Biosynthesis - Aromatic Polyketides, Isoprenoids, Alkaloids; Volume 209 of Topics in Current Chemistry - Springer

247 Pages·2016·3.06 MB·English

by  Leeper F.J.

Preface
The present book is the second of two volumes that provide state of the art
expert reviews of central topics in modern natural products chemistry and
secondary metabolism.Using specific examples,the previous volume emphasiz-
ed two revolutions in experimental techniques that completely transformed the
field ofnatural products chemistry from what it was in the 1950s.These were the
use ofstable isotopes in conjunction with modern NMR and mass spectrometry,
and more recently, the development of molecular biological techniques to
identify, purify and manipulate the enzymes responsible for the intricate
series ofsteps to complex natural compounds.The previous volume specifically
covered the use of isotopes in biosynthetic research and the formation of
enzyme cofactors,vitamin B and reduced polyketides.
12
This second volume describes the application ofthe same approaches (isotope
methodology and molecular biology) to the biosynthesis ofaromatic (unreduced)
polyketides,enzymes responsible for cyclization ofterpenoids (isoprenoids),and
biochemical generation of selected classes of alkaloids (prenylated tryptophan,
tropane,pyrrolizidine).The knowledge ofthe metabolic pathways and the tech-
niques to elucidate them opens the door to combinatorial biosynthesis as well as
to the production of targeted pharmaceutical agents utilizing a combination of
chemistry,molecular biology and protein biochemistry.Recent advances suggest
that it may soon be possible to rationally manipulate biochemical pathways to
produce any target molecule, including non-natural variants, in substantial
quantity.Genetically modified organisms containing mix-and-match combina-
tions of biosynthetic enzymes (natural and/or mutated) will allow formation of
large numbers ofnew compounds for biological evaluation.In addition,the avail-
ability of vast arrays of specialized enzymes in pure form may provide new
reagents for combinatorial chemistry and parallel synthesis in drug discovery
programs.
In the current volume,Ben Shenbegins with a review ofthe assembly ofun-
reduced polyketides leading to aromatic compounds.The current understand-
ing ofthe functions and interactions ofthe enzymes involved is gradually pro-
viding the rules for designing new compounds ofthis class as well as affording
a basis for biosynthesis of flavonoids via chalcone synthases. In chapter 2,
Edward Davisand Rodney Croteaudescribe the terpenoid synthases responsible
for formation of the huge array of mono-,sesqui- and diterpenes (over 30,000
terpene derivatives are known).In particular,detailed structural and functional
evaluation of four representative terpene synthases is provided. In the third
VIII Preface
chapter,Robert Williams,Emily Stocking and Juan Sanz-Cerverareview the bio-
synthesis of prenylated indole alkaloids and related substances derived from
tryptophan.Many ofthese compounds are potent mycotoxins that contaminate
food,but some,such as the ergot alkaloids (e.g.ergotamine,ergonovine) see
extensive application in medicine.In chapter 4 Thomas Hemscheidt describes
the current state ofknowledge on the biosynthesis oftropane and related alka-
loids,including cocaine.This well illustrates the difficulties that can be faced in
elucidating the sequence of reactions involved in a biosynthetic pathway,
especially when the intermediates are unstable and produced in very low
amounts.The last chapter,by Thomas Hartmann covers the pyrrolizidine alka-
loids.It not only describes the chemistry of the biosynthetic pathway but also
gives an account ofthe physiology and ecology involved in the distribution and
elaboration ofthe alkaloids within the producing plant,in the insects which eat
the plants,and even in the animals which eat the insects.This shows us that for
many natural products an understanding ofhow they are made is only a part of
the whole story.
Cambridge,January 2000 Finian J.Leeper
John C.Vederas
Biosynthesis of Aromatic Polyketides
Ben Shen
Department of Chemistry,University of California,One Shields Avenue,Davis,CA 95616,
USA.E-mail:[email protected]
Aromatic polyketides differ from other polyketides by their characteristic polycyclic aromatic
structures. These polyketides are widely distributed in bacteria, fungi, and plants, and
many ofthem are clinically valuable agents or exhibit other fascinating biological activities.
Analogous to fatty acids and reduced polyketide biosynthesis,aromatic polyketide biosynthe-
sis is accomplished by the polyketide synthases that catalyze sequential decarboxylative con-
densation between the starter and extender units to yield a linear poly-b-ketone intermediate.
The latter undergoes regiospecific reduction,aromatization,or cyclization to furnish the poly-
cyclic aromatic structures,which are further modified by tailoring enzymes to imbue them
with various biological activities.This review begins with a briefdiscussion on the architec-
tural organizations among various polyketide synthase genes and genetic contributions to
understanding polyketide synthases.It then presents a comprehensive account of the most
recent advances in the biochemistry and enzymology ofbacterial,fungal,and plant polyke-
tide synthases,with emphasis on in vitro studies.It concludes with a cautious summary ofthe
so-called design-rules to guide rational engineering ofpolyketide synthases for the synthesis
ofnovel aromatic polyketides.
Keywords. Aromatic polyketides, Bacterial polyketide synthase, Engineered biosynthesis,
Fungal polyketide synthase,Plant polyketide synthase
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2 Aromatic Polyketide Synthase Genes . . . . . . . . . . . . . . . . . 8
2.1 Bacterial Polyketide Synthase . . . . . . . . . . . . . . . . . . . . . . 8
2.2 Fungal Polyketide Synthase . . . . . . . . . . . . . . . . . . . . . . . 9
2.3 Plant Polyketide Synthase . . . . . . . . . . . . . . . . . . . . . . . . 10
3 Polyketide Synthase Biochemistry and Enzymology . . . . . . . . . 11
3.1 Bacterial Type II Polyketide Synthase . . . . . . . . . . . . . . . . . 11
3.1.1 Phosphopantetheinyl Transferase . . . . . . . . . . . . . . . . . . . . 12
3.1.2 Acyl Carrier Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.1.3 Malonyl CoA:Acyl Carrier Protein Transacylase . . . . . . . . . . . 16
3.1.4 b-Ketoacyl Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.1.5 Polyketide Aromatase and Cyclase . . . . . . . . . . . . . . . . . . . 21
3.1.6 In Vitro Reconstitution ofType II Polyketide Synthase . . . . . . . . 23
3.2 Fungal Polyketide Synthase . . . . . . . . . . . . . . . . . . . . . . . 28
Topics in Current Chemistry,Vol.209
© Springer-Verlag Berlin Heidelberg 2000
2 B.Shen
3.2.1 6-Methylsalicyclic Acid Synthase . . . . . . . . . . . . . . . . . . . . 29
3.2.2 The Aflatoxin Polyketide Synthase/Fatty Acid Synthase Complex . . 32
3.3 Plant Polyketide Synthase . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3.1 Chalcone Synthase and Stilbene Synthase . . . . . . . . . . . . . . . 34
3.3.2 Deoxychalcone Synthase . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3.3 Methylchalcone Synthase . . . . . . . . . . . . . . . . . . . . . . . . 37
3.3.4 2-Pyrone Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4 Genetic Engineering ofPolyketide Synthase
for Novel Aromatic Polyketides . . . . . . . . . . . . . . . . . . . . . 39
4.1 Expression System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.2 Chain Length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3 Starter Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.4 Ketoreduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.5 Cyclization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
5 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
List of Abbreviations
ACP acyl carrier protein
ACPS holo-acyl carrier protein synthase
Act actinorhodin
AF aflatoxin
ARO aromatase
AT acyl transferase
CHS chalcone synthase
CLF chain length factor
CoA coenzyme A
CYC cyclase
DMAC 3,8-dihydroxy-1-methyl anthraquinone-2-carboxylic acid
FAS fatty acid synthase
KR ketoreductase
KS b-ketoacyl:ACP synthase
MAT malonyl CoA:ACP acyltransferase
6MSAS 6-methylsalicyclic acid synthase
NAC N-acetylcysteamine
Orf open reading frame
PCP peptidyl carrier protein
PCR polymerase chain reaction
PKS polyketide synthase
PMSF phenylmethylsulfonyl fluoride
PPTase phosphopantetheinyl transferase
The Biosynthesis of Aromatic Polyketides 3
2PS 2-pyrone synthase
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
ST sterigmatocystin
STS stilbene synthase
Tcm tetracenomycin
TE thioesterase
1
Introduction
Polyketide metabolites are found in bacteria,fungi,and plants and represent one
ofthe largest groups ofnatural products [1–4].They are structurally classified
into four major groups:aromatics (e.g.,doxorubicin and tetracycline),macro-
lides (e.g.,erythromycin and rapamycin),polyethers (e.g.,monensin and sali-
nomycin),and polyenes (e.g.,amphotericin and candicidin),many ofwhich are
clinically valuable antibiotics or chemotherapeutic agents,or exhibit other phar-
macological activities [5–8].Despite their apparent structural diversity,poly-
ketides share a common mechanism ofbiosynthesis.The carbon backbone ofa
polyketide results from sequential condensation ofshort fatty acids like acetate,
propionate,or butyrate,in a manner resembling fatty acid biosynthesis,and this
process is catalyzed by polyketide synthases (PKSs).Much of the current re-
search on polyketide biosynthesis is driven by the unprecedented biochemistry
and enzymology ofthe PKSs that provide an excellent model for elucidating the
structure-function relationship of complex multienzyme systems and by the
great potential of generating novel polyketide libraries via combinatorial bio-
synthesis with engineered PKSs [7,9–21].
Following the convention of fatty acid synthases (FASs) [22–25],PKSs have
been classified into two types according to their enzyme architecture and gene
organization.Type I PKSs are multifunctional proteins consisting of domains
for individual enzyme activities and have been found in bacteria as well as in
fungi and plants.Type II PKSs are multienzyme complexes consisting ofdiscrete
proteins that are largely monofunctional and have so far only been found in bac-
teria.Although early isotope labeling experiments clearly demonstrated that
FASs and PKSs use similar substrates,it is the recent cloning ofPKS genes and
the biochemical characterization ofPKS enzymes that have provided a mecha-
nistic explanation ofhow PKSs achieve the vast structural diversity during poly-
ketide biosynthesis by varying the similar biosynthetic reactions ofFASs.Thus,
unlike fatty acid biosynthesis,in which the b-ketone group ofthe growing fatty
acid intermediate1undergoes full reduction to a methylene group2,3during
each cycle ofelongation (pathway A in Fig.1),the b-ketone group ofthe growing
polyketide intermediate4could either be left untouched (5,6),leading to aro-
matic polyketides (pathway B in Fig.1),or be subjected to no,partial,or full
reduction (7–10),depending on a given cycle ofelongation,leading to macroli-
des,polyethers,or polyenes (pathway C in Fig.1).The latter forms the mechanis-
tic basis for grouping macrolides,polyethers,and polyenes together as complex
or reduced polyketides.It has now been well established that the biosynthesis of