[Methods in Enzymology] Natural Product Biosynthesis by Microorganisms and Plants, Part A Volume 515 || Type III Polyketide Synthases in Microorganisms

CHAPTER SIXTEEN

Type III Polyketide Synthasesin MicroorganismsYohei Katsuyama, Yasuo Ohnishi1Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,Bunkyo-ku, Tokyo, Japan1Corresponding author: e-mail address: [email protected]

Contents

1.

MetISShttp

Introduction

hods in Enzymology, Volume 515 # 2012 Elsevier Inc.N 0076-6879 All rights reserved.://dx.doi.org/10.1016/B978-0-12-394290-6.00017-3

359
2. Methods of Study 363
2.1
In vitro enzyme assay of recombinant type III PKSs 363 2.2 Expression of type III PKS genes in heterologous hosts and characterization of
the compounds specifically produced in the recombinant strains
370 2.3 In vitro analysis of tailoring enzymes in type III PKS-mediated polyketide
biosynthesis
372 References 374
Abstract

Type III polyketide synthases (PKSs) are simple homodimers of ketosynthases which cata-lyze the condensation of one to severalmolecules of extender substrate onto a starter sub-strate through iterative decarboxylative Claisen condensation reactions. Type III PKSs havebeen found in bacteria and fungi, as well as plants. Microbial type III PKSs, which are in-volved in the biosynthesis of some lipidic compounds and various secondarymetabolites,have several interesting characteristics that are not shared by plant type III PKSs. Further,many compoundsproducedbymicrobial type III PKSshave significant biological functionsand/or important pharmaceutical activities. Thus, studies on this class of enzymes willexpand our knowledge of the biosynthetic machineries that generate natural productsand generate new findings about microbial physiology. The recent development ofnext-generation DNA sequencing has allowed for an increase in the number of microbialgenomes sequenced and the discovery of many microbial type III PKS genes. Here, wedescribe basic methods to study microbial type III PKSs whose genes are easy to clone.

1. INTRODUCTION

Type III polyketide synthases (PKSs) are simple homodimers of

ketosynthases which catalyze the condensation of one to several molecules

of extender substrate onto a starter substrate through iterative decarboxylative

359

http://dx.doi.org/10.1016/B978-0-12-394290-6.00017-3

360 Yohei Katsuyama and Yasuo Ohnishi

Claisen condensation reactions (Abe & Morita, 2010). Type III PKSs were

formerly believed to be specific to plants. However, as the characterization

of the 1,3,6,8-tetrahydroxynaphthalene (THN) synthase RppA, which is

involved in the biosynthesis of flaviolin and hexahydroxyperylenequinone

(HPQ) melanin (Fig. 16.1) in Streptomyces griseus (Funa, Ohnishi, Ebizuka,

& Horinouchi, 2002a, 2002b; Funa et al., 1999; Moore et al., 2002), it has

been realized that many type III PKSs are found in microorganisms

(Katsuyama & Horinouchi, 2010). Some of them are involved in the

biosynthesis of biologically important compounds (Fig. 16.1). For instance,

ArsB and ArsC from Azotobacter vinelandii are involved in the biosynthesis of

alkylresorcinol and alkylpyrone, respectively, which are important

components of the cyst cell wall (Funa, Ozawa, Hirata, & Horinouchi,

2006). SrsA is involved in the formation of alkylquinone (Fig. 16.1), which

confers penicillin resistance to S. griseus (Funabashi, Funa, & Horinouchi,

2008). Germicidin (Fig. 16.1) derivatives synthesized by Gcs (Song et al.,

2006) control the germination of Streptomyces coelicolor A3(2) spores (Aoki,

Matsumoto, Kawaide, & Natsume, 2011). 2,4-Diacetylphloroglucinol

(Fig. 16.1), synthesized by PhlD from Pseudomonas, has biocontrol activity

against soil-borne fungal plant pathogens (Bangera & Thomashow, 1999).

In addition to these genuine type III PKSs, the type III PKS domains of the

multidomain enzymes called “steely” from Dictyostelium discoideum are

responsible for the biosynthesis of the acylphloroglucinol and alkylresorcinol

scaffolds of the differentiation-inducing factors DIF-I (1-(3,5-dichloro-2,

6-dihydroxy-4-methoxyphenyl)-1-hexanone) (Fig. 16.1) and MPBD

(4-methyl-5-pentylbenzene-1,2-diol), respectively (Austin et al., 2006;

Ghosh et al., 2008). Type III PKSs also provide building blocks for the

biosynthesis of other secondary metabolites, such as kendomycin (type I

PKS) and balhimycin (nonribosomal peptide synthetase) (Fig. 16.1) (Pfeifer

et al., 2001; Wenzel, Bode, Kochems, & Muller, 2008).

In reactions catalyzed by type III PKSs, the polyketide intermediates are

further cyclized by aldol condensation,Claisen condensation, or lactonization

(Fig. 16.2). The various type III PKSs differ in starter substrate specificity,

extender substrate specificity, number of extender substrates to be condensed,

and cyclization reactions. Thus, type III PKSs are capable of synthesizing a

wide variety of natural products. Compared to type I and type II PKSs, type

III PKSs have a simple structure and catalyze various reactions in a single cat-

alytic center. Therefore, the modulating mechanisms (programming) of the

reactions should be much more complicated in type III PKSs than in type I

and type II PKSs. Microbial type III PKSs have some features that are not

OH

OH

OH

OH

OH

OH

OH

O

OO

O

O

O

OH

HO

OH

MPBD2,4-Diacetylphloroglucinol

HO R

HO

O

DIF-1

O

Alkyl-O-dihydrogeranyl-methoxyhydroquinone

OH

OH

Cl

Cl

OH

OH

O

O

O

O

O

O

OO

Germicidin A

Flaviolin

Alkylquinone

Furaquinocin D

OH

O

O O

OO

OO O

O

O

NH

HN

H H

H

N

H

H2N

NNN

HOOC

HO

O

O

O

O

O

OO

Cl

Cl

O

Balhimycin

O

OH

HN

OHOH

OH

HO

HO

HO

O

Kendomycin

HO

OH H2N

HO

O R

O

O O

O OHOO

HO

HPQ

HO

HO

HO

OH

Naphterpin

Furanonaphthoquinone I

Figure 16.1 Natural products synthesized through biosynthetic pathways catalyzed bymicrobial type III polyketide synthases. Bold lines indicate scaffolds synthesized by theseenzymes. R, alkyl.

361Type III PKSs in Microorganisms

CoA

CoA(Enz) (Enz) (Enz)

CoASH

CoA

CO2

(ACP) (Enz)S

S CoA CoAS S

CoAS

CoAS

CoA CoA(Enz) (Enz)

ArsC ArsC SrsAFtpA

S S

CoA(Enz)

S

CoA(Enz)

RppA

S

OO

OO

O

O

O

O

O

O O O

O O

OO

HOR

R

R

R

O

O O O O

O

O

R

R

SR

R

R

R

R

R R

R

O

O

O

O

O

O

OO

O

O

O

O

O

OOH

OH

O O

OO

O

OH

OO

OO O

O

HO

O O

PhlD StlB ArsB

OH

HO OH

OHPhloroglucinol Acylphloroglucinol Alkylresorcinol

Pentaketidealkylresorcylic acid

3,5-Dihydroxyphenyl-acetyl-CoA

HO

HO

HO

HO

Tetrahydroxynaphthalene

HO

HO

HO HO

ORASDpgA

OH

OH

OH

Triketidealkylpyrone

Tetraketidealkylpyrone

2-Alkyl-3-methyl-resorcylic acid

OH

OH

OH

OH OH

OH

n� n

n

�

n �

malonyl-CoA Polyketide intermediate(extendersubstrate)

O O

Acyl-CoA(starter substrate)

Figure 16.2 Reactions catalyzed by microbial type III PKSs.


shared by plant type III PKSs. First, many microbial type III PKSs seem to use

an acyl–acyl carrier protein (ACP) as starter substrate, whereas most plant type

III PKSs use acyl-CoA as starter substrate (Austin et al., 2006; Funa, Funabashi,

Yoshimura, & Horinouchi, 2005; Ghosh et al., 2008; Gruschow, Buchholz,

Seufert, Dordick, & Sherman, 2007; Hayashi, Kitamura, Funa, Ohnishi, &

Horinouchi, 2011; Miyanaga, Funa, Awakawa, & Horinouchi, 2008; Song

et al., 2006). In some cases, type III PKS genes form a cluster with ACP or

fatty acid biosynthetic genes (Funa, Funabashi, Yoshimura et al., 2005;

Hayashi et al., 2011; Miyanaga et al., 2008). The type III PKSs discovered

from D. discoideum are even fused with a type I fatty acid synthase and act as

a domain of these multidomain enzymes (Austin et al., 2006; Ghosh et al.,

2008). Also, some type III PKSs, such as Gcs and SrsA, whose genes do not

form clusters with ACP or fatty acid biosynthetic genes, may incorporate


acyl-ACP as starter substrate, as suggested by the in vitro study of Gruschow

et al. (2007) and the in vivo study of Song et al. (2006). In these cases, the

enzymes are most likely to incorporate acyl-ACPs synthesized by the fatty

acid biosynthetic pathway (Song et al., 2006). Further, some microbial type

III PKSs can incorporate methylmalonyl-CoA and ethylmalonyl-CoA as

extender substrates, whereas to the best of our knowledge, all known plant

type III PKSs, except the PstrCHS from Pinus strobus, do not incorporate

methylmalonyl-CoA or ethylmalonyl-CoA as physiological substrates (Abe

& Morita, 2010; Schroder et al., 1998). Gcs catalyzes a single condensation

of a beta-ketoacyl-ACP starter unit with an ethylmalonyl-CoA extender

unit (Song et al., 2006). SrsA and FtpA catalyze three condensations using a

fatty acyl-ACP (or CoA) as starter unit, and two malonyl-CoAs and one

methylmalonyl-CoA as extender substrates (Funabashi et al., 2008, Hayashi

et al., 2011; Nakano, Funa, Ohnishi, & Horinouchi, 2012). Interestingly,

the order of incorporation of these extender substrates is highly regulated:

SrsA and FtpA use methylmalonyl-CoA as the first extender substrate,

followed by two molecules of malonyl-CoA as the second and third

extender substrates. Little is known about the regulation mechanism of the

condensation order of the extender units in type III PKSs. These unusual

features of the microbial type III PKSs are likely to become interesting

topics in the study of the enzymology of type III PKSs.

The recent development of next-generation DNA sequencing has

allowed for an increase in the number of microbial genomes sequenced

and the discovery of many microbial type III PKS genes. However, there

are still many type III PKSs to be identified. Future studies on type III PKSs

will provide important insights into the properties of these enzymes and their

role in the biosynthesis of natural products. Further, their study will generate

new findings about the physiology of microorganisms, because many com-

pounds produced by microbial type III PKSs have biologically important

functions. Here, we describe basic methods for the study of microbial type

III PKSs whose genes are easy to clone.

2. METHODS OF STUDY

2.1. In vitro enzyme assay of recombinant type III PKSs
As type III PKSs are composed of a simple homodimer and catalyze reactions
without the need of a cofactor or other agents, in vitro enzyme assays are a

basic and useful tool to characterize them. Here, we describe several

methods for in vitro enzyme assays of type III PKSs.


2.1.1 Production and purification of recombinant type III PKSsType III PKS genes are usually expressed in Escherichia coli BL21(DE3) using

the pET system (Novagen), and the enzymes can be produced in active form

in the soluble fraction (Achkar, Xian, Zhao, & Frost, 2005; Awakawa,

Fujita, Hayakawa, Ohnishi, & Horinouchi, 2011; Funa, Awakawa,

& Horinouchi, 2007; Funa et al., 1999, 2006; Ghosh et al., 2008;

Li, Gruschow, Dordick, & Sherman, 2007). If the expression level is high

enough without induction, the enzyme can be produced easily by

cultivating the recombinant E. coli strain in Luria Bertani (LB) broth (1%

peptone, 0.5% yeast extract, and 0.5% NaCl) containing appropriate

antibiotics, at 26 �C overnight. Otherwise, expression of the cloned type

III PKS gene is induced by isopropyl b-D-thiogalactopyranoside (IPTG)

and the enzyme is produced using the following method.

1. The recombinant E. coli strain is inoculated into 100 mL LB broth con-

taining appropriate antibiotics.

2. The strain is cultivated at 37 �C until the OD600 reaches 0.4–0.6.

3. The culture is incubated at 16–26 �C, and 0.05–0.5 mM IPTG is added.

4. Cultivation is continued for a further 4–16 h.

The pCold system (Takara) is also useful for the production of type III PKSs in

E. coli (Nakano, Ozawa, Akanuma, Funa, & Horinouchi, 2009). Another al-

ternative is the production of type III PKSs as fusion proteins with maltose-

bindingproteins, byusingpMAL-c2x (Izumikawaetal., 2003).However,with

these systems some type III PKSs are poorly produced in soluble form inE. coli.

In such cases,Streptomyces lividans is useful as an alternative host. The expression

plasmids pSH19 (Herai et al., 2004) and pIJ6021 (Takano,White, Thompson,

& Bibb, 1995) can be used to produce the enzymes (Funabashi et al., 2008;

Hayashi et al., 2011). Following is a description of the method using the

pSH19 system (Funabashi et al., 2008). This method is based on the

induction of the NitR-repressing nitA promoter by e-caprolactam.

1. A pair of primers for the cloning of a type III PKS gene is designed. One

primer should contain the following sequences: AGCAACGGAGGT

ACGGAC, which contains the Shine–Dalgarno sequence for nitA,

polyhistidine tag sequence (for adding apolyhistidine tag at theNterminus

of the recombinant enzyme) and the first 20–24 nucleotides of the target

gene,while theother primer should contain a complementary sequence to

the last 20–24 nucleotides of the target gene. To add a polyhistidine tag to

the C terminus of the enzyme, a polyhistidine tag-coding sequence in the

former primer should be removed and added to the latter primer. Both


primers should contain a restriction site for cloning.The target gene is am-

plified by PCR using the primer set and the DNA fragment is digested

with the restriction enzymes for cloning into pSH19. The obtained plas-

mid is introduced into S. lividans by protoplast transformation (Kieser,

Bibb, Buttner, Chater, & Hopwood, 2000).

2. The recombinant S. lividans strain is cultivated in yeast extract-malt ex-

tract (YEME) medium with 5 mg/L of thiostrepton at 30 �C for 2 days.

The YEMEmedium contains 0.3% yeast extract, 0.3%malt extract, 0.5%

peptone, 1% glucose, and 34% sucrose; 0.2 mL/100 mL of 2.5 M

MgCl2�6H2O and 2.5 mL/100 mL of 20% glycine are added after

autoclaving. The pH of the medium is 7.0–7.2.

3. e-Caprolactam (final concentration 0.1%) is added to induce expression

of the nitA promoter. The strain is incubated at 30 �C for a further 60 h.

4. The cells are harvested.

As an alternative, the method using the pIJ6021 system is described below

(Takano et al., 1995). This method is based on induction of the tipA pro-

moter by thiostrepton.

1. The target type III PKS gene is cloned under the control of the tipA pro-

moter on pIJ6021.

2. The recombinant S. lividans strain harboring the pIJ6021-derived plas-

mid is cultivated in 100 mL YEME medium containing 5 mg/L kana-

mycin at 30 �C for 2 days.

3. 5 mg/L thiostrepton is added to the culture to induce the tipA promoter.

4. The culture is incubated at 30 �C for a further 60 h.

5. The cells are harvested.

Enzymes fused with a polyhistidine tag, expressed in either E. coli or

S. lividans, can be easily purified by Ni2þ affinity chromatography using

Ni sepharose (GE healthcare) or Ni-NTA superflow resin (QIAGEN),

applying the following method.

1. The harvested cells are resuspended into lysis buffer containing 50 mM

Tris–HCl (pH 8.0), 150 mM NaCl, 5 mM imidazole, and 10% glycerol.

2. The cells are disrupted by sonication and cell debris is removed by cen-

trifugation and filtration.

3. Ni sepharose (GE healthcare) or Ni-NTA superflow resin (QIAGEN) is

added to the supernatant and the sample is incubated at 4 �C for 1 h.

4. The resin is applied to a gravity-flow column and washed with 5�column-volume of wash buffer containing a low concentration of

imidazole.


5. The polyhistidine-tagged enzyme is eluted with buffer containing a high

concentration of imidazole.

6. The amount and purity of the enzyme are checked with SDS-PAGE.

7. The sample is dialyzed against an appropriate buffer (e.g., lysis buffer

without imidazole).

8. The resulting solution is concentrated by ultrafiltration.

9. The concentrated enzyme sample can usually be stored at �80 �C after

being rapidly frozen with liquid nitrogen. It is recommended to check

the enzyme activity before and after freezing. The microbial type III

PKS SrsA becomes inactive on freezing (our unpublished observation).

2.1.2 In vitro enzyme assayIn general, microbial type III PKSs are most active at pH 7–8 and 30 �C. Fora preliminary study, products can be easily analyzed by radio-thin layer chro-

matography (TLC) using [2-14C]malonyl-CoA and various acyl-CoAs as

extender and starter substrates, respectively (Funa et al., 2002a, 2002b,

2006, 2007; Funabashi et al., 2008). Following is an example of an in

vitro enzyme assay of a type III PKS producing alkylresorcinols, followed

by analysis of reaction products by radio-TLC.

1. 100 mL of a reaction mixture containing 100 mM of each acyl-CoA

(C2–C20), 100 mM [2-14 C]malonyl-CoA, and 4 mM enzyme in

100 mM Tris–HCl buffer (pH 8.0) are incubated at 30 �C for 30 min.

2. The reaction is quenched by adding 20 mL of 6 M HCl.

3. The products are extracted with ethyl acetate.

4. The organic layer is evaporated to dryness.

5. The resulting compounds are dissolved in 15 mL of methanol, applied to

TLC analysis using a silica gel 60 WF254 TLC plate (Merck), and devel-

oped in benzene/acetone/acetic acid (85:15:1, v/v/v).

6. The products are visualized using an image analyzer.

The previously described radio-TLC analysis is useful to estimate the sub-

strate and reaction specificities of the enzyme examined. Typically, products

in a similar reaction with nonlabeled malonyl-CoA are also analyzed by

reverse phase liquid chromatography mass spectrometry (LC–MS) or high

performance liquid chromatography (HPLC) equipped with a C18 or C4

column using water/acetonitrile/trifluoroacetic acid (TFA) or formic acid

as the mobile phase (Funa et al., 2002a, 2002b, 2006, 2007; Funabashi

et al., 2008). If products are released as CoA-bound forms, as in the

DpgA reaction, the following method is applicable (Chen, Tseng,

Hubbard, & Walsh, 2001).


1. 200 mL of a reaction mixture containing 200 mM malonyl-CoA and

5 mM DpgA are incubated at 24 �C for 1 h.

2. The reaction is quenched by adding 4 mL of 50% TFA.

3. The precipitated protein is removed by centrifugation.

4. The supernatant is analyzed using reverse phase LC–MS or HPLC

equipped with a C18 column.

Several microbial type III PKSs produce alkylresorcinols or alkylresorcylic

acids from acyl-CoA and malonyl-CoA derivatives (Awakawa et al.,

2011; Funa et al., 2006; Funabashi et al., 2008, Hayashi et al., 2011,

Katsuyama and Horinouchi, 2010; Nakano et al., 2012). Because

alkylresorcylic acids are nonenzymatically converted to alkylresorcinols, a

production profile analysis considering different reaction times is

necessary to determine which of these compounds is actually produced

by these type III PKSs.

When a type III PKS incorporates both malonyl-CoA and

methylmalonyl-CoA, it is difficult to deduce the condensation order of

the extender substrates only from the structure of the compounds. For in-

stance, alkylresorcinols synthesized from the condensation of one methyl-

malonyl-CoA after the condensation of two malonyl-CoAs show the same

structure as alkylresorcinols synthesized from the condensation of two

malonyl-CoAs after the condensation of one methylmalonyl-CoA. In such

cases, it is useful to use [13C3]malonyl-CoA to deduce the order of incorpo-

ration of malonyl-CoA and methylmalonyl-CoA (Hayashi et al., 2011;

Nakano et al., 2012). The synthesized alkylresorcinol can be analyzed by

LC–MS and the order of the extender substrates can be deduced from

changes in the molecular weight of the product, because alkylresorcinol

formation involves removal of the carboxyl group derived from the last

extender substrate. Therefore, if methylmalonyl-CoA is incorporated

first, the molecular weight increases by 3 Da and if methylmalonyl-CoA

is incorporated last, the molecular weight increases by 4 Da.

Kinetic parameters are usually measured by observing product forma-

tion. Products can be quantified in HPLC analysis by comparing the peak

area of UV absorbance with that of authentic samples. When no authentic

compound is available, quantification can be established by measuring the

radioactivity of polyketides synthesized from [2-14C]malonyl-CoA. It is

possible to measure the amount of CoA by using alpha-ketoglutarate dehy-

drogenase (KDH), which catalyzes the formation of succinyl-CoA and

NADH from CoA, NADþ, and alpha-ketoglutarate (Molnos, Gardiner,

Dale, & Lange, 2003). Thus, when coupled with the KDH reaction,


formation of free CoA from acyl-CoA or malonyl-CoA in the PKS reaction

can be detected by monitoring the increase of absorbance at 340 nm corre-

lated with NADH formation. Achkar et al. (2005) analyzed the kinetic pa-

rameters of PhlD by this method. The reaction mixture used in this study

contained 2 mM alpha-ketoglutarate, 0.3 mM NADþ, and 0.1–0.3 U of

KDH in addition to other components required for the reaction of PhlD.

Direct transfer of an acyl moiety from ACP to a type III PKS can be

observed by SDS-PAGE followed by autoradiography when 14C-labeled

substrates are used (Hayashi et al., 2011; Miyanaga et al., 2008). This

method is summarized below.

1. 14C-labeled fatty acyl-ACP is prepared by the methods described in

Section 2.1.3.

2. 1 mM of type III PKS is incubated with the 14C-labeled fatty acyl-ACP at

30 �C for 20 min.

3. The reaction mixture is analyzed by SDS-PAGE and the 14C-labeled

proteins are visualized on the gel with an image analyzer.

2.1.3 Substrate preparationMalonyl-CoA and many fatty acid CoA esters are commercially available.

However, this is not the case for some CoA esters, such as branched-chain

or long-chain fatty acids. These CoA esters should be synthesized by the

method reported by Blecher (1981), using N-hydroxysuccinimide esters,

which is summarized below.

1. Fatty acid (3 mmol), N-hydroxysuccinimide (3 mmol), water-soluble

carbodiimide (WSC, 3.3 mmol), and a catalytic amount of

4-dimethylaminopyridine are dissolved in dry dichloromethane. The

mixture is incubated at room temperature for 12 h, with stirring, before

quenching with ice.

2. The aqueous layer is extracted with dichloromethane. The dichlo-

romethane extract is washed with brine, dried over anhydrous sodium

sulfate, and the solvent is removed by evaporation.

3. The resulting fatty acid succinimide ester is purified by silica gel chroma-

tography using chloroform/methanol as the mobile phase, and analyzed

by NMR.

4. Thioglycolic acid (0.2 mmol) and sodium bicarbonate (0.8 mmol) are

added to 5 mL water containing CoASH (23 mmol). The fatty acid

succinimide ester (0.8 mmol) is dissolved in 5 mL of tetrahydrofuran

and added to the solution containing CoA. Tetrahydrofuran is added


to the mixture till it forms a single phase. The mixture is stirred at 4 �Cfor 16 h under an argon atmosphere.

5. Tetrahydrofuran is removed by evaporation and the remaining

succinimide ester is removed by washing the resultant aqueous phase

with chloroform. The CoA ester is purified by reversed-phase prepara-

tive HPLC equipped with a C4 column using acetonitrile and 25 mM

KH2PO4 as the mobile phase. Acetonitrile is removed by evaporation

and the resulting aqueous solution containing the CoA ester is desalted

using reversed-phase preparative HPLC equipped with a C18 column.

If the CoA ester synthesis proves difficult, an N-acetylcysteamine (NAC)

thioester can be used as a substitute. NAC thioesters can be synthesized using

dicyclohexylcarbodiimide (DCC) or WSC. The method is summarized

below (Oguro, Akashi, Ayabe, Noguchi, & Abe, 2004).

1. Fatty acid (3 mmol), NAC (3 mmol), WSC (3.3 mmol), and a

catalytic amount of 4-dimethylaminopyridine are dissolved in dry

dichloromethane. The mixture is incubated at room temperature for

12 h, with stirring, before quenching by ice.

2. The aqueous layer is extracted with dichloromethane. The dichlo-

romethane extract is washed with brine, dried over anhydrous sodium

sulfate, and the solvent is removed by evaporation.

3. The resulting fatty acid–NAC ester is purified by silica gel chromatog-

raphy using chloroform/methanol as solvent, and analyzed by NMR.

Several microbial type III PKSs, such as ArsB, ArsC, Gcs, and FtpA, incor-

porate acyl-ACP esters, instead of CoA esters, as starter substrates in vivo.

Although CoA esters can be incorporated by these enzymes in in vitro

enzyme reactions, it is necessary to synthesize ACP esters to further study

them. In a report by Gruschow et al. (2007), ACP esters were synthesized

by an enzymatic reaction using Sfp. Sfp is a phosphopantetheinyl transferase

that transfers the phosphopantetheinyl moiety fromCoA onto the serine res-

idue of apo-ACP, forming holo-ACP. This enzyme has broad substrate

specificity and is capable of catalyzing the transfer of a phosphopantetheinyl

moiety of various acyl-CoAs onto ACP in the absence of free CoA. By using

this enzyme, acyl-ACP could be synthesized from acyl-CoA and apo-ACP.

Sfp and apo-ACP are prepared as recombinant proteins in E. coli using the

pET system and purified by Ni2þ affinity chromatography. The method to

synthesize acyl-ACP using Sfp and apo-ACP is as follows.

1. A reaction mixture containing 30 mM apo-ACP, 1 mM Sfp, 0.3 mM

acyl-CoA, 10 mM MgCl2, and 0.1M Tris–HCl (pH 8.1) is incubated

at 30 �C for 45 min.


2. The synthesized acyl-ACP is purified by ion exchange chromatography.

3. The presence of acyl-ACP can be confirmed by MALDI-TOF-MS or

reverse phase LC–MS equipped with a 300 A C4 column (e.g., Jupiter

300 C4 column, Phenomenex) using water/acetonitrile/formic acid or

TFA as the mobile phase.

It is also possible to synthesize acyl-ACP using the Ftp system. Coincubation

of FtpD (a fatty acyl-AMP ligase), holo-ACP (holo-FtpC), and ATP has

resulted in the formation of acyl-ACP (acyl-FtpC) (Hayashi et al., 2011).

This system may be applied to the synthesis of different acyl-ACPs.

2.2. Expression of type III PKS genes in heterologous hosts andcharacterization of the compounds specifically producedin the recombinant strains

The heterologous expression of type III PKS genes or whole gene clusters

containing type III PKS genes is a powerful tool to explore in vivo functions

of these enzymes and gene clusters. S. lividans, Pseudomonas putida, E. coli,

Bacillus subtilis, and Aspergillus oryzae have been used as heterologous hosts

(Achkar et al., 2005; Awakawa et al., 2011; Cortes et al., 2002; Gross

et al., 2006; Hayashi et al., 2011; Nakano et al., 2009; Seshime, Juvvadi,

Kitamoto, Ebizuka, Fujii, 2010; Seshime, Juvvadi, Kitamoto, Ebizuka,

Nonaka, et al., 2010).

2.2.1 Heterologous expression in E. coliFor characterizing the phloroglucinol synthase PhlD and phl gene cluster

from Pseudomonas fluorescens, Achkar et al. (2005) expressed phlD and part

or the whole of the phl gene cluster in E. coli by using the pET system

(Novagen). Following is an example of the method for heterologous expres-

sion of a type III PKS gene or gene cluster in E. coli using the pET system.

1. E. coli BL21(DE3) harboring a pET-derived vector is cultivated in 10 mL

LB broth containing appropriate antibiotics at 37 �C for 12 h.

2. 10 mL of this culture is inoculated into 500 mL of Terrific Broth (1.2%

peptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, and

0.4% glycerol) containing appropriate antibiotics.

3. The strain is cultivated at 37 �C until OD600 reaches 1.0–1.2.

4. IPTG (final concentration 0.5 mM) is added to the culture to induce ex-

pression of the target gene(s) and the strain is cultivated at 23 �C for 4 h.

5. The cells are harvested by centrifugation (15,000� g, 4 �C, 4 min) and

resuspended in 500 mL of M9 medium containing appropriate antibi-

otics, as well as 0.5 mM IPTG.


6. The strain is cultivated at 30 �C and 15 mL of culture broth are harvested

at 24 h intervals.

2.2.2 Heterologous expression in PseudomonasPseudomonas spp. are Gram-negative bacteria which are often used as a host

for heterologous expression. Gross et al. (2006) used the broad-host-range

vector pJB861, containing the Pm promoter, for the expression of a type III

PKS gene from myxobacteria in P. putida. This method seems to be useful

for the analysis of type III PKSs and some polyketide modification enzymes

from high-GC Gram-negative bacteria such as Pseudomonas spp. Following

is an example of the method for heterologous expression of a type III PKS

gene or gene cluster in P. putida using the pJB861 system.

1. P. putida KT2440 harboring a pJB861-derived plasmid is cultivated in

10 mL LB broth containing kanamycin for 12 h.

2. 5 mL of this culture are inoculated into 500 mL of LB broth containing

kanamycin and incubated at 30 �C until the OD600 reaches 0.6.

3. The temperature of the culture is lowered to 16 �C to reduce the pro-

duction of insoluble protein.

4. Cultivation continues at 16 �C for 48 h.

2.2.3 Heterologous expression in StreptomycesS. lividans is a useful host for the heterologous expression of type III PKS

genes not only from actinomycetes (Awakawa et al., 2011; Funabashi

et al., 2008) but also from myxobacteria (Hayashi et al., 2011). The

expression plasmid pIJ6021, which contains the thiostrepton-inducible

tipA promoter, has been used for this purpose. Three gene clusters

containing a type III PKS gene have been successfully characterized by

heterologous expression in S. lividans using the pIJ6021 system (Awakawa

et al., 2011; Funabashi et al., 2008; Hayashi et al., 2011). When multiple

genes should be expressed by this system, it is useful to place each gene

under the control of its own tipA promoter. Following is the description

of the method for heterologous expression of a type III PKS gene or

gene cluster in S. lividans using the pIJ6021 system.

1. The recombinant S. lividans strain harboring the pIJ6021-derived

plasmid is cultivated in 100 mL YEME medium containing 5 mg/L

kanamycin at 30 �C for 1 day.

2. 5 mg/L thiostrepton is added to the culture to induce the tipA promoter.

3. The culture is incubated at 30 �C for a further 60 h.


2.2.4 Heterologous expression in B. subtilisB. subtilis seems to be one of the best hosts for the heterologous expression of

a type III PKS gene or gene cluster from low-GC Gram-positive bacteria.

The xylB promoter on pWH1530 has been used to overexpress bpsA and the

bpsAB operon in B. subtilis (Nakano et al., 2010). The protocol used in the

report by Nakano et al. (2010) involves the steps described below.

1. The recombinant B. subtilis strain harboring the pWH1530-derived plas-

mid is cultivated in 100 mL LB broth containing 10 mg/L tetracycline at

37 �C for 2 h.

2. 5 mg/mL xylose is added to the culture to induce expression from the

xylA promoter.

3. The strain is cultivated for a further 8 h.

2.2.5 Heterologous expression in A. oryzaeFor the overexpression of fungal type III PKS genes (cysA and cysB) in

A. oryzae, pTAex3 containing the amyB promoter has been used

(Seshime, Juvvadi, Kitamoto, Ebizuka, & Fujii, 2010; Seshime, Juvvadi,

Kitamoto, Ebizuka, Nonaka, et al., 2010). The overproduction of

polyketides is established by incubating the recombinant A. oryzae strain

in Czapek-Dox-maltose medium (1% polypeptone, 2% maltose, 0.3%

NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCl, and 0.0018%

FeSO4�7H2O) at 30 �C for 3 days.

2.3. In vitro analysis of tailoring enzymes in type IIIPKS-mediated polyketide biosynthesis

In general, polyketide intermediates synthesized by type III PKSs are further

modified by postpolyketide modification enzymes (tailoring enzymes).

Examples of these tailoring enzymes are SrsBC (Funabashi et al., 2008;

Nakano et al., 2012), FtpBE (Hayashi et al., 2011), ChlA and DmtA

(Neumann, Walsh, & Kay, 2010), DpgBCD (Chen et al., 2001),

AgqBCD (Awakawa et al., 2011), NphB and Fur7 (Kumano, Tomita,

Nishiyama, & Kuzuyama, 2010; Kuzuyama, Noel, & Richard, 2005),

MomA (Funa, Funabashi, Yoshimura, et al., 2005), and P450mel (Funa,

Funabashi, Ohnishi, & Horinouchi, 2005). In most cases, these enzymes

are analyzed by coexpression of their genes with type III PKS genes,

applying the heterologous expression methods described above.

However, it is also very important to analyze these enzymes in vitro.

There are several methods to achieve this, as we describe below.


2.3.1 OxidaseAn oxidase gene often forms an operon with a microbial type III PKS gene;

three oxidases of different classes have been characterized: MomA, belong-

ing to the cupin superfamily, catalyzes monooxygenation of THN to form

flaviolin (Funa, Funabashi, Yoshimura, et al., 2005); DpgC, the cofactor-

independent oxidase, is involved in (S)-3,5-dihydroxyphenylglycine

biosynthesis (Chen et al., 2001); and P450mel is a cytochrome P450 that

catalyzes oxidative biaryl coupling of THN to yield HPQ (Funa, Funabashi,

Ohnishi, et al., 2005). The biosynthetic gene clusters of furano-

naphthoquinone I and kendomycin include a MomA and a DpgC

homologue, respectively (Haagen et al., 2006; Wenzel et al., 2008).

Cytochrome P450 was also discovered in the biosynthetic gene cluster

of furaquinocins (Kawasaki et al., 2006). Thus, these three classes of

enzymes seem to be commonly involved in the modification of

polyketides synthesized by type III PKSs.

Both momA and dpgC can be expressed using the pET system, and the

recombinant enzymes are then purified with Ni2þ affinity chromatography.

MomA can be analyzed by incubation with substrate in a buffer containing

100 mM sodium phosphate (pH 7.5) at 30 �C for 15 s. For the in vitroDpgC

assay, 8 mM of purified enzyme is incubated with 2 mM of

3,5-dihydroxyphenylacetyl-CoA in Tris–HCl buffer (pH 7.5) at 24 �Cfor 1 h. Nevertheless, because cytochrome P450 requires a heme cofactor,

it has not been possible to produce it using this method. Following is

an example of the method for heterologous production of a P450

monooxygenase in E. coli using the pET system.

1. E. coli BL21(DE3) harboring the pET-derivative plasmid is cultivated in

M9 medium containing 100 mM FeSO4 and an appropriate antibiotic

until the OD600 reaches 0.4–0.6.

2. 80 mg/L of 5-aminolevulinic acid and 0.1 mM IPTG are added and

incubated at 22 �C for 1 day.

3. The cells are harvested and disrupted by sonication. Then, the cell debris

is removed by centrifugation, and the protein from the supernatant is pu-

rified using Ni2þ affinity chromatography.

The resulting enzyme can be analyzed by incubation with 1 mM NADPH,

0.5 U of spinach ferredoxin-NADP reductase, 40 mg of spinach ferredoxin,

and 400 mM of substrate in a buffer containing 100 mM sodium phosphate

(pH 7.3), 1 mM dithiothreitol, 1 mM EDTA, and 10% glycerol at 30 �C for

30 min.


2.3.2 PrenyltransferasesThere are two different types of prenyltransferases known to modify

polyketides synthesized by type III PKSs. One is the NphB-type

prenyltransferase (Kumano et al., 2010; Kuzuyama et al., 2005) and the

other is the UbiA-type prenyltransferase, represented by AgqD (Awakawa

et al., 2011). NphB-type prenyltransferase can be easily expressed using the

pET system and purified with Ni2þ affinity chromatography (Kumano

et al., 2010; Kuzuyama et al., 2005). The catalytic activity of the enzyme can

be determined by incubating it with a buffer containing 50 mM

Hepes–NaOH (pH 7.5), 5 mM MgCl2, 1 mM substrate, 5 mM geranyl

pyrophosphate (GPP) (or other prenyl pyrophosphate) at 30 �C for 2–16 h.

Reaction products can be extracted with ethyl acetate and analyzed by

LC–MS. In contrast, UbiA-type prenyltransferase is a membrane-bound

protein, making it difficult to purify the active enzyme from the membrane

fraction. AgqD can be analyzed in vitro using the AgqD-containing

membrane fraction as described below (Awakawa et al., 2011).

1. The recombinant AgqD protein is produced in S. lividans using the

pIJ6021 system.

2. The cells are harvested by centrifugation and resuspended in a buffer

containing 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10% glycerol,

and a protease inhibitor cocktail.

3. The cells are disrupted by sonication and cell debris is removed by

centrifugation.

4. Cell membranes are pelleted by ultracentrifugation (235,000� g).

5. Cell membranes are resuspended in a buffer containing 10 mMTris–HCl

(pH 8.0), 150 mM NaCl, and 10% glycerol.

6. The resulting cell membranes are incubated with prenyl pyrophosphates

(dimethylallyl pyrophosphate (DMAP), geranyl pyrophosphate (GPP),

farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP),

200 mM) anda substrate in50 mMTris–HClbuffer (pH8.0) at37 �Cfor3 h.

7. The reaction products are then extracted with ethyl acetate and analyzed

by LC–MS.

REFERENCESAbe, I., & Morita, H. (2010). Structure and function of the chalcone synthase superfamily of

plant type III polyketide synthases. Natural Product Reports, 27, 809–838.Achkar, J., Xian, M., Zhao, H., & Frost, J. W. (2005). Biosynthesis of phloroglucinol. Journal

of the American Chemical Society, 127, 5332–5333.Aoki, Y., Matsumoto, D., Kawaide, H., & Natsume, M. (2011). Physiological role of

germicidins in spore germination and hyphal elongation in Streptomyces coelicolor A3(2). The Journal of Antibiotics, 64, 607–611.


Austin, M. B., Saito, T., Bowman, M. E., Haydock, S., Kato, A., Moore, B. S., et al. (2006).Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type Ifatty acid-type III polyketide synthase. Nature Chemical Biology, 2, 494–502.

Awakawa, T., Fujita, N., Hayakawa, M., Ohnishi, Y., & Horinouchi, S. (2011). Character-ization of the biosynthesis gene cluster for alkyl-O-dihydrogeranyl-methoxyhy-droquinones in Actinoplanes missouriensis. Chembiochem, 12, 39–48.

Bangera, M. G., & Thomashow, L. S. (1999). Identification and characterization of a genecluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudo-monas fluorescens Q2-87. Journal of Bacteriology, 181, 3155–3163.

Blecher, M. (1981). Synthesis of long-chain fatty acyl-CoA thioesters usingN-hydroxysuccinimide esters. Methods in Enzymology, 72, 404–408.

Chen, H., Tseng, C. C., Hubbard, B. K., & Walsh, C. T. (2001). Glycopeptide antibioticbiosynthesis: Enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine. Proceedings of the National Academy of Sciences of the United Statesof America, 98, 14901–14906.

Cortes, J., Velasco, J., Foster, G., Blackaby, A. P., Rudd, B. A., & Wilkinson, B. (2002).Identification and cloning of a type III polyketide synthase required for diffusible pig-ment biosynthesis in Saccharopolyspora erythraea. Molecular Microbiology, 44, 1213–1224.

Funa, N., Awakawa, T., & Horinouchi, S. (2007). Pentaketide resorcylic acid synthesis bytype III polyketide synthase fromNeurospora crassa.The Journal of Biological Chemistry, 282,14476–14481.

Funa, N., Funabashi, M., Ohnishi, Y., & Horinouchi, S. (2005). Biosynthesis of hexa-hydroxyperylenequinone melanin via oxidative aryl coupling by cytochrome P-450in Streptomyces griseus. Journal of Bacteriology, 187, 8149–8155.

Funa, N., Funabashi, M., Yoshimura, E., & Horinouchi, S. (2005). A novel quinone-forming monooxygenase family involved in modification of aromatic polyketides. TheJournal of Biological Chemistry, 280, 14514–14523.

Funa, N., Ohnishi, Y., Ebizuka, Y., & Horinouchi, S. (2002a). Properties and substrate spec-ificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus.The Journal of Biological Chemistry, 277, 4628–4635.

Funa, N., Ohnishi, Y., Ebizuka, Y., & Horinouchi, S. (2002b). Alteration of reaction andsubstrate specificity of a bacterial type III polyketide synthase by site-directed mutagen-esis. The Biochemical Journal, 367, 781–789.

Funa, N., Ohnishi, Y., Fujii, I., Shibuya, M., Ebizuka, Y., & Horinouchi, S. (1999). A newpathway for polyketide synthesis in microorganisms. Nature, 400, 897–899.

Funa, N., Ozawa, H., Hirata, A., & Horinouchi, S. (2006). Phenolic lipid synthesis bytype III polyketide synthases is essential for cyst formation in Azotobacter vinelandii.Proceedings of the National Academy of Sciences of the United States of America, 103,6356–6361.

Funabashi, M., Funa, N., & Horinouchi, S. (2008). Phenolic lipids synthesized by type IIIpolyketide synthase confer penicillin resistance on Streptomyces griseus. The Journal ofBiological Chemistry, 283, 13983–13991.

Ghosh, R., Chhabra, A., Phatale, P. A., Samrat, S. K., Sharma, J., Gosain, A., et al. (2008).Dissecting the functional role of polyketide synthases inDictyostelium discoideum: Biosyn-thesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol. TheJournal of Biological Chemistry, 283, 11348–11354.

Gross, F., Luniak, N., Perlova, O., Gaitatzis, N., Jenke-Kodama, H., Gerth, K., et al. (2006).Bacterial type III polyketide synthases: Phylogenetic analysis and potential for the pro-duction of novel secondary metabolites by heterologous expression in pseudomonads.Archives of Microbiology, 185, 28–38.

Gruschow, S., Buchholz, T. J., Seufert, W., Dordick, J. S., & Sherman, D. H. (2007). Sub-strate profile analysis and ACP-mediated acyl transfer in Streptomyces coelicolor type IIIpolyketide synthases. Chembiochem, 8, 863–868.


Haagen, Y., Gluck, K., Fay, K., Kammerer, B., Gust, B., & Heide, L. (2006). A gene clusterfor prenylated naphthoquinone and prenylated phenazine biosynthesis in Streptomycescinnamonensis DSM 1042. Chembiochem, 7, 2016–2027.

Hayashi, T., Kitamura, Y., Funa, N., Ohnishi, Y., & Horinouchi, S. (2011). Fatty acyl-AMPligase involvement in the production of alkylresorcylic acid by aMyxococcus xanthus typeIII polyketide synthase. Chembiochem, 12, 2166–2176.

Herai, S., Hashimoto, Y., Higashibata, H., Maseda, H., Ikeda, H., Omura, S., et al. (2004).Hyper-inducible expression system for streptomycetes. Proceedings of the National Academyof Sciences of the United States of America, 101, 14031–14035.

Izumikawa,M., Shipley, P. R., Hopke, J. N., O’Hare, T., Xiang, L., Noel, J. P., et al. (2003).Expression and characterization of the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene synthase from Streptomyces coelicolor A3(2). Journal of IndustrialMicrobiology & Biotechnology, 30, 510–515.

Katsuyama, Y., & Horinouchi, S. (2010). Microbial type III polyketide synthases. Compre-hensive Natural Products II, 1, 147–170.

Kawasaki, T., Hayashi, Y., Kuzuyama, T., Furihata, K., Itoh, N., Seto, H., et al. (2006). Bio-synthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: Identi-fication and heterologous expression of the gene cluster. Journal of Bacteriology, 188,1236–1244.

Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., & Hopwood, D. A. (2000). PracticalStreptomyces genetics.Norwich Research Park, Colney, Norwich NR4 7UH, England:John Innes Centre.

Kumano, T., Tomita, T., Nishiyama, M., & Kuzuyama, T. (2010). Functional characteriza-tion of the promiscuous prenyltransferase responsible for furaquinocin biosynthesis:Identification of a physiological polyketide substrate and its prenylated reaction products.The Journal of Biological Chemistry, 285, 39663–39671.

Kuzuyama, T., Noel, J. P., & Richard, S. B. (2005). Structural basis for the promiscuous bio-synthetic prenylation of aromatic natural products. Nature, 435, 983–987.

Li, S., Gruschow, S., Dordick, J. S., & Sherman, D. H. (2007). Molecular analysis of the roleof tyrosine 224 in the active site of Streptomyces coelicolor RppA, a bacterial type III poly-ketide synthase. The Journal of Biological Chemistry, 282, 12765–12772.

Miyanaga, A., Funa, N., Awakawa, T., & Horinouchi, S. (2008). Direct transfer of startersubstrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipidsynthesis. Proceedings of the National Academy of Sciences of the United States of America, 105,871–876.

Molnos, J., Gardiner, R., Dale, G. E., & Lange, R. (2003). A continuous coupled enzymeassay for bacterial malonyl-CoA: Acyl carrier protein transacylase (FabD). Analytical Bio-chemistry, 319, 171–176.

Moore, B. S., Hertweck, C., Hopke, J. N., Izumikawa, M., Kalaitzis, J. A., Nilsen, G., et al.(2002). Plant-like biosynthetic pathways in bacteria: From benzoic acid to chalcone.Journal of Natural Products, 65, 1956–1962.

Nakano, C., Funa, N., Ohnishi, Y., & Horinouchi, S. (2012). TheO-methyltransferase SrsBcatalyzes the decarboxylative methylation of alkylresorcylic acid during phenolic lipidbiosynthesis by Streptomyces griseus. Journal of Bacteriology, 194, 1544–1551.

Nakano, C., Ozawa, H., Akanuma, G., Funa, N., & Horinouchi, S. (2009). Biosynthesis ofaliphatic polyketides by type III polyketide synthase and methyltransferase in Bacillus sub-tilis. Journal of Bacteriology, 191, 4916–4923.

Neumann, C. S., Walsh, C. T., & Kay, R. R. (2010). A flavin-dependent halogenase cat-alyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducingfactor 1. Proceedings of the National Academy of Sciences of the United States of America, 107,5798–5803.


Oguro, S., Akashi, T., Ayabe, S., Noguchi, H., & Abe, I. (2004). Probing biosynthesis ofplant polyketides with synthetic N-acetylcysteamine thioesters. Biochemical and Biophys-ical Research Communications, 325, 561–567.

Pfeifer, V., Nicholson, G. J., Ries, J., Recktenwald, J., Schefer, A. B., Shawky, R. M., et al.(2001). A polyketide synthase in glycopeptide biosynthesis: The biosynthesis of the non-proteinogenic amino acid (S)-3,5-dihydroxyphenylglycine. The Journal of BiologicalChemistry, 276, 38370–38377.

Schroder, J., Raiber, S., Berger, T., Schmidt, A., Schmidt, J., Soares-Sello, A. M., et al.(1998). Plant polyketide synthases: A chalcone synthase-type enzyme which performsa condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylatedchalcones. Biochemistry, 37, 8417–8425.

Seshime, Y., Juvvadi, P. R., Kitamoto, K., Ebizuka, Y., & Fujii, I. (2010). Identification ofcsypyrone B1 as the novel product ofAspergillus oryzae type III polyketide synthase CsyB.Bioorganic & Medicinal Chemistry, 18, 4542–4546.

Seshime, Y., Juvvadi, P. R., Kitamoto, K., Ebizuka, Y., Nonaka, T., & Fujii, I. (2010).Aspergillus oryzae type III polyketide synthase CsyA is involved in the biosynthesis of3,5-dihydroxybenzoic acid. Bioorganic & Medicinal Chemistry Letters, 20, 4785–4788.

Song, L., Barona-Gomez, F., Corre, C., Xiang, L., Udwary, D. W., Austin, M. B., et al.(2006). Type III polyketide synthase beta-ketoacyl-ACP starter unit andethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genomemining. Journal of the American Chemical Society, 128, 14754–14755.

Takano, E., White, J., Thompson, C. J., & Bibb, M. J. (1995). Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp. Gene, 166,133–137.

Wenzel, S. C., Bode, H. B., Kochems, I., & Muller, R. (2008). A type I/type III polyketidesynthase hybrid biosynthetic pathway for the structurally unique ansa compoundkendomycin. Chembiochem, 9, 2711–2721.

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[Methods in Enzymology] Natural Product Biosynthesis by Microorganisms and Plants, Part A Volume 515 || Type III Polyketide Synthases in Microorganisms