Synthesis of polyketides from diketides

Cell-free systems which effect the production of polyketides employing modular polyketide synthases are described. Libraries of new and/or known polyketides may also be produced in cell-free systems employing aromatic PKS, modular PKS or both.

TECHNICAL FIELD 
The present invention relates generally to polyketides and polyketide 
synthases. In particular, the invention pertains to novel methods of 
producing polyketides and libraries of polyketides using a cell-free 
system. 
BACKGROUND OF THE INVENTION 
Polyketides are a large, structurally diverse family of natural products. 
Polyketides possess a broad range of biological activities including 
antibiotic and pharmacological properties. For example, polyketides are 
represented by such antibiotics as tetracyclines and erythromycin, 
anticancer agents including daunomycin, immunosuppressants, for example 
FK506 and rapamycin, and veterinary products such as monensin and 
avermectin. 
Polyketides occur in most groups of organisms and are especially abundant 
in a class of mycelial bacteria, the actinomycetes, which produce various 
polyketides. Polyketide synthases (PKSs) are multifunctional enzymes 
related to fatty acid synthases (FASs). PKSs catalyze the biosynthesis of 
polyketides through repeated (decarboxylative) Claisen condensations 
between acylthioesters, usually acetyl, propionyl, malonyl or 
methylmalonyl. Following each condensation, they introduce structural 
variability into the product by catalyzing all, part, or none of a 
reductive cycle comprising a ketoreduction, dehydration, and 
enoylreduction on the .beta.-keto group of the growing polyketide chain. 
PKSs incorporate enormous structural diversity into their products, in 
addition to varying the condensation cycle, by controlling the overall 
chain length, choice of primer and extender units and, particularly in the 
case of aromatic polyketides, regiospecific cyclizations of the nascent 
polyketide chain. After the carbon chain has grown to a length 
characteristic of each specific product, it is released from the synthase 
by thiolysis or acyltransfer. Thus, PKSs consist of families of enzymes 
which work together to produce a given polyketide. It is the controlled 
variation in chain length, choice of chain-building units, and the 
reductive cycle, genetically programmed into each PKS, that contributes to 
the variation seen among naturally occurring polyketides. 
Two general classes of PKSs exist. These classifications are well known. 
See, for example, Hopwood, D. A. and Khosla, C., Secondary Metabolites: 
Their Function and Evolution (1992) Wiley Chichester (Ciba Foundation 
Symposium 171) pp. 88-112. 
One class, known as Type I or modular PKSs, is represented by the PKSs 
which catalyze the biosynthesis of complex polyketides such as 
erythromycin and avermectin. These "modular" PKSs include assemblies of 
several large multifunctional proteins carrying, between them, a set of 
separate active sites for each step of carbon chain assembly and 
modification (Cortes, J. et al. Nature (1990) 348:176; Donadio, S. et al. 
Science (1991) 252:675; MacNeil, D. J. et al. Gene (1992) 115:119). The 
active sites required for one cycle of condensation and reduction are 
clustered as "modules" (Donadio et al. Science (1991), supra; Donadio, S. 
et al. Gene (1992) 111:51). For example, 6-deoxyerythronolide B synthase 
(DEBS) consists of the three multifunctional proteins, DEBS 1, DEBS 2, and 
DEBS 3 (Caffrey, P. et al. FEBS Letters (1992) 304:225), each of which 
possesses two modules. (See FIG. 1.) 
As described below, a module contains at least the minimal activities 
required for the condensation of an extender unit onto a growing 
polyketide chain; the minimal activities required are a ketosynthase (KS), 
an acyl transferase (AT) and an acyl carrier protein (ACP). Additional 
activities for further modification reactions such as a reductive cycle or 
cyclization may also be included in a module. Structural diversity occurs 
in this class of PKSs from variations in the number and type of active 
sites in the PKSs. This class of PKSs displays a one-to-one correlation 
between the number and clustering of active sites in the primary sequence 
of the PKS and the structure of the polyketide backbone. 
The second class of PKSs, the aromatic or Type II PKSs, has a single set of 
iteratively used active sites (Bibb, M. J. et al. EMBO J. (1989) 8:2727; 
Sherman, D. H. et al. EMBO J. (1989) 8:2717; Fernandez-Moreno, M. A. et 
al. J. Biol. Chem. (1992) 267:19278). Streptomyces is an actinomycete 
which is an abundant producer of aromatic polyketides. In each 
Streptomyces aromatic PKS so far studied, carbon chain assembly requires 
the products of three open reading frames (ORFs). (See FIG. 2.) ORF1 
encodes a ketosynthase (KS) and an acyltransferase (AT) active site 
(KS/AT); ORF2 encodes a chain length determining factor (CLF), a protein 
similar to the ORF1 product but lacking the KS and AT motifs; and ORF3 
encodes a discrete acyl carrier protein (ACP). Some gene clusters also 
code for a ketoreductase (KR) and a cyclase, involved in cyclization of 
the nascent polyketide backbone. However, it has been found that only the 
KS/AT, CLF, and ACP, need be present in order to produce an identifiable 
polyketide. 
Fungal PKSs, such as the 6-methylsalicylic acid PKS, consist of a single 
multidomain polypeptide which includes all the active sites required for 
the biosynthesis of 6-methylsalicylic acid (Beck, J. et al. Eur. J. 
Biochem. (1990) 192:487-498; Davis, R. et al. Abstr. of the Genetics of 
Industrial Microorganism Meeting, Montreal, abstr. P288 (1994)). Fungal 
PKSs incorporate features of both modular and aromatic PKSs. 
Streptomyces coelicolor produces the blue-pigmented polyketide, 
actinorhodin. The actinorhodin gene cluster (act), has been cloned 
(Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462; Malpartida, F. 
and Hopwood, D. A. Mol. Gen. Genet. (1986) 205:66) and completely 
sequenced (Fernandez-Moreno et al. J. Biol. Chem. (1992), supra; Hallam, 
S. E. et al. Gene (1988) 74:305; Fernandez-Moreno, M. A. et al. Cell 
(1991) 66:769; Caballero, J. et al. Mol. Gen. Genet. (1991) 230:401). The 
cluster encodes the PKS enzymes described above, a cyclase and a series of 
tailoring enzymes involved in subsequent modification reactions leading to 
actinorhodin, as well as proteins involved in export of the antibiotic and 
at least one protein that specifically activates transcription of the gene 
cluster. Other genes required for global regulation of antibiotic 
biosynthesis, as well as for the supply of starter (acetyl-CoA) and 
extender (malonyl-CoA) units for polyketide biosynthesis, are located 
elsewhere in the genome. 
The act gene cluster from S. coelicolor has been used to produce 
actinorhodin in S. parvulus. Malpartida, F. and Hopwood, D. A. Nature 
(1984) 309:462. 
Bartel et al. J. Bacteriol. (1990) 172:4816-4826, recombinantly produced 
aloesaponarin II using S. galilaeus transformed with an S. coelicolor act 
gene cluster consisting of four genetic loci, actI, actIII, actIV and 
actVII. Hybrid PKSs, including the basic act gene set but with ACP genes 
derived from granaticin, oxytetracycline, tetracenomycin and frenolicin 
PKSs, have also been designed which are able to express functional 
synthases. Khosla, C. et al. J. Bacteriol. (1993) 175:2197-2204. Hopwood, 
D. A. et al. Nature (1985) 314:642-644, describes the production of hybrid 
aromatic polyketides, using recombinant techniques. Sherman, D. H. et al. 
J. Bacteriol. (1992) 174:6184-6190, reports the transformation of various 
S. coelicolor mutants, lacking different components of the act PKS gene 
cluster, with the corresponding granaticin (gra) genes from S. 
violaceoruber, in trans. 
Although the above described model for complex polyketide biosynthesis by 
modular (Type I) PKSs has been substantiated by radioisotope and stable 
isotope labeling experiments, heterologous expression, directed 
mutagenesis, and in vitro studies on partially active proteins, cell-free 
enzymatic synthesis of complex polyketides has proved unsuccessful despite 
more than 30 years of intense efforts (Caffrey et al. FEBS Letters (1992), 
supra; Aparicio, J. F. et al. J. Biol. Chem. (1994) 269:8524; Bevitt, D. 
J. et al. Eur. J. Biochem. (1992) 204:39; Caffrey, P. et al. Eur. J. 
Biochem. (1991) 195:823); Leadlay, P. F. et al. Biochem. Soc. Trans. 
(1993) 21:218; Marsden, A. F. A. et al. Science (1994) 263:378; 
Wawszkiewicz, E. J. et al. Biochemische Z. (1964) 340:213; Corcoran, J. W. 
et al. in Proc. 5th Int. Congr. Chemother. (Vienna, 1967), Abstracts of 
Communications, p. 35; Corcoran, J. W. et al. in Antibiotics IV. 
Biosynthesis (1982) Corcoran, J. W., Ed. (Springer-Verlag, New York) p. 
146; Roberts, G. FEBS Lett. (1983) 159:13; Roberts, G. et al. Biochemical 
Soc. Trans. (1984) 12:642; Hunaiti, A. A. et al. Antimicrob. Agents. 
Chemother. (1984) 25:173). This is due, in part, to the difficulty of 
isolating fully active forms of these large, poorly expressed 
multifunctional proteins from naturally occurring producer organisms and, 
in part, to the relative lability of intermediates formed during the 
course of polyketide biosynthesis. For example, the three DEBS proteins 
have been purified individually from the natural producer organism, 
Saccharopolyspora erythraea (Caffrey et al. FEBS Letters (1992), supra; 
Aparicio et al. J. Biol. Chem. (1994), supra; Bevitt et al. Eur. J. 
Biochem. (1992), supra; Caffrey et al. Eur. J. Biochem. (1991), supra; 
Leadlay et al. Biochem. Soc. Trans. (1993); Marsden et al. Science (1994), 
supra). Studies on the purified enzymes facilitated clarification of their 
stereospecificity, showing that 2S-methylmalonyl-CoA is the extender 
substrate for all 6 acyltransferase sites (Marsden et al. Science (1994), 
supra), thereby implying that the differing configurations of the 
methyl-branched centers result from selective epimerization of specific 
enzyme-bound intermediates. However, the lack of a full turnover assay 
prevented these investigators from probing the mechanisms of the enzyme 
complex in greater detail. 
In an attempt to overcome some of these limitations, modular PKS subunits 
have been expressed in heterologous hosts such as E. coli (Aparicio et al. 
J. Biol. Chem. (1994), supra; Bevitt et al. Eur. J. Biochem. (1992), 
supra; Caffrey et al. Eur. J. Biochem. (1991), supra; Leadlay et al. 
Biochem. Soc. Trans. (1993);) and S. coelicolor (Kao, C. M. et al. Science 
(1994) 265:509; International Publication No. WO 95/08548 (published Mar. 
30, 1995)). Whereas the proteins expressed in E. coli are not fully 
active, heterologous expression in S. coelicolor resulted in production of 
active protein as demonstrated by the production of 6-deoxyerythronolide 
("6-DEB") in vivo. Cell-free enzymatic synthesis of polyketides from 
simpler PKSs such as the 6-methylsalicylate synthase (Dimroth, P. et al. 
Eur. J. Biochem. (1970) 13:98; Beck, J. et al. Eur. J. Biochem. (1990) 
192:487); Spencer J. B. et al. Biochem. J. (1992) 288:839), chalcone 
synthase (Lanz, T. et al. J. Biol. Chem. (1991) 266:9971 (1991)), and the 
tetracenomycin synthase (Shen, B. et al. Science (1993) 262:1535) has been 
reported. 
However, no one to date has described the cell-free enzymatic synthesis of 
polyketides from modular PKSs, or has used a cell-free system to produce 
libraries containing a multiplicity of different polyketides. 
SUMMARY OF THE INVENTION 
The present invention provides methods to produce both novel and known 
polyketides. In one embodiment, a cell-free system comprising a modular 
PKS effects synthesis of a polyketide when incubated with an appropriate 
substrate set. 
In another embodiment, the invention is directed to a method of 
synthesizing a library containing a multiplicity of different polyketides 
by use of cell-free systems and to a matrix of cell-free subsystems for 
the production of these libraries. 
Thus, in one aspect, the invention is directed to a method comprising 
providing one or more proteins comprising at least two modules of a 
modular polyketide synthase in a cell-free system; adding to said system 
at least one starter unit and at least one extender unit; incubating said 
cell-free system containing said starter unit and extender unit under 
conditions wherein said polyketide is synthesized; and optionally 
recovering the polyketide from the cell-free system. 
In another aspect, the invention is directed to a matrix for the production 
of a polyketide library which comprises a series of cell-free subsystems 
each containing one or more polyketide synthase proteins comprising 
enzymatic activities that effect the coupling of at least one extender 
unit to a starter unit, including a growing polyketide chain; each said 
subsystem containing at least one starter unit and at least one extender 
unit; and wherein at least one enzymatic activity or at least one extender 
unit or at least one starter unit or is different as between each 
subsystem. 
The invention in another aspect is directed to methods to prepare libraries 
of polyketides using these matrices. 
In yet another aspect, the invention is directed to method to produce a 
desired polyketide which method comprises: providing a system comprising a 
functional modular polyketide synthase (PKS), or a functional portion 
thereof, wherein said PKS cannot be loaded with a natural first-module 
starter unit, or wherein, once loaded, cannot catalyze the condensation of 
an extender unit to the first-module starter unit to produce a polyketide 
intermediate; adding to said system a starter unit that is a substrate for 
the PKS; incubating the system containing said PKS and said starter unit 
substrate under conditions wherein said polyketide is synthesized; and 
optionally recovering the polyketide. 
In still another aspect, the invention is directed to a functional modular 
polyketide synthase system, or a functional portion thereof, which cannot 
be loaded with a natural first-module starter unit, or which, once loaded, 
cannot catalyze the condensation of an extender unit to the first-module 
starter unit to produce a polyketide intermediate.

DETAILED DESCRIPTION OF THE INVENTION 
The invention provides cell-free systems for the synthesis of novel and 
known polyketides and of polyketide libraries. In the case of modular 
polyketide synthases, cell-free production of polyketides using these 
enzymes has not heretofore been accomplished. Although, as described 
above, cell-free synthesis of polyketides by some aromatic synthases has 
been achieved, these systems have not been used in constructing libraries 
of polyketides, said libraries being useful as sources of compounds to be 
screened for pharmacological or other activities. 
The use of cell-free systems for the construction of such libraries has 
several advantages. First, permeability problems are eliminated, so that 
substrates can be used which might otherwise be ineffective due to failure 
to permeate the cell. Second, variability in product due to differential 
permeability is eliminated. Third, alternative metabolic events are 
minimized or eliminated so that the reaction proceeds cleanly to convert 
substrates to polyketide products. Fourth, there are greater possibilities 
for regulating the conditions under which the polyketide synthase genes 
are expressed and the polyketides are produced. For example, cofactors 
which are ordinarily useful in the synthesis of a given polyketide, such 
as NADPH, can be supplied or withheld. Finally, it is possible to use 
"unnatural" substrates for a given synthase since cellular mechanisms for 
providing the substrate to the synthase are eliminated. As a result of 
using cell-free systems to create libraries, a greater variety of 
polyketides may be synthesized than would have been possible had 
production been limited to intracellular synthesis. 
Given a particular cell-free system containing polyketide synthase 
proteins, the nature of the polyketide ultimately produced will depend on 
the substrates provided and on the conditions with respect to cofactors, 
etc. In order to explore the possibilities for a given cell-free system 
with a given complement of PKS proteins, it will be advantageous to 
subdivide the cell-free system into "subsystems" with variation in these 
factors. In order to be workable, the cell-free system or subsystem must 
contain polyketide synthase proteins with enzymatic activities sufficient 
to effect the condensation of an "extender unit" onto a "starter unit," 
where a "starter unit" may include a growing polyketide chain. Because the 
cell-free system offers greater promiscuity of starter and extender units, 
a number of subsystems containing a variety of starter and extender units, 
as well as differing conditions may result in a corresponding variety of 
polyketides. 
As used in the present application, a "starter unit" refers to a substance 
to which additional Claisen condensations may be effected. The starter 
unit may be one which is natively regarded as a starter, or may be what 
would in the native state be an intermediate growing polyketide chain. A 
"first-module starter unit" is an acyl thioester that can be loaded onto 
the appropriate active site of the first module of a PKS. A "natural 
starter unit" is an acyl thioester which upon extension by the PKS 
produces the natural polyketide product. An "unnatural starter unit" is an 
acy thioester which upon extension by the PKS produces a polyketide 
product other than that normally produced by the PKS during metabolism. 
A relaxed specificity of modular PKS for starter units under in vitro 
conditions has been reported by Pieper et al. Nature (1995) 378:263-266. 
Known starter units include, for example acetyl-CoA, propionyl-CoA, 
butyryl-CoA, isobutyryl-CoA, cyclohexanoyl-CoA, aminohydroxy benzoyl-CoA, 
and intermediate polyketide chains. In addition, an extender unit may be 
used as a source of starter units (see Pieper et al. Biochem. (1996) 
35:2054-2060). Thus, in a system capable of producing a polyketide, the 
starter unit and the extender unit used therein may be the same or 
different. 
The starter unit is then extended by virtue of the activity of the synthase 
contained in the cell-free system or subsystem. Extender units are added 
to the carboxy terminus of a growing polyketide and the nature of the 
extender unit is determined by the acyl transferase (AT) activity. 
Suitable extender units include malonyl-CoA, methylmalonyl-CoA and 
ethylmalonyl-CoA. sequence comparisons have identified the characteristics 
of malonyl-CoA-specific AT and methylmalonyl-CoA-specific AT (Haydock et 
al. FEBS Lett. (1995) 374:246-248. When methylmalonyl-CoA or 
ethylmalonyl-CoA is used as the extender unit, a chiral center is 
generated in the condensation. 
The reductive cycle which occurs in either aromatic or modular PKS systems 
depends both on the presence of suitable ketoreductase (KR) activity as 
well as the reaction conditions in an in vitro system. The absence of 
reductive activity yields a ketone; reduction generates an alcohol 
containing a chiral center. If a dehydratase (DH) activity is also 
present, an alkene results which eliminates the chiral center. 
Additionally, an enoyl reductase activity may be present which reduces the 
.beta.-keto group to a methylene group. Thus, for a single condensation of 
an extender unit, there are five theoretically possible reductive cycle 
outcomes. This variation is effected both by the cofactor conditions and 
by the nature of the proteins in the cell-free system to be employed. 
Various other catalytic activities resulting in cyclization, aromatization, 
chain-length limitation, and the like are determined mainly by the nature 
of the synthase proteins. 
Thus, the availability of cell-free systems for the production of ketides 
provides a unique opportunity to generate libraries of polyketides by 
varying the nature of the synthase, the nature of the extender unit, the 
nature of the starter unit and the nature of the conditions. A simple 
matrix can be envisioned whereby cell-free systems containing varying 
synthase catalytic activities, but at a minimum the capability to extend a 
starter unit, including a growing polyketide chain through an additional 
Claisen condensation, can be employed. Each of these cell-free systems can 
be subdivided into subsystems in which the remaining variables are 
manipulated to effect the eventual outcome of synthesis. Thus, a series of 
subsystems containing identical polyketide synthase activities can be 
supplied different starter units, different extender units, and incubated 
under different conditions so as to result in a multiplicity of 
polyketides. Similar variation can be employed with respect to subsystems 
of cell-free systems containing different PKS activities, thus resulting 
in a matrix wherein one dimension may be envisioned as varying the nature 
of the cell-free system itself and the other dimension comprises variation 
in the substrates and conditions. 
A. Definitions 
In describing the present invention, the following terms will be employed, 
and are intended to be defined as indicated below. 
By a "cell-free system" is intended a cell lysate, cell extract or other 
preparation in which substantially all of the cells in the preparation 
have been disrupted or otherwise processed so that all or selected 
cellular components, e.g., organelles, proteins, nucleic acids, the cell 
membrane itself (or fragments or components thereof), or the like, are 
released from the cell or resuspended into an appropriate medium and/or 
purified from the cellular milieu. Cell-free systems include, of course, 
reaction mixtures prepared from purified or isolated proteins and suitable 
reagents and buffers. 
By a "cell-free subsystem" is meant either a portion of a cell-free 
system--i.e., the cell-free system that results when a given composition 
is effectively subdivided into two or more separate compartments for 
independent catalysis, or is a reaction mixture which contains the same 
complement of polyketide synthase enzymatic activity. Thus, a "subsystem" 
of a given "cell-free system" may differ in composition by virtue of 
differing substrates or conditions, but contains the same catalytic 
polyketide synthase activities. 
By "purified" or "isolated" is meant, when referring to a polypeptide or 
nucleotide sequence, that the indicated molecule is separate and discrete 
from the whole organism from which the molecule is normally associated in 
nature. Thus, a protein contained in a cell free extract would constitute 
a "purified" or "isolated" protein, as would a protein further purified 
from a cell-free extract. In addition, a "purified" or "isolated" protein 
refers to a protein which has been synthetically or recombinantly produced 
and, optionally, purified from the host cell. An "isolated" nucleotide 
sequence is a nucleotide sequence separate and discrete from the whole 
organism with which the sequence is found in nature; or a sequence devoid, 
in whole or part, of sequences normally associated with it in nature; or a 
sequence, as it exists in nature, but having heterologous sequences (as 
defined below) in association therewith. 
A single "module" of a modular PKS gene cluster or a modular polyketide 
synthase refers to sufficient portions of the gene cluster to encode, or 
sufficient portions of the polyketide synthase to include, at least the 
activities required to effect the condensation of a single extender unit 
onto a starter unit or a growing polyketide chain. Thus, the minimal 
activities required include a ketosynthase (KS) an acyltransferase (AT) 
and an acyl carrier protein (ACP). All three of these activities are 
required for the condensation of a single extender unit onto the growing 
polyketide chain. At least one module for the effective synthesis of a 
polyketide must contain an additional AT and ACP in order to effect the 
initial condensation. In addition, and optionally, the module may include 
a ketoreductase activity (KR), a cyclase, a dehydratase (DH) an enoyl 
reductase (ER) and/or a thioesterase (TE). 
In native forms of aromatic polyketide synthases, portions of the required 
activities may occur on different proteins. In the case of aromatic 
polyketide synthases also, a ketosynthase (KS), an acyl transferase (AT) 
and an acyl carrier protein (ACP) must be present to effect the 
condensation of a single extender unit onto a starter unit or a growing 
polyketide. Various activities associated with reduction, cyclization, 
aromatization and further derivatization may also be present. There must 
also be at least one chain-length limiting factor (CLF). 
The phrases "PKS gene cluster" and "PKS gene set" are used interchangeably 
to mean any set of PKS genes capable of producing a functional PKS when 
under the direction of one or more compatible control elements, as defined 
below, in a host cell. A functional PKS is one which catalyzes the 
condensation of at least one extender unit onto a growing 
polyketide--i.e., has at least one functional module, or extension 
function either in vivo or in vitro. A "PKS gene cluster" thus need not 
include all of the genes found in the corresponding cluster in nature. 
Furthermore, the cluster can include PKS genes derived from a single 
species, or may be hybrid in nature with, e.g., a coding sequence derived 
from a cluster for the synthesis of a particular polyketide replaced with 
a corresponding coding sequence from a cluster for the synthesis of 
another polyketide. Hybrid clusters can include genes derived from either 
or both modular and aromatic PKSs. The genes included in the gene cluster 
need not be the native genes, but can be mutants or analogs thereof. 
Mutants or analogs may be prepared by the deletion, insertion or 
substitution of one or more nucleotides of the coding sequence. Techniques 
for modifying nucleotide sequences, such as site-directed mutagenesis, are 
described in, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, 
supra; Nucleic Acid Hybridization, supra. 
A "PKS gene cluster" may also contain genes coding for modifications to the 
core polyketide produced by the PKS, including, for example, genes 
encoding post-polyketide synthesis enzymes derived from natural products 
pathways such as O-methyltransferases and glycosyltransferases. A "PKS 
gene cluster" may further include genes encoding hydroxylases, methylases 
or other alkylases, oxidases, reductases, glycotransferases, lyases, ester 
or amide syntheses, and various hydrolases such as esterases and amidases. 
As explained further below, the genes included in the PKS gene cluster need 
not be on the same plasmid or, if present on the same plasmid, can be 
controlled by the same or different control sequences. 
A "host cell" is a cell and the progeny and cultures thereof derived from a 
procaryotic microorganism or a eucaryotic cell line cultured as a 
unicellular entity, which can be, or has been, used as a recipient for 
recombinant vectors bearing the PKS gene clusters of the invention. It is 
understood that the progeny of a single parental cell may not necessarily 
be completely identical in morphology or in genomic or total DNA 
complement to the original parent, due to accidental or deliberate 
mutation. Progeny of the parental cell which are sufficiently similar to 
the parent to be characterized by the relevant property, such as the 
presence of a nucleotide sequence encoding a desired PKS, are included in 
the definition, and are covered by the above terms. 
The term "heterologous" as it relates to nucleic acid sequences such as 
coding sequences and control sequences, denotes sequences that are not 
normally associated with a region of a recombinant construct, and/or are 
not normally associated with a particular cell. Thus, a "heterologous" 
region of a nucleic acid construct is an identifiable segment of nucleic 
acid within or attached to another nucleic acid molecule that is not found 
in association with the other molecule in nature. For example, a 
heterologous region of a construct could include a coding sequence flanked 
by sequences not found in association with the coding sequence in nature. 
Another example of a heterologous coding sequence is a construct where the 
coding sequence itself is not found in nature (e.g., synthetic sequences 
having codons different from the native gene). Similarly, a host cell 
transformed with a construct which is not normally present in the host 
cell would be considered heterologous for purposes of this invention. 
Allelic variation or naturally occurring mutational events do not give 
rise to heterologous DNA, as used herein. 
A "coding sequence" or a sequence which "encodes" a protein or peptide is a 
nucleic acid sequence which is transcribed into mRNA (in the case of DNA) 
or translated into a polypeptide (in the case of mRNA) in vitro or in vivo 
when placed under the control of appropriate regulatory sequences. 
"Control sequences" refers collectively to promoter sequences, ribosome 
binding sites, polyadenylation signals, transcription termination 
sequences, upstream regulatory domains, enhancers, and the like, which 
collectively provide for the transcription and/or translation of a coding 
sequence in a host cell. Not all of these control sequences need always be 
present in a recombinant vector so long as the desired gene is capable of 
being transcribed and translated. 
"Operably linked" refers to an arrangement of elements wherein the 
components so described are configured so as to perform their usual 
function. Thus, control sequences operably linked to a coding sequence are 
capable of effecting the expression of the coding sequence. The control 
sequences need not be contiguous with the coding sequence, so long as they 
function to direct the expression thereof. Thus, for example, intervening 
untranslated yet transcribed sequences can be present between a promoter 
sequence and the coding sequence and the promoter sequence can still be 
considered "operably linked" to the coding sequence. 
A "library" or "combinatorial library" of polyketides is intended to mean a 
collection of a multiplicity of different polyketides. The differences in 
the members of the library may result from their being produced by 
different PKS cell-free systems that contain any combination of native, 
homolog or mutant genes from aromatic, modular or fungal PKSs. The 
differences in the members of the library may also result from the use of 
different starter units, extender units and conditions. The PKSs in the 
cell-free systems used to generate the library may be derived from a 
single system, such as act, fren, gra, tcm, whiE, gris, ery, or the like, 
and may optionally include genes encoding tailoring enzymes which are 
capable of catalyzing the further modification of a polyketide. 
Alternatively, the combination of synthase activities can be rationally or 
stochastically derived from an assortment of synthases, e.g., a synthase 
system can be constructed to contain the KS/AT component from an act PKS, 
the CLF component from a gra PKS and a ACP component from a fren PKS. The 
synthase can optionally include other enzymatic activities as well. 
The variety of polyketides in the library may thus result from varying the 
nature of the synthase or varying the nature of the substrates used to 
construct the polyketides or both. Preferably, the library is produced as 
a result of culturing a matrix which varies the nature of the synthase 
systems in one dimension and the nature of the substrates and/or 
incubation conditions in the other. The library of polyketides thus 
produced can be tested or screened for biological, pharmacological or 
other activity. 
"Optional" or "optionally" means that the subsequently described event or 
circumstance may or may not occur, and that the description includes 
instances where said event or circumstance occurs and instances where it 
does not. For example, the phrase "optionally further purified" means that 
further purification may or may not be performed and that the description 
includes both the performance and the lack of performance of such further 
purification. 
B. General Methods 
The polyketides produced by the invention methods can be screened for use 
as therapeutic agents to treat a number of disorders, depending on the 
type of polyketide in question. For example, several of the polyketides 
produced by the present method will find use as immunosuppressants, as 
anti-tumor agents, as well as for the treatment of viral, bacterial and 
parasitic infections. 
By use of the cell-free systems of the invention, a wide variety of 
polyketides can be synthesized as candidates. As explained above, the use 
of cell-free technology permits greater flexibility in choice of substrate 
and less interference in the synthesis of the desired polyketide from 
competing metabolic reactions. The ability to produce polyketides in a 
cell-free system also provides a powerful tool for characterizing PKSs and 
the mechanism of their actions. 
The practice of the present invention will employ, unless otherwise 
indicated, conventional methods of chemistry, microbiology, molecular 
biology and recombinant DNA techniques within the skill of the art. Such 
techniques are explained fully in the literature. See, e.g., Sambrook, et 
al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: 
A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide 
Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. 
Hames & S. Higgins, eds., Current Edition); Transcription and Translation 
(B. Hames & S. Higgins, eds., Current Edition); H. O. House, Modern 
Synthetic Reactions, Second Edition (Menlo Park, Calif.: The 
Benjamin/Cummings Publishing Company, 1972); and J. March, Advanced 
organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: 
Wiley-Interscience, 1992). 
All publications, patents and patent applications cited herein, whether 
supra or infra, are hereby incorporated by reference in their entirety. 
As used in this specification and the appended claims, the singular forms 
"a," "an" and "the" include plural references unless the content clearly 
dictates otherwise. Thus, reference to "a polyketide synthase" includes 
mixtures of polyketide synthases, reference to "a PKS enzyme" includes 
mixtures of such enzymes, and the like. 
1. Recombinant Production of PKS 
The invention, for the production and isolation of a significant quantity 
of functional modular PKS enzymes, in particular, makes use of host cells 
transformed with recombinant vectors for the production of these enzymes. 
Aromatic and hybrid PKS may be produced in this way as well. The host 
cells may be genetically engineered cells which have their naturally 
occurring PKS genes substantially deleted. 
Host cells for the production of the functional PKS enzymes effective in 
cell-free systems can be derived from any organism with the capability of 
harboring a recombinant PKS gene cluster, and can be derived from either 
procaryotic or eucaryotic organisms. However, preferred host cells are 
those constructed from the actinomycetes, a class of mycelial bacteria 
which are abundant producers of a number of polyketides. A particularly 
preferred genus for use in production of the PKSs is Streptomyces. Thus, 
for example, S. ambofaciens, S. avermitilis, S. azureus, S. cinnamonensis, 
S. coelicolor, S. curacoi, S. erythraeus, S. fradiae, S. galilaeus, S. 
glaucescens, S. hygroscopicus, S. lividans, S. parvulus, S. peucetius, S. 
rimosus, S. roseofulvus, S. thermotolerans, S. violaceoruber, among 
others, will provide convenient host cells, with S. coelicolor being 
preferred. (See, e.g., Hopwood, D. A. and Sherman, D. H. Ann. Rev. Genet. 
(1990) 24:37-66; O'Hagan, D. The Polyketide Metabolites (Ellis Horwood 
Limited, 1991), for a description of various polyketide-producing 
organisms and their natural products.) 
The above-described cells can be genetically engineered by deleting the 
naturally occurring PKS genes therefrom, using standard techniques, such 
as by homologous recombination. (See, e.g., Khosla, C. et al. Molec. 
Microbiol. (1992) 6:3237). For example, native strains of S. coelicolor 
produce a PKS which catalyzes the biosynthesis of the aromatic polyketide 
actinorhodin. The strain, S. coelicolor CH999 (as described in WO 
95/08548, supra), was constructed by deleting, via homologous 
recombination, the entire natural act cluster from the chromosome of S. 
coelicolor CH1 (Khosla et al. Molec. Microbiol. (1992), supra), a strain 
lacking endogenous plasmids and carrying a stable mutation that blocks 
biosynthesis of another pigmented S. coelicolor antibiotic, 
undecylprodigiosin. 
The host cells described above can be transformed with one or more vectors, 
collectively encoding at least a set of functional PKS activities 
sufficient to effect condensation of an extender unit, or a cocktail 
comprising a random assortment of PKS associated sequences with this 
activity. The vector(s) can include native or hybrid combinations of PKS 
subunits or cocktail components, or mutants thereof. 
In order to produce the PKS for practice of the cell-free synthesis, 
recombinant vector(s) can be constructed that include genes from a single 
PKS aromatic or modular gene cluster, or may comprise hybrid PKS gene 
clusters with, e.g., a gene or part of a gene from one cluster replaced by 
the corresponding portion from another gene cluster. For example, it has 
been found that ACPs are readily interchangeable among different aromatic 
synthases without an effect on product structure. Furthermore, a given KR 
can recognize and reduce polyketide chains of different chain lengths. 
Accordingly, these coding sequences are freely interchangeable in the 
constructs described herein. Thus, the PKS gene clusters used to produce 
the PKS enzymes can be derived from any combination of PKS gene sequences 
which ultimately function to produce a PKS that condenses at least one 
extender unit into a growing polyketide. 
Examples of hybrid clusters include clusters with coding sequences derived 
from two or more of the act gene cluster, the whiE gene cluster, 
frenolicin (fren), granaticin (gra), tetracenomycin (tcm), 
6-methylsalicylic acid (6-msas), oxytetracycline (otc), tetracycline 
(tet), erythromycin (ery), griseusin (gris), nanaomycin, medermycin, 
daunorubicin, tylosin, carbomycin, spiramycin, avermectin, monensin, 
nonactin, curamycin, rifamycin and candicidin synthase gene clusters, 
among others. A number of hybrid gene clusters have been constructed 
having components derived from the act, fren, tcm, gris and gra gene 
clusters (see, WO 95/08548). Several of the hybrid clusters were able to 
functionally express both novel and known polyketides in S. coelicolor 
CH999. However, other hybrid gene clusters, as described above, can easily 
be produced and screened using the disclosure herein, for the production 
of identifiable polyketides. 
The recombinant vectors, harboring the gene clusters or random assortment 
of PKS genes, modules, active sites or portions thereof described above, 
can be conveniently generated using techniques known in the art. For 
example, the PKS subunits of interest can be obtained from an organism 
that expresses the same, using recombinant methods, such as by screening 
cDNA or genomic libraries, derived from cells expressing the gene, or by 
deriving the gene from a vector known to include the same. The gene can 
then be isolated and combined with other desired PKS subunits, using 
standard techniques. If the gene in question is already present in a 
suitable expression vector, it can be combined in situ, with, e.g., other 
PKS subunits, as desired. The gene of interest can also be produced 
synthetically, rather than cloned. The nucleotide sequence can be designed 
with the appropriate codons for the particular amino acid sequence 
desired. In general, one will select preferred codons for the intended 
host in which the sequence will be expressed. The complete sequence can be 
assembled from overlapping oligonucleotides prepared by standard methods 
and assembled into a complete coding sequence. See, e.g., Edge (1981) 
Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) 
J. Biol. Chem. 259:6311. 
Mutations can be made to the native PKS subunit sequences and such mutants 
used in place of the native sequence, so long as the mutants are able to 
function with other PKS subunits to collectively catalyze the synthesis of 
an identifiable polyketide. Such mutations can be made to the native 
sequences using conventional techniques such as by preparing synthetic 
oligonucleotides including the mutations and inserting the mutated 
sequence into the gene encoding a PKS subunit using restriction 
endonuclease digestion. (See, e.g., Kunkel, T. A. (1985) Proc. Natl. Acad. 
Sci. USA 82:448; Geisselsoder et al. (1987) BioTechniques 5:786.) 
Alternatively, the mutations can be effected using a mismatched primer 
(generally 10-20 nucleotides in length) which hybridizes to the native 
nucleotide sequence (generally cDNA corresponding to the RNA sequence), at 
a temperature below the melting temperature of the mismatched duplex. The 
primer can be made specific by keeping primer length and base composition 
within relatively narrow limits and by keeping the mutant base centrally 
located. Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer 
extension is effected using DNA polymerase, the product cloned and clones 
containing the mutated DNA, derived by segregation of the primer extended 
strand, selected. Selection can be accomplished using the mutant primer as 
a hybridization probe. The technique is also applicable for generating 
multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. 
Acad. Sci USA (1982) 79:6409. PCR mutagenesis will also find use for 
effecting the desired mutations. 
Random mutagenesis of the nucleotide sequences obtained as described above 
can be accomplished by several different techniques known in the art, such 
as by altering sequences within restriction endonuclease sites, inserting 
an oligonucleotide linker randomly into a plasmid, by irradiation with 
X-rays or ultraviolet light, by incorporating incorrect nucleotides during 
in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing 
synthetic mutants or by damaging plasmid DNA in vitro with chemicals. 
Chemical mutagens include, for example, sodium bisulfite, nitrous acid, 
hydroxylamine, agents which damage or remove bases thereby preventing 
normal base-pairing such as hydrazine or formic acid, analogues of 
nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 
2-aminopurine, or acridine intercalating agents such as proflavine, 
acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNA 
fragments are treated with chemicals, transformed into E. coli and 
propagated as a pool or library of mutant plasmids. 
Large populations of random enzyme variants can be constructed in vivo 
using "recombination-enhanced mutagenesis" as described in U.S. Pat. No. 
5,521,077 to Khosla et al. 
The gene sequences, native or mutant, which collectively encode PKS 
protein(s) at least sufficient to catalyze condensation of an extender 
unit, can be inserted into one or more expression vectors, using methods 
known to those of skill in the art. In order to incorporate a random 
assortment of PKS genes, modules, active sites or portions thereof into am 
expression vector, a cocktail of same can be prepared and used to generate 
the expression vector by techniques well known in the art and described in 
detail below. Expression vectors will include control sequences operably 
linked to the desired PKS coding sequence. Suitable expression systems for 
use with the present invention include systems which function in 
eucaryotic and procaryotic host cells. However, as explained above, 
procaryotic systems are preferred, and in particular, systems compatible 
with Streptomyces spp. are of particular interest. Control elements for 
use in such systems include promoters, optionally containing operator 
sequences, and ribosome binding sites. Particularly useful promoters 
include control sequences derived from PKS gene clusters which result in 
the production of functional PKS enzymes, such as one or more act 
promoters, tcm promoters, spiramycin promoters, and the like. However, 
other bacterial promoters, such as those derived from sugar metabolizing 
enzymes, such as galactose, lactose (lac) and maltose, will also find use 
in the present constructs. Additional examples include promoter sequences 
derived from biosynthetic enzymes such as tryptophan (trp), the 
.beta.-lactamase (bla) promoter system, bacteriophage lambda PL, and T5. 
In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 
4,551,433), which do not occur in nature also function in bacterial host 
cells. 
Other regulatory sequences may also be desirable which allow for regulation 
of expression of the PKS genes relative to the growth of the host cell. 
Regulatory sequences are known to those of skill in the art, and examples 
include those which cause the expression of a gene to be turned on or off 
in response to a chemical or physical stimulus, including the presence of 
a regulatory compound. Other types of regulatory elements may also be 
present in the vector, for example, enhancer sequences. 
Selectable markers can also be included in the recombinant expression 
vectors. A variety of markers are known which are useful in selecting for 
transformed cell lines and generally comprise a gene whose expression 
confers a selectable phenotype on transformed cells when the cells are 
grown in an appropriate selective medium. Such markers include, for 
example, genes which confer antibiotic resistance or sensitivity to the 
plasmid. Alternatively, several polyketides are naturally colored and this 
characteristic provides a built-in marker for selecting cells successfully 
transformed by the present constructs, i.e., that express a functional PKS 
that can be isolated and used to catalytically prepare polyketides in a 
cell-free system. 
The various PKS subunits of interest, or the cocktail of PKS genes, 
modules, active sites, or portions thereof, can be cloned into one or more 
recombinant vectors as individual cassettes, with separate control 
elements, or under the control of, e.g., a single promoter. The PKS 
subunits or cocktail components can include flanking restriction sites to 
allow for the easy deletion and insertion of other PKS subunits or 
cocktail components so that hybrid PKSs can be generated. The design of 
such unique restriction sites is known to those of skill in the art and 
can be accomplished using the techniques described above, such as 
site-directed mutagenesis and PCR. 
Using these techniques plasmid pRM5 was constructed as a shuttle vector for 
the production of the PKS enzymes for use in a cell-free system described 
herein. Plasmid pRM5 includes the genes encoding the actinorhodin PKS 
subunits flanked by PacI and NsiI restriction sites. A new nucleotide 
sequence encoding a PKS flanked by PacI and NsiI sites can be easily 
substituted for the actinrhodin PKS genes. The shuttle plasmid also 
contains the act KR gene (actIII), the cyclase gene (actVII), and a 
putative dehydratase gene (actIV), as well as a ColEI replicon (to allow 
transformation of E. coli), an appropriately truncated SCP2* (low copy 
number) Streptomyces replicon, and the actII-ORF4 activator gene from the 
act cluster, which induces transcription from act promoters during the 
transition from growth phase to stationary phase in the vegetative 
mycelium. pRM5 carries the divergent actI/actIII promoter pair. 
Methods for introducing the recombinant vectors of the present invention 
into suitable hosts are known to those of skill in the art and typically 
include the use of CaCl.sub.2 or other agents, such as divalent cations 
and DMSO. DNA can also be introduced into bacterial cells by 
electroporation. 
The cells modified to contain expression systems for functional PKS 
proteins are then cultured under conditions wherein these proteins are 
produced. 
2. Preparation of the Cell-Free System 
If the polyketide synthase proteins for use in the cell-free system are to 
be prepared recombinantly as described above, the cells producing the 
relevant PKS proteins are optionally harvested and disrupted if the 
desired proteins have been intracellularly produced. However, if the 
expression system secretes the protein into growth media, the protein can 
be purified directly from the media. 
If the protein is not secreted, it can be isolated from cell lysates. This 
is generally accomplished by first preparing a crude extract which lacks 
cellular components and several extraneous proteins. The desired proteins 
can then be further purified i.e. by column chromatography, HPLC, 
immunoadsorbent techniques or other conventional methods well known in the 
art. The selection of the appropriate growth conditions and recovery 
methods are within the skill of the art. 
For example, cells that express the PKS of interest can be grown to produce 
a predetermined number of cells. The cells may be disrupted by sonication, 
freeze-thaw cycles or other like techniques by which the cell membrane is 
breached to form a crude cell-free preparation. The crude cell-free 
preparation may be used at this stage as a source of PKS or may be further 
processed by centrifugation, filtration or the like, to form a cell 
supernatant. Optionally, nucleic acids may be removed from the cell 
supernatant by, for example, precipitation with polyethyleneimine, or 
other like agent which does not disturb the enzymatic activity of the PKS. 
The preparation may be used at this stage as a source of PKS. Optionally, 
the PKS may be further purified by techniques known to those of skill in 
the art. 
For use in the construction of libraries of polyketides, in addition to 
recombinantly produced polyketide synthase proteins, isolated native forms 
may in some instances be used. 
The purified PKS can be used to catalytically synthesize polyketides in a 
cell-free system as exemplified below. The cell-free system includes 
purified PKS, in an appropriate buffer, and the substrates required for 
the catalytic synthesis of polyketides. Depending on the PKS, starter 
substrate units can include, e.g., acetyl-CoA, malonamyl-CoA, 
propionyl-CoA, butyryl-CoA, isobutyryl-CoA, isovaleryl-CoA, aromatic 
coenzyme A thioesters such as benzoyl-CoA, aminobenzoyl-CoA, aminohydroxy 
benzoyl-CoA, and the like, heterocyclics such as thiophenecarboxyl-CoA, 
and the like, and partially synthesized polyketides. Alternatively, the 
coenzyme A thioesters may be replaced by corresponding N-acetylcysteamine 
thioesters. Extender units include, for example, malonyl-CoA, 
methylmalonyl-CoA, ethylmalonyl-CoA, and other like molecules well known 
to those of skill in the art. 
It has not been possible, heretofore, to provide cell-free systems for the 
synthesis of polyketides using isolated or purified modular polyketide 
synthases. According to the present invention, such cell-free systems are 
provided even for the production of polyketides produced by these complex 
synthases. 
The polyketides that are prepared using the cell-free system disclosed 
herein may be isolated and identified using any of a variety of techniques 
known in the art (see, e.g., WO 95/08548) including thin layer 
chromatography, high performance liquid chromatography, analytical and/or 
preparative gel electrophoresis, column chromatography, gas 
chromatography, nuclear magnetic resonance ("NMR"), mass spectrometry, or 
other conventional methods well known in the art. 
3. Additional Background Information on PKS and Use in Library Design 
The cell-free preparations described above are particularly useful in 
constructing polyketide libraries that contain a multiplicity of different 
polyketides. It will be useful to review the variations that can be 
included by virtue of varying the proteins containing the PKS catalytic 
activities required for synthesis. Although hybrid systems can be obtained 
which combine coding sequences derived from aromatic and modular and 
fungal PKS, it may be helpful to describe in more detail the mode of 
action of these PKSs and the combinatorial possibilities. For ease of 
explanation, the aromatic, modular and fungal PKS systems are discussed 
separately. 
Generally, polyketide synthesis occurs in three stages. In the first stage, 
catalyzed by the PKS, a nascent polyketide backbone is generated from 
monomeric CoA thioesters. In the second stage this backbone is 
regiospecifically cyclized. While some cyclization reactions are 
controlled by the PKS itself, others result from activities of downstream 
enzymes. In the final stage, the cyclized intermediate is modified further 
by the action of mechanistically diverse "tailoring enzymes," giving rise 
to the natural product. 
a) Aromatic PKS 
Background: For aromatic PKS, polyketide biosynthesis begins with a primer 
unit loading on to the active site of the condensing enzyme, .beta.-keto 
acyl synthase/acyl transferase (KS/AT). An extender unit (usually 
malonate) is then transferred to the pantetheinyl arm of the acyl carrier 
protein (ACP). The KS/AT catalyzes the condensation between the ACP-bound 
malonate and the starter unit. Additional extender units are added 
sequentially until the nascent polyketide chain has grown to a desired 
chain length determined by the protein chain length factor (CLF), perhaps 
together with the KS/AT. Thus, the KS, CLF and the ACP form a minimal set 
to generate a polyketide backbone. The nascent polyketide chain is then 
subjected to regiospecific ketoreduction by a ketoreductase (KR) if it 
exists. Cyclases (CYC) and aromatases (ARO) later catalyze regiospecific 
ring formation events through intramolecular aldol condensations. The 
cyclized intermediate may then undergo additional regiospecific and/or 
stereospecific modifications (e.g., O-methylation, hydroxylation, 
glycosylation, etc.) controlled by downstream tailoring enzymes). 
Acetyl-CoA is the usual starter unit for most aromatic polyketides. 
However, malonamyl-CoA (Gatenbeck, S. Biochem. Biophy. Res. Commun. (1961) 
6:422-426) and propionyl-CoA (Paulick, R. C. et al. J. Am. Chem. Soc. 
(1976) 98:3370-3371) are primers for many members of the tetracycline and 
anthracycline classes of polyketides, respectively. Daunorubicin PKS can 
also accept acetyl-CoA, butyryl-CoA, and isobutyryl-CoA as starter units. 
(Oki, T. et al. J. Antibiot. (1981) 34:783-790; Yoshimoto, A. et al. J. 
Antibiot. (1993) 46:1758-1761). 
The act KR can productively interact with all minimal PKSs studied thus far 
and is both necessary and sufficient to catalyze a C-9 ketoreduction. 
Although homologous KRs have been found in other PKS clusters, they 
catalyze ketoreduction with the same regiospecificity. However, the 
structures of frenolicin, griseusin and daunorubicin suggest that an 
additional C-17 ketoreduction occurs in these biosynthetic pathways. 
Likewise, several angucyclines undergo a C-15 ketoreduction, which occurs 
before the nascent polyketide chain is cyclized (Gould, S. J. et al. J. 
Am. Chem. Soc. (1992) 114:10066-10068). The ketoreductases responsible for 
C-15 and C-17 reductions have not yet been identified; however, two 
homologous KRs have been found in the daunorubicin PKS cluster (Grimm, A. 
et al. Gene (1994) 151:1-10; Ye, J. et al. J. Bacteriol. (1994) 
176:6270-6280). It is likely that they catalyze the C-9 and C-17 
reductions. 
The formation of the first two six-membered rings in the biosynthesis of 
most naturally occurring bacterial aromatic polyketides is controlled by 
PKS subunits; further ring closures are controlled by additional cyclases 
and modifying enzymes. The structural diversity introduced via these 
reactions appears to be greater than via the first two cyclizations. 
However, certain preferred patterns are observed, which suggests that at 
least some of these downstream cyclases may be useful for the construction 
of combinatorial libraries. For example, the pyran ring in 
isochromanequinones is invariably formed via cyclization between C-3 and 
C-15; two stereochemically distinct classes of products are observed. In 
anthracyclines and tetracyclines a third aldol condensation usually occurs 
between C-3 and C-16, whereas in unreduced tetracenomycins and related 
compounds it occurs between C-5 and C-18, and in angucyclines it occurs 
between C-4 and C-17. Representative gene(s) encoding a few of these 
enzymes have already been cloned (Fernandez-Moreno, M. A. et al. J. Biol. 
Chem. (1994) 269:24854-24863; Shen, B. et al. Biochemistry (1993) 
32:11149-11154). At least some cyclases might recognize chains of altered 
lengths and/or degrees of reduction, thereby increasing the diversity of 
aromatic polyketide combinatorial libraries. 
In the absence of downstream cyclases, polyketide chains undergo 
non-enzymatic reactions. Recently, some degree of predictability has 
emerged within this repertoire of possibilities. For instance, hemiketals 
and benzene rings are two common moieties seen on the methyl end. 
Hemiketals are formed with an appropriately positioned enol and can be 
followed by a dehydration. Benzene rings are formed with longer uncyclized 
methyl terminus. On the carboxyl terminus, a .gamma.-pyrone ring formed by 
three ketide units is frequently observed. Spontaneous decarboxylations 
occur on free carboxyl ends activated by the existence of a 
.beta.-carbonyl. 
A cyclized intermediate can undergo various types of modifications to 
generate the final natural product. The recurrence of certain structural 
motifs among naturally occurring aromatic polyketides suggests that some 
tailoring enzymes, particularly group transferases, may be combinatorially 
useful. Two examples are discussed below. 
O-methylation is a common downstream modification. Although several 
SAM-dependent O-methyltransferase genes have been found in PKS gene 
clusters (Decker, H. et al. J. Bacteriol. (1993) 175:3876-3886), their 
specificities have not been systematically studied as yet. Perhaps some of 
them could be useful for combinatorial biosynthesis. For instance, 
O-11-methylation occurs in several members of the anthracycline, 
tetracenomycin, and angucycline classes of aromatic polyketides. 
Library Design: The following set of design rules permits rationally or 
stochastically manipulating early biosynthetic steps in aromatic 
polyketide pathways including chain synthesis, C-9 ketoreduction, and the 
formation of the first two aromatic rings. If each biosynthetic degree of 
freedom is independent of all others, then it should be possible to design 
a single combinatorial library of N.sub.1 .times.N.sub.2 .times. . . . 
N.sub.i .times. . . . N.sub.n-1 .times.N.sub.n clones, where N.sub.i is 
the number of ways in which the ith degree of freedom can be exploited. In 
practice however, not all enzymatic degrees of freedom are independent. 
Therefore, to minimize redundancy, it is preferable to design several 
sub-libraries of PKS enzyme-producing clones. 
(1) Chain length. In the aromatic synthases, polyketide carbon chain length 
is dictated by the minimal PKS. Within the minimal PKS, the acyl carrier 
protein can be interchanged without affecting specificity, whereas the 
chain length factor is crucial. Although some ketosynthase/chain length 
factor combinations are functional, others are not; therefore, 
biosynthesis of a polyketide chain of specified length can be insured with 
a minimal PKS in which both the ketosynthase and chain length factor 
originate from the same PKS gene cluster. So far, chain lengths of 16 
(octaketide), 18 (nonaketide), 20 (decaketide), and 24 carbons 
(dodecaketide) can be generated with minimal PKSs from the act, fren, tcm, 
and, whiE PKS clusters, respectively (McDaniel et al. Science (1993), 
supra; McDaniel et al. J. Am. Chem. Soc. (1993), supra; McDaniel et al. 
Proc. Natl. Acad. Sci. USA (1994), supra). The whiE minimal PKS can also 
generate 22-carbon backbones in the presence of a KR, suggesting a degree 
of relaxed chain length control as found for the fren PKS. 
(2) Ketoreduction. Ketoreduction requires a ketoreductase. The act KR can 
catalyze reduction of the C-9 carbonyl (counting from the carboxyl end) of 
a nascent polyketide backbone of any length studied so far. Furthermore, 
the act KR is compatible with all the minimal PKSs mentioned above. 
Homologous ketoreductases have been identified in other PKS clusters 
(Sherman, D. H., et al. EMBO J. (1989) 8:2717-2725; Yu, T. -W. et al. J. 
Bacteriol. (1994) 176:2627-2534; Bibb, M. J. et al. Gene (1994) 
142:31-39). These enzymes may catalyze ketoreduction at C-9 as well since 
all the corresponding natural products undergo this modification. In 
unusual circumstances, C-7 ketoreductions have also been observed with the 
act KR. 
(3) Cyclization of the first ring. Although the minimal PKS alone can 
control formation of the first ring, the regiospecific course of this 
reaction may be influenced by other PKS proteins. For example, most 
minimal PKSs studied so far produce polyketides with C-7/C-12 cyclizations 
when present alone. In contrast, the tcm minimal PKS alone generates both 
C-7/C-12 and C-9/C-14 cyclized products. The presence of a ketoreductase 
with any minimal PKS restricts the nascent polyketide chain to cyclize 
exclusively with respect to the position of ketoreduction: C-7/C-12 
cyclization for C-9 ketoreduction and C-5/C-10 cyclization for C-7 
ketoreduction (McDaniel, R. et al. J. Am. Chem. Soc. (1993) 
115:11671-11675; McDaniel, R. et al. Proc. Natl. Acad. Sci. USA (1994) 
91:11542-11546; McDaniel, R. et al. J. Am. Chem. Soc. (1994) 
116:1085510859). Likewise, use of the TcmN enzyme alters the 
regiospecificity to C-9/c-14 cyclizations for unreduced polyketides of 
different lengths, but has no effect on reduced molecules. 
(4) First ring aromatization. The first ring in unreduced polyketides 
aromatizes non-catalytically. In contrast, an aromatizing subunit is 
required for reduced polyketides. There appears to be a hierarchy in the 
chain length specificity of these subunits from different PKS clusters. 
For example, the act ARO will recognize only 16-carbon chains (McDaniel et 
al. Proc. Natl. Acad. Sci. USA (1994), supra), the fren ARO recognizes 
both 16- and 18-carbon chains, while the gris ARO recognizes chains of 16, 
18, and 20 carbons. 
(5) Second ring cyclization. C-5/C-14 cyclization of the second ring of 
reduced polyketides may be achieved with an appropriate cyclase. While the 
act CYC can cyclize octa- and nonaketides, it does not recognize longer 
chains. No equivalent C-5/C-14 cyclase with specificity for decaketides or 
longer chains has been identified, although the structures of natural 
products such as griseusin imply their existence. In the case of 
sufficiently long unreduced chains with a C-9/C-14 first ring, formation 
of a C-7/C-16 second ring is catalyzed by the minimal PKS (McDaniel et al. 
Proc. Natl. Acad. Sci. USA (1994), supra). 
(6) Additional cyclizations. The KS/AT, CLF, ACP, KR, ARO, and CYC subunits 
of the PKS together catalyze the formation of an intermediate with a 
defined chain length, reduction pattern, and first two cyclizations. While 
the biosynthesis of naturally occurring polyketides typically requires the 
activity of downstream cyclases and other modifying enzymes to generate 
the characteristic biologically active product, subsequent reactions in 
the biosynthesis of engineered polyketides described here and in our 
earlier work occur in the absence of specific enzymes and are determined 
by the different physical and chemical properties of the individual 
molecules. Presumably reflecting such chemical possibilities and 
constraints, consistent patterns have been observed, leading to some 
degree of predictability. Two common moieties formed by the uncyclized 
methyl terminus of polyketide chains are hemiketals and benzene rings. 
Formation of a hemiketal occurs in the presence of an appropriately 
positioned enol and can be followed by a dehydration since both the 
hydrated and dehydrated forms are often isolated (McDaniel, R. et al. 
Science (1993) 262:15461550; McDaniel, R. et al. J. Am. Chem. Soc. (1994) 
116:1085510859; Fu, H. et al. J. Am. Chem. Soc. (1994) 116:41664170), 
while benzene ring formation occurs with longer unprocessed methyl ends 
(Fu et al. J. Am. Chem. Soc. (1994), supra). The most frequently observed 
moiety at the carboxyl terminus of the chain is a .gamma.-pyrone ring 
formed by three ketide units (McDaniel et al. J. Am. Chem. Soc. (1994), 
supra; Fu et al. J. Am. Chem. Soc. (1994), supra; Fu, H., et al. 
Biochemistry (1994) 33:9321-9326; Fu, H. et al. Chem. & Biol. (1994) 
1:205-210; Zhang, H. -l. et al. J. Org. Chem. (1990) 55:1682-1684); if a 
free carboxylic acid remains, decarboxylation typically occurs if a 
.beta.-carbonyl exists (McDaniel et al. Science (1993), supra; McDaniel, 
R., Ebert-Khosla, S., Hopwood, D. A. & Khosla, C. J. Am. Chem. Soc. 
(1993), supra; Kao, C. M. et al. J. Am. Chem. Soc. (1994) 
116:11612-11613). Many aldol condensations can be predicted as well, 
bearing in mind that the methyl and carboxyl ends tend preferentially to 
cyclize independently but will co-cyclize if no alternative exists 
(McDaniel et al. Proc. Natl. Acad. Sci. USA (1994), supra. These 
non-enzymatic cyclization patterns observed in vivo are also consistent 
with earlier biomimetic studies (Griffin, D. A. et al. J. Chem. Soc. 
Perkin Trans. (1984) 1:1035-1042). 
Taken together with the structures of other naturally occurring bacterial 
aromatic polyketides, the design rules presented above can be extrapolated 
to estimate the extent of molecular diversity that might be generated via 
in vivo combinatorial biosynthesis of, for example, reduced and unreduced 
polyketides. For reduced polyketides, the identified degrees of freedom 
include chain length, aromatization of the first ring, and cyclization of 
the second ring. For unreduced ones, these include chain length and 
regiospecificity of the first ring cyclization. The number of accessible 
structures is the product of the number of ways in which each degree of 
freedom can be varied. Chains of five different lengths have so far been 
manipulated (16-, 18- 20-, 22- and 24-carbon lengths). From the structure 
and deduced biosynthetic pathways of the dynemicin anthraquinone (Tokiwa, 
Y. et al. J. Am. Chem. Soc. (1992) 114:4107-4110), simaomicin (Carter, G. 
T. et al. J. Org. Chem. (1989) 54:4321-4323), and benastatin (Aoyama, T. 
et al. J. Antibiot. (1992) 45:1767-1772), the isolation of minimal PKSs 
that generate 14-, 26-, and possibly 28-carbon backbones, respectively, is 
anticipated, bringing the potential number to eight. Cloning of such 
minimal PKSs can be accomplished using the genes for minimal PKSs which 
have previously been isolated, such as the actI genes (Sherman et al. EMBO 
J. (1989), supra; Yu et al. J. Bacteriol. (1994), supra; Bibb et al. Gene 
(1994), supra; Malpartida, F. et al. Nature (1987) 325:818-821). Reduced 
chains can either be aromatized or not; a second ring cyclase is optional 
where the first ring is aromatized. The regiospecificity of the first 
cyclization of an unreduced chain can be varied, depending on the presence 
of an enzyme like TcmN. 
For example, for reduced polyketides the relevant degrees of freedom 
include the chain length (which can be manipulated in at least seven 
ways), the first ring aromatization (which can be manipulated in at least 
two ways), and the second ring cyclization (which can be manipulated in at 
least two ways for aromatized intermediates only). For unreduced 
polyketides, the regiospecificity of the first cyclization can also be 
manipulated. Thus, the combinatorial potential for reduced polyketides is 
at least 7.times.3=21; for unreduced polyketides the combinatorial 
potential is at least 7.times.2=14. Moreover, these numbers do not include 
additional minor products, on the order of 5 to 10 per major product, that 
are produced in the recombinant strains through non-enzymatic or 
non-specific enzyme catalyzed steps. Thus, the number of polyketides that 
can be generated from combinatorial manipulation of only the first few 
steps in aromatic polyketide biosynthesis is on the order of a few 
hundred. Thus, genetically engineered biosynthesis represents a 
potentially unlimited source of chemical diversity for drug discovery. 
b) Modular PKS 
Background: As illustrative of synthesis by modular PKS, polyketide 
biosynthesis by DEBS begins with the first acyltransferase (AT) activity 
in module 1 loading the starter unit onto the module 1 condensing 
activity, the .beta.-ketoacylsynthase (KS). The second AT of module 1 
loads the first extender unit onto the pantetheinyl arm of the 
acyl-carrier protein (ACP) activity. The KS catalyzes the decarboxylative 
condensation between the ACP-bound malonyl unit and the primer unit. The 
resulting diketide is then reduced by the ketoreductase (KR) activity of 
module 1, which converts the .beta.-keto group into an alcohol. In more 
complex modules, such as DEBS module 4, additional reductive cycle 
activities (a dehydratase (DH) and an enoylreductase (ER)) come into play 
after the module's KR performs the initial ketoreduction. The module 1 
product is then passed to the module 2 KS. The module 2 AT loads the 
second extender unit onto the module 2 ACP, and the module 2 KS then 
performs the condensation to produce a triketide which is reductively 
processed. Additional modules come into play, each adding and processing 
another extender unit onto the growing polyketide chain. The final length 
of the polyketide chain is determined by the number of modules present in 
the PKS (six in the case of DEBS), and the reductive outcome at any 
position is determined by the complement of reductive cycle activities 
present in the corresponding module. After elaboration of the polyketide 
chain, the molecule is subjected to regiospecific cyclization by a 
thioesterase (TE) activity fused to the end of DEBS module 6. The 
macrolide product is then tailored by downstream enzymes, e.g., 
hydroxylases, oxidases, methyltransferases, glycosylases, and the like, to 
produce the final natural product. 
Library Construction: The following set of design rules applies for 
rationally or stochastically manipulating early biosynthetic steps in 
modular polyketide biosynthetic pathways. The manipulative elements 
include: 
(1) Starter Unit. The relaxed specificity of modular PKSs for the starter 
unit under in vitro conditions has been reported (Pieper et al. 1995, 
supra). 
(2) Extender Unit. The nature of the extender unit used by a given module 
is determined by the AT activity. Sequence comparisons have clearly 
identified the characteristics of malonyl-CoA-specific AT and 
methylmalonyl-CoA-specific AT activities (Haydock et al. 1995, supra) At 
activities using methylmalonyl-CoA or ethylmalonyl-CoA generate a chiral 
center having one of two possible stereochemistries. For the two common 
extender units, malonyl-CoA and methylmalonyl-CoA, there are thus three 
possible structural outcomes. 
(3) Reductive Cycle. The state of reduction of the .beta.-keto group formed 
by KS-catalyzed condensation is governed by the set of reductive cycle 
activities present within the module. Thus, absence of any reductive 
activity yields a ketone function, while presence of only a KR activity 
generates an alcohol group having one of two possible stereochemistries. 
The presence of both KR and DH activities results in the formation of an 
alkene; if a stereocenter had been generated by the AT activity, the 
chirality at that position is lost. The presence of the full complement of 
KR, DH, and ER activities results in complete reduction of the .beta.-keto 
group to a methylene group. There are thus 5 theoretically possible 
reductive cycle outcomes at any module. 
(4) Cyclizations. The linear polyketide chain may cyclize through a number 
of possible mechanisms. The DEBS thioesterase (TE) activity demonstrates a 
broad capacity to lactonize hydroxy-acids (Aggarwal et al. 1995, supra). 
Also, several known natural products, e.g., avermectin, mevinolin, appear 
to be formed through Diels-Alder cyclizations of polyketide chains 
containing multiple alkene groups. Cyclizations of alcohols onto ketones 
to form ketals and spiroketals is also commonly observed. 
For any single module, therefore, there are at least 14 theoretical 
structural outcomes when only the two common extender units are 
considered. If all manipulable elements can be simultaneously controlled, 
there are 
EQU S.times.(14).sup.N 
possible polyketide chains which can be produced from an N-module PKS using 
S starter units. For a 6-module PKS such as DEBS, 14.sup.6, or more than 
7.5.times.10.sup.6 polyketide chains could be produced using a single 
starter unit. Furthermore, enzymes that catalyze downstream modifications, 
e.g., cyclizations, group-transfer reactions, oxidoreductions, and the 
like, can be studied along the lines presented herein and elsewhere. It is 
therefore possible that at least some of these degrees of freedom can be 
combinatorially exploited to generate libraries of synthetic products with 
structural diversity that is comparable to that observed in nature. 
Although modular PKSs have not been extensively analyzed, the one-to-one 
correspondence between active sites and product structure, together with 
the incredible chemical diversity observed among naturally occurring 
"complex" polyketides, indicates that the combinatorial potential within 
these multienzyme systems could be considerably greater than that for 
aromatic PKSs. For example, a wider range of primer units including 
aliphatic monomers (acetate, propionate, butyrate, isovalerate, etc.), 
aromatics (aminohydroxybenzoic acid), alicyclics (cyclohexanoic acid), and 
heterocyclics (pipecolic acid) are found in various macrocyclic 
polyketides. Recent studies have shown that modular PKSs have relaxed 
specificity for their starter units (Kao et al. Science (1994), supra). 
The degree of .beta.-ketoreduction following a condensation reaction can 
also be altered by genetic manipulation (Donadio et al. Science (1991), 
supra; Donadio, S. et al. Proc. Natl. Acad. Sci. USA (1993) 90:7119-7123). 
Likewise, the size of the polyketide product can be varied by designing 
mutants with the appropriate number of modules (Kao, C. M. et al. J. Am. 
Chem. Soc. (1994) 116:11612-11613). Modular PKSs also exhibit considerable 
variety with regards to the choice of extender units in each condensation 
cycle, although it remains to be seen to what extent this property can be 
manipulated. Lastly, these enzymes are particularly well-known for 
generating an extensive range of asymmetric centers in their products in a 
highly controlled manner. Thus, the combinatorial potential within modular 
PKS pathways could be virtually unlimited. 
c) Glycosylation 
Both aromatic and complex polyketides are often glycosylated. In many cases 
(e.g., doxorubicin and erythromycin) absence of the sugar group(s) results 
in considerably weaker bioactivity. There is tremendous diversity in both 
the types and numbers of sugar units attached to naturally occurring 
polyketide aglycones. In particular, deoxy- and aminosugars are commonly 
found. Regiochemical preferences can be detected in many glycosylated 
natural products. Among anthracyclines, O-17 is frequently glycosylated, 
whereas among angucyclines, C-10 is usually glycosylated. 
Glycosyltransferases involved in erythromycin biosynthesis may have 
relaxed specificities for the aglycone moiety (Donadio, S. et al. Science 
(1991) 252:675-679). An elloramycin glycosyltransferase may be able to 
recognize an unnatural NDP-sugar unit and attach it regiospecifically to 
an aromatic polyketide aglycone (Decker, H. et al. Angew. Chem. (1995), in 
press). These early results suggest that glycosyltransferases derived from 
secondary metabolic pathways have unique properties and may be attractive 
targets for use in the generation of combinatorial libraries. 
d) Fungal PKS 
Like the actinomycetes, filamentous fungi are a rich source of polyketide 
natural products. The fact that fungal PKSs, such as the 6-methylsalicylic 
acid synthase (6-MSAS) and the mevinolin synthase, are encoded by single 
multi-domain proteins (Beck et al. Eur. J. Biochem. (1990), supra; Davis, 
R. et al. Abstr. Genet. Ind. Microorg. Meeting, supra) indicates that they 
may also be targeted for combinatorial mutagenesis. Moreover, fungal PKSs 
can be functionally expressed in S. coelicolor CH999 using the genetic 
strategy outlined above and described in WO 95/08548, supra. Chain lengths 
not observed in bacterial aromatic polyketides (e.g., tetraketides, 
pentaketides and hexaketides) have been found among fungal aromatic 
polyketides (O'Hagan, D. The Polyketide Metabolites (Ellis Horwood, 
Chichester, U.K., 1991). Likewise, the cyclization patterns of fungal 
aromatic polyketides are quite different from those observed in bacterial 
aromatic polyketides (Id.). In contrast with modular PKSs from bacteria, 
branched methyl groups are introduced into fungal polyketide backbones by 
S-adenosylmethionine-dependent methyltransferases; in the case of the 
mevinolin PKS (Davis, R. et al. Abstr. Genet. Ind. Microorg. Meeting, 
supra), this activity is encoded as one domain within a monocistronic PKS. 
It is now possible to experimentally evaluate whether these and other 
sources of chemical diversity in fungal polyketides are indeed amenable to 
combinatorial manipulation. 
e) Summary: 
The number of potentially novel polyketides that can be catalytically 
produced by PKS gene products in a cell-free system increases 
geometrically as new degrees of freedom are exploited and/or protein 
engineering strategies are brought to bear on the task of creating enzyme 
subunits with specificities not observed in nature. For example, 
non-acetate starter units can be incorporated into polyketide backbones 
(e.g., propionate in daunorubicin and malonamide in oxytetracycline). 
Furthermore, enzymes that catalyze downstream cyclizations and late-step 
modifications, such as group transfer reactions and oxidoreductions 
commonly seen in naturally occurring polyketides, can be studied along the 
lines presented here and elsewhere. It is therefore possible that at least 
some of these degrees of freedom can be combinatorially exploited to 
generate libraries of synthetic products with structural diversity that is 
comparable to that observed in nature. 
C. Experimental 
Below are examples of specific embodiments for carrying out the present 
invention. The examples are offered for illustrative purposes only, and 
are not intended to limit the scope of the present invention in any way. 
Efforts have been made to ensure accuracy with respect to numbers used 
(e.g., amounts, temperatures, etc.), but some experimental error and 
deviation should, of course, be allowed for. 
The Examples provided below describe recombinant production of a modular 
PKS and methods for in vitro synthesis of polyketides by recombinant DEBS 
and by an active deletion mutant. The latter mutant, designated "DEBS 
1+2+TE", contains the first two modules from DEBS 1 fused to the 
thioesterase domain normally found at the C-terminal end of module 6 of 
DEBS (FIG. 1). Both DEBS and DEBS 1+2+TE have been successfully expressed 
in S. coelicolor CH999, purified, and used in a cell-free system for the 
in vitro catalytic synthesis of, respectively, 6-dEB (1) 
##STR1## 
and (2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid 
.delta.-lactone (2) 
##STR2## 
Three open reading frames (eryAI, eryAII, and eryAII) encode the DEBS 
polypeptides and span 32 kb in the ery gene cluster of the 
Saccharopolyspora erythraea genome. The genes are organized in six 
repeated units, each designated a "module" (see FIG. 1). Each module 
encodes a set of active sites that, during polyketide biosynthesis, 
catalyzes the condensation of an additional monomer onto the growing 
chain. Each module includes an acyltransferase (AT), .beta.-ketoacyl 
carrier protein synthase (KS), and acyl carrier protein (ACP) as well as a 
subset of reductive active sites (.beta.-ketoreductase (KR), dehydratase 
(DH), enoyl reductase (ER)). The number of reductive sites within a module 
corresponds to the extent of g-keto reduction in each condensation cycle. 
The thioesterase (TE) encoded at the end of module appears to catalyze 
lactone formation. 
Due to the large sizes of eryAI, eryAII, and eryAIII, and the presence of 
multiple active sites, these genes can be conveniently cloned into a 
plasmid suitable for expression in a host cell, such as the genetically 
engineered host cell CH999, using an in vivo recombination technique. This 
technique, described in WO 95/08548 utilizes derivatives of the plasmid 
pMAK705 (Hamilton et al. (1989) J. Bacteriol. 171:4617) to permit in vivo 
recombination between a temperature-sensitive donor plasmid, which is 
capable of replication at a first, permissive temperature and incapable of 
replication at a second, non-permissive temperature, and recipient 
plasmid. The eryA genes thus cloned gave pCK7, a derivative of pRM5 
(McDaniel et al. (1993) Science 262:1546). A control plasmid, pCK7f, was 
constructed to carry a frameshift mutation in eryAI. pCK7 and pCK7f 
possess a ColEI replicon for genetic manipulation in E. coli as well as a 
truncated SCP2* (low copy number) Streptomyces replicon. These plasmids 
also contain the divergent actI/actIII promoter pair and actII-ORF4, an 
activator gene, which is required for transcription from these promoters 
and activates expression during the transition from growth to stationary 
phase in the vegetative mycelium. High-level expression of PKS genes 
occurs at the onset of stationary phase of mycelial growth. 
EXAMPLE 1 
Production of S. coelicolor CH999 
An S. coelicolor host cell, genetically engineered to remove the native act 
gene cluster, and termed CH999, was constructed using S. coelicolor CH1 
(Khosla et al. Molec. Microbiol. (1992), supra) as described in 
WO95/08548, incorporated herein by reference. S. coelicolor CH999 lacks 
the entire ACT gene cluster. 
EXAMPLE 2 
Production of the Recombinant Vector pRM5 
Shuttle plasmids are used to express recombinant PKSs in CH999. Such 
plasmids typically include a colEI replicon, an appropriately truncated 
SCP2* Streptomyces replicon, two act-promoters to allow for bidirectional 
cloning, the gene encoding the actII-ORF4 activator which induces 
transcription from act promoters during the transition from growth phase 
to stationary phase, and appropriate marker genes. Restriction sites have 
been engineered into these vectors to facilitate the combinatorial 
construction of PKS gene clusters starting from cassettes encoding 
individual subunits (or domains) of naturally occurring PKSs. Among the 
many advantages of this method are that (i) all relevant biosynthetic 
genes are plasmid-borne and therefore amenable to facile manipulation and 
mutagenesis in E. coli and (ii) the entire library of PKS gene clusters 
can be expressed in the same bacterial host. 
pRM5 was the shuttle plasmid used for expressing PKSs in CH999. It includes 
a ColEI replicon to allow genetic engineering in E. coli, an appropriately 
truncated SCP2* (low copy number) Streptomyces replicon, and the 
actII-ORF4 activator gene from the act cluster, which induces 
transcription from act promoters during the transition from growth phase 
to stationary phase in the vegetative mycelium. pRM5 carries the divergent 
actI/actIII promoter pair, together with convenient cloning sites to 
facilitate the insertion of a variety of engineered PKS genes downstream 
of both promoters. pRM5 lacks the par locus of SCP2 *; as a result the 
plasmid is slightly unstable (approx. 2% loss in the absence of 
thiostrepton). This feature was deliberately introduced in order to allow 
for rapid confirmation that a phenotype of interest could be unambiguously 
assigned to the plasmid-borne mutant PKS. The recombinant PKSs from pRM5 
are expressed approximately at the transition from exponential to 
stationary phase of growth, in good yields. pRM5 was constructed as 
described in WO95/08548. 
EXAMPLE 3 
Construction of Expression Vectors for, and Expression of Aromatic PKS 
WO95/08548 describes the construction of expression vectors using the pRM5 
host plasmid using portions of the aromatic polyketide synthase gene 
clusters of actinorhodin (act), granaticin (gra) and tetracenomycin (tcm) 
gene clusters. A number of hybrid clusters are described. These hybrid 
clusters were introduced into S. coelicolor CH999 and expressed to produce 
the relevant polyketide synthases which in turn produce a variety of 
polyketides. Additional constructs using genes derived from the frenolicin 
B (fren) PKS gene cluster were also prepared. 
EXAMPLE 4 
Production of Modular PKS 
Expression plasmids containing recombinant modular DEBS PKS genes were 
constructed by transferring DNA incrementally from a temperature-sensitive 
"donor" plasmid, i.e., a plasmid capable of replication at a first, 
permissive temperature and incapable of replication at a second, 
non-permissive temperature, to a "recipient" shuttle vector via a double 
recombination event, as described in WO95/08548, and in Kai et al. Science 
(1994) 265:509. pCK7, contains the complete eryA gene. A control plasmid, 
pCK7f, which contains a frameshift error in eryAI, was constructed in a 
similar manner. pCK7 and pCK7f were transformed into E. coli ET12567 
(MacNeil (1988) J. Bacteriol. 170:5607) to generate unmethylated plasmid 
DNA and subsequently moved into S. coelicolor CH999 using standard 
protocols (Hopwood et al. (1985) Genetic manipulation of Streptomyces. A 
laboratory manual. The John Innes Foundation: Norwich). 
Upon growth of CH999/pCK7 on R2YE medium, two polyketides were produced. In 
addition, three high-molecular-weight proteins (&gt;200 kDa) presumably 
DEBS1, DEBS2 and DEBS3 (Caffrey et al. FEBS Lett. (1992) 304:225) were 
also observed in crude extracts of CH999/pCK7 via sodium dodecyl 
sulfate-polyacrylamide gel electrophoresis ("SDS-PAGE"). No polyketide 
products were observed from CH999/pCK7F. 
EXAMPLE 5 
Recombinant Production of a Mutant DEBS PKS 
In this Example a deletion mutant PKS was constructed that consists of 
DEBS1 fused to the TE of DEBS3 ("DEBS 1+2+TE"); plasmid pCK12 contained 
the genes encoding the DEBS 1+2+TE. 
The DEBS 1+2+TE PKS contained a fusion of the carboxy-terminal end of the 
acyl carrier protein of module 2 (ACP-2) to the carboxy-terminal end of 
the acyl carrier protein of module 6 (ACP-6) (see FIG. 1). Thus ACP-2 is 
essentially intact in this PKS and is followed by the amino acid sequence 
naturally found between ACP-6 and the TE. pCK12 is identical to pCK7 (Kao 
et al. Science (1994), supra) with the exception of a deletion between the 
carboxy-terminal ends of ACP-2 and ACP-6. The fusion occurs between 
residues L3455 of DEBS1 and Q2891 of DEBS3. An SpeI site is present 
between these two residues so that the DNA sequence at the fusion is 
CTCACTAGTCAG. 
EXAMPLE 6 
Preparation of Cell-Free DEBS from pCK7 and pCK12 
The DEBS preparation was carried out as follows. S. coelicolor CH999/pCK12 
or CH999/pCK7 cells were harvested after a growth of 55 h in liquid 
cultures. Typically, 8-10 grams of cells (wet cell weight) were disrupted 
using sonication (5.times.30 s bursts). The resultant cell slurry was 
ultracentrifuged (2 h at 192,000.times.g) and nucleic acids precipitated 
with 0.2% polyethyleneimine (Step 1) yielding about 200 mg total protein. 
All 3 DEBS proteins were precipitated in a 55% saturated ammonium sulfate 
solution. The incubation buffer (buffer I) used thereafter contained 150 
mM sodium phosphate buffer (pH 7.1), 15% glycerol, 2 mM dithiothreitol 
("DTT"), and 2 mM ethylene diamine tetraacetic acid ("EDTA"). After 
desalting on Sephadex G25 M (Step 2), about 30 mg protein (15-20 mg/mL) 
was applied to an Agarose BioGel A size exclusion column (140 mL). 
Fractions containing DEBS proteins were pooled and concentrated to 1 mg/mL 
on YM 30 ultrafiltration membranes (Step 3). 
DEBS proteins were detected by their high molecular weights of 330 kDa 
(DEBS 3), 370 kDa (DEBS 1) and 380 kDa (DEBS 2) by SDS-PAGE; these 
proteins were absent in cell extracts of a variety of control strains. The 
apparent molecular weights of the DEBS proteins were also evaluated by gel 
filtration on a Superose 6 HR 10/30 column (Pharmacia), using 
thyroglobulin (669 kDa) and apoferritin (443 kDa) as high molecular weight 
markers. (see FIG. 3A). 
Recombinant DEBS proteins isolated from cell extracts were partially 
purified as described above. Size exclusion chromatography of a crude 
extract containing the three DEBS subunits on Biogel A and Superose 6 
(upper size exclusion limit 15 MDa and 1.5 MDa, respectively) revealed 
that DEBS 1 and 2 associate more tightly with each other than with DEBS 3. 
Moreover, DEBS 1 and 2 (370 kDa and 380 kDa, respectively) elute over a 
wide range of fractions corresponding to M.sub.r between 10 and 1 MDa, 
indicating that they might form a multimeric complex, which partially 
dissociates during gel filtration. DEBS 3, however, is not present in this 
extremely large M.sub.r range. From size calibration experiments on a 
Superose 6 column, DEBS 3 (330 kDa) mostly elutes as a dimer (similar to 
thyroglobulin, 669 kDa). Upon concentration, the Biogel A column fractions 
(see FIG. 3A) containing DEBS 1 and 2 alone were found be inactive in 
vitro. However, when pooled with a concentrated fraction containing DEBS 3 
alone (FIG. 3A), the reconstituted complex of the three proteins showed 
comparable activity to the DEBS 1, 2, and 3 preparation derived via 
ammonium sulfate precipitation of the crude cell extract. These 
purification results suggest that activity of DEBS requires the formation 
of a high molecular weight oligomeric complex, possibly a trimer of 
dimers. The formation of homodimers by purified (but not fully active) 
DEBS subunits has been reported (Aparicio et al. J. Biol. Chem. (1994), 
supra; Bevitt et al. Eur. T. Biochem. (1992), supra; Caffrey et al. Eur. 
J. Biochem. (1991), supra; and Leadlay et al. Biochem. Soc. Trans. (1993), 
supra). 
The covalent modification of the cell-free DEBS preparations by .sup.14 
C-labeled starter units in the absence of chain extension reactions is 
depicted in FIG. 3B. Partially purified DEBS preparations (40 mg total 
protein) were incubated with the substrates [1-.sup.14 C]butyryl-CoA (160 
.mu.M), [1-14C]acetyl-CoA (40 .mu.M), or [1-.sup.14 C]propionyl-CoA (20 
.mu.M) including a 30-min preincubation with iodoacetamide (1 mM). After, 
denaturation and separation of the proteins in a SDS-PAGE (5%), the 
separated proteins were electrotransferred onto a nitrocellulose membrane, 
.sup.14 C-labeled proteins were exposed to an X-ray film for 5 days. 
EXAMPLE 7 
Cell-Free Synthesis of 6-dEB and 
(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid .delta.-lactone 
In order to establish an in vitro assay system for polyketide synthesis, 
partially purified preparations of the complete DEBS 1, 2, and 3 system 
and of DEBS 1+2+TE, prepared as described in Example 6, were incubated 
with their natural substrates [1-.sup.14 C]propionyl-CoA, 
(2RS)-methylmalonyl-CoA, and NADPH. 
The DEBS 1+2+TE and DEBS 1, 2, and 3 preparations described in the Example 
6 (purification Step 2) were adjusted to a concentration of 8 mg total 
protein/mL buffer. Incubations were carried out at 28.degree. C. with the 
[1-1.sup.4 C]propionyl-CoA (specific activity 50 Ci/mol, 10 .mu.M), 
methylmalonyl-CoA (250 .mu.M), and NADPH (500 .mu.M) dissolved in buffer I 
in a volume of 250 .mu.L for 3 h. Thereafter, the incubation mix was 
extracted with 2.times.2 mL ethyl acetate, the ethyl acetate was 
evaporated of, and the product was analyzed by thin layer chromatography 
("TLC") in 60% ethyl acetate/40% hexane followed. The TLC plate was 
exposed to an X-ray film for 2 days. 
Lanes Ia and Ib of the autoradiogram (FIG. 4) show extracts of DEBS 1+2+TE 
including [1-.sup.14 C]propionyl-CoA and NADPH but excluding 
methylmalonyl-CoA (Ia) and including all 3 substrates (Ib). Lanes IIa, IIb 
and IIc of the autoradiogram (FIG. 4) show incubations of DEBS 1, 2, and 3 
including [1-.sup.14 C]propionyl-CoA and NADPH but excluding 
methylmalonyl-CoA (IIa), including all 3 substrates (IIb) and 
preincubation of DEBS 1, 2, and 3 with cerulenin (100 .mu.M) for 15 min, 
followed by addition of all 3 substrates (IIc). Ethyl acetate/hexane 
(50:50) was used as the solvent system. In lane Ib the major labeled 
component is identical in R.sub.f (0.30) to authentic triketide (2) (see 
arrow), while in lane IIb, the least polar labeled product is identical in 
R.sub.f (0.40) to authentic 6-dEB (1) (see arrow). In lanes Ib and IIb the 
concentrations of the minor products (but not 6-dEB nor the triketide 
lactone) vary substantially as a function of the DTT concentration in the 
reaction buffer; the structures of these DTT-dependent products are under 
investigation. Control experiments were also performed with DEBS 1+2+TE, 
with complete DEBS in the absence of NADPH, and with comparable cell-free 
preparations from CH999 and from CH999/pSEK38 (a recombinant strain that 
expresses the actinorhodin PKS gene cluster). In all four controls, 
neither 6-dEB nor the triketide lactone were detected. The intense, more 
polar band, evident lanes IIa, IIb and IIc was also present in all the 
above null controls including extracts obtained from CH999 alone. The 
identities of the enzymatically generated [1.sup.4 C]-(1) and [.sup.14 
C]-(2) were each confirmed by dilution of the respective TLC-purified 
product with authentic unlabeled carrier and recrystallization to constant 
activity. Thus labeled 6-dEB, from incubation of [1-.sup.14 
C]propionyl-CoA with DEBS 1, 2, and 3, mixed with 15.4 mg of (1), was 
recrystallized 4 times from ether/hexane. After each recrystallization, 
two to three portions of each sample were analyzed by liquid scintillation 
counting: 2132.+-.16 dpm/mg (1st recryst); 2117.+-.23 dpm/mg (2nd 
recryst); 2125.+-.2 dpm/mg (3rd recryst); 2141.+-.17 dpm/mg (4th recryst); 
(mean .sup.14 C act. 2129.+-.9 dpm/mg). Similarly labeled triketide, from 
incubation of [1-.sup.14 C]propionyl-CoA with DEBS 1+2+TE, was mixed with 
20.4 mg of unlabeled (2) and recrystallized 4 times from ether/hexane: 
4528.+-.306 dpm/mg (1st recryst); 4725.+-.80 dpm/mg (2nd recryst); 
4662.+-.74 dpm/mg (3rd recryst); 4706.+-.60 dpm/mg (4th recryst); (mean 
.sup.14 C act. 4655.+-.77 dpm/mg). 
These results indicate that each cell-free DEBS protein preparation 
synthesized a .sup.14 C-labeled product with TLC R.sub.f values identical 
to those of reference samples of either 6-dEB (1) or 
(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid 6-lactone (2), 
respectively, as evidenced by TLC-autoradiography (FIG. 4). The identities 
of [.sup.14 C]-(1) and [.sup.14 C]-(2) were confirmed by dilution of each 
of the TLC-purified radiolabeled products with authentic unlabeled carrier 
6-dEB or triketide lactone and repeated recrystallization of each sample 
to constant activity. The formation of each lactone product showed an 
absolute requirement for the relevant protein preparation as well as for 
methylmalonyl-CoA and NADPH and was inhibited by both N-ethylmaleimide and 
cerulenin, both well-known inhibitors of the condensation reactions of 
fatty acid biosynthesis (Plate, C. A. et al. J. Biol. Chem. (1970) 
245:2868; D'Agnolo, G. et al. Biochim. Biophys. Acta (1973) 326:155; 
Kauppinen, S. et al. Carlsberg Res. Commun. (1988) 53:357-370). Based on 
the observed radiochemical yield of purified product, the formation of 
6-dEB catalyzed by DEBS 1, 2, and 3 was estimated to be 33 pmol/mg total 
protein. By comparison, the formation of (2) by DEBS 1+2+TE was 600 
pmol/mg total protein. 
The specificity of labeling in the triketide lactone product was 
unambiguously confirmed by preparative scale incubation of [1-.sup.13 
C)propionyl-CoA with DEBS 1+2+TE in the presence of methylmalonyl-CoA and 
NADPH. Analysis of the derived product (2a) (see FIG. 4 (a)) by 100 MHz 
.sup.13 C NMR showed an enhanced peak at 81.3 ppm corresponding to 
enrichment at the predicted site, C-5, in 
(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid .delta.-lactone 
(Kao, C. M. et al. J. Am. Chem. Soc. (1994), supra). 
EXAMPLE 8 
Substrate Specificity of DEBS 1+2+TE In a Cell-Free System 
The in vitro assays were carried out as described in Example 6, 
substituting [1-.sup.14 C]propionyl-CoA by either [1-.sup.14 C]butyryl-CoA 
(160 .mu.M) or (1-.sup.14 C]acetyl-CoA, 40 .mu.M. Incubations of DEBS 
1+2+TE (purification Step 2) were performed excluding methylmalonyl-CoA or 
including methylmalonyl-CoA and NADPH in addition to the appropriate 
.sup.14 C-labeled primer substrate. Alternatively, DEBS 1+2+TE was 
preincubated with 1 mM N-ethylmaleimide before addition of all 3 
substrates. Control experiments carried out in the absence of NADPH as 
well as with an equivalent protein preparation from S. coelicolor CH999 
did not yield the observed labeled products. Ethyl acetate/hexane (60:40) 
was used as the solvent system. 
Development of the above radiochromatographic assay has allowed a 
preliminary analysis of the substrate specificity of these multifunctional 
enzyme complexes. For example, DEBS 1+2+TE appears to exhibit a relaxed 
specificity for primer unit analogs, as shown by both protein acylation 
and product formation. In addition to the expected acylation by [1-.sup.14 
C]propionyl-CoA, both DEBS 1+2+TE and DEBS 1 (from the mixture of DEBS 1, 
2, and 3) also form covalent adducts with [1-.sup.14 C]acetyl-CoA and 
[1-.sup.14 C]butyryl-CoA (see FIG. 3B). Since the protein labeling is 
unaffected by the presence of active site thiol inhibitors such as 
iodoacetamide, the substrates are presumably bound to the "loading" 
acyltransferase domain at the N-terminal end of DEBS 1. All three acyl-CoA 
substrates appear to react with DEBS 1 or DEBS 1+2+TE with comparable 
efficiencies. (The apparently lower intensity of the butyryl-CoA-labeled 
band in FIG. 3B is due to the 10-fold lower specific activity of 
[1-.sup.14 C]butyryl-CoA.) In the presence of methylmalonyl-CoA and NADPH, 
both acetyl-CoA and butyryl-CoA serve as surrogate polyketide chain 
initiators for DEBS 1+2+TE, giving rise to compound (4) (see FIG. 6B) the 
previously described C.sub.8 analog of compound (2), and what is presumed 
to be compound (5) (see FIG. 6B), the C.sub.10 homolog of (2), 
respectively, as judged by thin layer chromatography-autoradiography (see 
FIG. 6A). 
In addition to the above CoA thioesters, DEBS 1+2+TE enzyme can also 
process the N-acetylcysteamine thioester of the polyketide chain 
elongation intermediate, (2S,3R)-2-methyl-3-hydroxypentanoyl-NAC thioester 
(3). Thus incubation of [1-.sup.14 C]-(3) (Cane, D. E. et al. J. Am. Chem. 
Soc. (1981) 103:5960; Cane, D. E. et al. Tetrahedron (1983) 39:3449; Cane, 
D. E. et al. J. Am. Chem. Soc. (1986) 108:4957; Cane, D. E. et al. J. Am. 
Chem. Soc. (1987) 109:1255; Cane, D. E. et al, Tetrahedron Lett. (1991) 
32:5457; and Cane, D. E. et al. J. Antibiot. (1995) 48:647-651 (1995)) 
with DEBS 1+2+TE in the presence of methylmalonyl-CoA and NADPH gave rise 
to a labeled product which had the chromatographic mobility expected for 
the triketide lactone (2) and which could be recrystallized to constant 
activity when diluted with unlabeled triketide lactone, as described 
above. The specificity of labeling was unambiguously confirmed by 
preparative scale incubation of [2,3-.sup.13 C.sub.2 ]-(3) with DEBS 
1+2+TE, methylmalonyl-CoA and NADPH. The .sup.13 C NMR spectrum of the 
resulting triketide lactone (2b) (see FIG. 4) displayed a pair of enhanced 
and coupled doublets (J.sub.CC =34.5 Hz) centered at 36.7 and 81.3 ppm, 
corresponding to enrichment at each of the expected sites of labeling, C-4 
and C-5, respectively. This result is consistent with the previously 
reported incorporation of [2,3-.sup.13 C.sub.2 ]-(3) into the erythromycin 
macrolide (Cane et al. J. Am. Chem. Soc. (1981), supra; Cane et al. 
Tetrahedron (1983), supra; Cane et al. J. Am. Chem. Soc. (1986), supra; 
Cane et al. J. Am. Chem. Soc. (1987), supra; Cane et al. Tetrahedron Lett. 
(1991), supra) in intact cell experiments, as well as the results of 
numerous experiments in which NAC thioesters of advanced intermediates of 
polyketide chain elongation have been shown to be incorporated into other 
polyketides. 
The Examples provided herein lend further credence to the speculation that 
these NAC thioesters are directly loaded on to the appropriate active site 
in the PKS, and do not require the participation of additional proteins or 
cofactors. This experiment also confirms directly the ability of the 
macrolide synthase to recognize exogenously added chain elongation 
intermediates and load them correctly on the cognate PKS module for 
further processing to the natural product. 
EXAMPLE 9 
Cell-Free Synthesis of 2,4-Dimethyl-5-ethyl-3-hydroxy-2-pyrone 
The DEBS1+2+TE preparation described in Example 6 (purification step 2) was 
used as described in Example 7 but without the addition of NADPH to the 
reaction mixture. This reaction produced 
2,4-dimethyl-5-ethyl-3-hydroxy-2-pyrone as demonstrated by NMR analysis. 
The following Examples provide methods for inhibiting the synthesis of 
polyketides in a modular PKS from natural first-module starter units or 
from such starter units derived from the decarboxylation of extender units 
in so far as such substrates compete with unnatural starter units. The 
method involves inhibiting the loading of the first module of a PKS with a 
natural starter unit by inactivating a key active site on which starter 
units are loaded, for example, by deleting the KS1 or otherwise rendering 
KS1 nonfunctional or, alternatively, by deleting or rendering 
nonfunctional the ACP1. In a cell-free system, wherein the synthesis of a 
polyketide from unnatural starter units is desired, this method provides 
the advantage of minimizing undesirable competitive polyketide synthesis 
based on the presence of the natural starter and/or extender units. In 
addition, the method spares the extender units which would otherwise be 
supplied in a cell-free system at a considerable cost. This method is also 
of particular importance in an in vivo system in which the production of 
desired polyketides from unnatural substrates may be inhibited by the 
presence of natural substrates, thereby precluding the efficient use of 
the unnatural starter units to yield the desired product. 
EXAMPLE 10 
Construction, Expression and Analysis of [rKS1*]-DEBS1+2+TE 
In the absence of added propionyl-CoA, DEBS1+2+TE can form the propionyl 
starter unit through decarboxylation of methylmalonyl-loaded enzyme. This 
reaction requires a functional module 1 KS activity, which decarboxylates 
loaded methylmalonate in order to condense the extender unit with the 
starter unit. In the absence of supplied propionyl-CoA starter unit, the 
decarboxylated extender unit can be transferred backwards, allowing the 
loading of a second methylmalonyl-CoA extender and subsequent formation of 
a diketide. As this back-formation of propionyl units is undesirable when 
alternative starter units are being supplied to the system, a mutant of 
DEBS1+2+TE in which the module 1 KS has been inactivated through 
site-directed mutagenesis was prepared. The KS1 sequence was altered such 
that the active site cysteine residue (in the signature sequence 
cys-ser-ser-ser-leu) was replaced by alanine. The resulting expression 
plasmid, designated pKAO179, encodes a 2-module PKS which is inactive 
under the standard reaction conditions (propionyl-CoA, methylmalonyl-CoA, 
and NADPH). Inactivation of KS1 thus prevents the back formation of 
propionyl units, but also prevents diketide formation. When this protein 
is supplied with diketide thioester, i.e., 
(2S,3R)-2-methyl-3-hydroxy-pentanoyl N-acetylcysteamine thioester, 
methylmalonyl-CoA, and NADPH, however, the triketide product 
(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoic .delta.-lactone is 
produced. This construct allows the in vitro production of triketides 
having unusual starter units through use of the corresponding diketide 
thioesters, uncontaminated by the normal triketide product. 
EXAMPLE 11 
In Vivo Production of Novel Polyketides by Fermentation Using [FKS1*]DEBS 
Mutants 
As described in Example 10, the module 1 .beta.-ketoacylsynthase (KS1) 
activity of DEBS can be inactivated through site-directed mutagenesis. 
This mutation can be introduced into any combination of modules to produce 
a set of DEBS-backed PKSs which are incompetent for polyketide synthesis 
unless supplied with a suitable diketide thioester, e.g., 
2-methyl-3-hydroxypentanoyl N-acetylcysteamine thioester or analogs. The 
method described in Example 10 can be extended to allow for the in vivo 
production of novel polyketides through feeding of the appropriate 
diketide thioester analogs to actively growing cultures of S. coelicolor 
CH999 containing [KS1 ]-DEBS-based expression plasmids. The corresponding 
diketide as a free carboxylic acid may also be fed to the cultures if the 
cellular thioesterification system is functional on the acid, and if the 
cells are permeable to the acid. For example, the in vivo production of 
(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid .delta.-lactone 
is described above. 
A culture of S. coelicolor CH999/pKAO179 is established by inoculation of 
200 mL of SMM medium (5% PEG-800, 0.06% MgSO.sub.4, 0.2% (NH.sub.4).sub.2 
SO.sub.4, 25 mM TES, pH 7.2, 25 mM KH.sub.2 PO.sub.4, 25 mM K.sub.2 
HPO.sub.4, 1.6% glucose, 0.5% casamino acids, trace elements) with spores. 
The culture is incubated at 30.degree. C. with shaking at 325 rpm. A 
solution of (2S,3R)-2-methyl-3-hydroxypentanoyl N-acetlycysteamine 
thioester (100 mg) and 4-pentynoic (15 mg) in 1 mL of methylsulfoxide is 
added to the culture in three parts: after 50 hours (400 mL); after 62 
hours (300 mL); and after 86 hours (300 mL). After a total of 144 hours, 
the culture is centrifuged to remove mycelia. The fermentation broth is 
saturated with NaCl and extracted with ethyl acetate (5.times.100 mL). The 
combined organic extract is dried over Na.sub.2 SO.sub.4, filtered, and 
concentrated. Silica gel chromatography yields 
(2R,3S,4S,5R)-2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid .delta.-lactone. 
This method provides a means for large-scale production of novel modular 
polyketides containing unnatural starter units, uncontaminated by the 
polyketide containing the native propionate starter unit. 
The cell-free results reported here, in conjunction with the availability 
of facile mutagenesis tools, provide a novel approach to the study of 
modular PKS structure and mechanisms. The considerable yield of fully 
active protein from the recombinant source described will permit detailed 
analysis of this multifunctional catalyst by radioisotopic methods as well 
as by NMR and mass spectroscopy. Given that DEBS can accept a variety of 
substrates as primers, it will be possible to make quantitative 
assessments of substrate specificity by determination of the relevant 
steady state kinetic parameters and to further probe mechanistic details. 
In addition, cell-free systems such as the one reported here provide a 
completely novel route for the controlled synthesis of novel polyketides 
which might otherwise not be accessible via in vivo engineered 
biosynthesis. 
Thus, novel methods for producing polyketides, are disclosed. Although 
preferred embodiments of the subject invention have been described in some 
detail, it is understood that obvious variations can be made without 
departing from the spirit and the scope of the invention as defined by the 
appended claims.