT didomain (3µM) toward the ACP
domain at 2mM malonyl-CoA…………………………………………………………… 34
Figure 12: Starter unit selection by PKS_WJ…………………………………………… 36
Figure 13: (A) HPLC analysis (280nm) of reaction mixtures. (B) (i) C7-C12 cyclized
polyketides produced by bacterial minimal PKSs alone without aid of cyclases. (ii) Natural
aromatic polyketides with C7-C12-cyclized first ring. (C) Cyclization of the nonaketide
backbone to yield 7………………………………………………………………………. 38
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Figure 14: (A) HPLC analysis (430nm) of SEK26 63 biosynthesis by using E. coli BAP1. (B)
High-cell density, feb-batch fermentation production of 63……………………………… 41
Figure 15: (A) Biosynthesis of fungal aromatic polyketides. (B) C6-C11/C4-C13 cyclization
regioselectivity has been observed in various fungal aromatic polyketide………………. 51
Figure 16: Phylogenetic analysis of PT domains from 30 NRPKSs that have been related to
known aromatic polyketides……………………………………………………………… 53
Figure 17: (A) Construction and SDS-PAGE analysis of PKSH1. (B) LC-MS (325nm) analysis
of the polyketides synthesized by PKSH1 in vitro……………………………………….. 57
Figure 18: The biosynthesis of 68 and the 1H-13C HMBC correlations of 68…………… 59
Figure 19: (A) Construction and SDS-PAGE analysis of PKSH2. (B) LC-MS (325 nm) analysis
of the polyketides synthesized by PKSH2 in vitro……………………………………….. 61
Figure 20: (A) VrtA has been related to the biosynthesis of viridicatumtoxin in P. aethiopicum.
(B) LC-MS (325 nm) analysis of the polyketides synthesized by PKS4 minimal PKS domains
with AptA-PT (trace i) and VrtA-PT (trace ii) in vitro…………………………………… 62
Figure 21: Biosynthetic pathway of 65 and related shunt products……………………… 72
Figure 22: Induction of the ada gene cluster…………………………………………….. 75
Figure 23: LC analysis (400 nm) of polyketides synthesized by coexpression of different
combinations of ada and apt genes in S. cerevisiae BJ5464-NpgA……………………… 78
Figure 24: Biosynthetic pathway of 70 and related shunt products……………………… 81
Figure 25: In vitro reconstitution of AptB activities……………………………………... 83
Figure 26: Structural modeling and mutational analysis of AptB……………………….. 87
Figure 27: Proposed enzymatic mechanism of MβL-TE………………………………… 89
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