Combinatorial approaches to polyketide biosynthesis

162

Combinatorial

approaches

to polyketide

biosynthesis

Peter F Leadlay Polyketides natural

are a large and structurally

products

based

The polyketide already

synthases

pathways

individual

genes

these

other polyketide

individual

the constituent

as domains

in a giant multienzyme

the growing

polyketide

approach

here therefore

individual

enzymatic assembly

A key recent genuinely

complex

complex

reduced

the fusing

together

enzyme

an antiparasitic

compound

that makes antimicrobial

so that the

a library of altered

do work as predicted, that recruits

of

such synthases

has been to demonstrate

enzymes

linked

along which

ways as can be devised,

broad-specificity

the In

A combinatorial

from several

line produces

advance

hybrid

requires

can

compounds.

are actually

chain is passed.

domains

in as many productive enzyme

synthesise

enzymes

biosynthetic

to increase

aromatic

which

polyketides,

have

libraries,

work has shown

components

such hybrid

synthases

polyketides

they encode

Recent

boxylative (Claisen) condensation. Where FASs typically use acetyl-CoA as the starter unit and malonyl-CoA as the

acid units.

from different

to make novel products. of obtaining

family of

small combinatorial

so that the enzymes

together,

how to choose chances

diverse

of carboxylic

that make aromatic

been used to generate

by expressing interact

on chains

products.

that such for example

the chain starter

has been grafted

a

unit for

onto a synthase

macrolides.

extender unit, PKSs often use acetyl or propionyl-CoA to provide the starter unit and malonyl-, methylmalonyland occasionally ethylmalonyl-CoA as the source of extender units. After each condensation, an FAS catalyses reduction of the P-oxoacylthioester by way of ketoreduction, dehydration, and further reduction. In contrast, PKSs either leave the reactive p-0x0 groups substantially intact (the aromatic PKSs), or catalyse a variable extent of reduction (the so-called modular PKSs, so that after each cycle of chain extension there may be no reduction, or there may be formation of a hydroxyacyl thioester, enoylthioester, or fully saturated acylthioester, and the outcome may vary between different cycles. This inherent variability, together with the greater variety of monomer units used, and the controlled variation in chain length, explains the far greater structural diversity of polyketides compared to fatty acids. In addition, the scabilisation of the fully formed polyketide chain by cyclisation, and its further modification by downstream enzymes, for example through oxidation, reduction, glycosylation, and methylation, provide further potential for the generation of diverse products.

Addresses Department of Biochemistry and Cambridge Recognition, CB2

University of Cambridge,

Centre for Molecular Tennis Court Road, Cambridge

1 QW, UK; e-mail: pfll [email protected]

Current Opinion

in Chemical

Biology

1997,

1 :162-l

66

http://biomednet.com/elecref/1367593100100162 0 Current Biology Ltd ISSN

1367-5931

Abbreviations ACP AT CLF CoA DEBS

acyl carrier protein acyltransferase chain length factor

FAS

coenzyme A 6-deoxyerythronolide fatty acid synthase

KS PKS

ketosynthase polyketide synthase

B synthase

Introduction Polyketides are a major class of natural products, synthesised principally by soil microorganisms like Str-eptomwes and related filamentous bacteria, but also by fungi and plants. They include a considerable number of clinically valuable antibiotics, antifungals, antiparasitic and antitumour compounds, cholesterol-lowering agents, and immunosuppressants. Like the related fatty acid synthases (FASs), polyketide synthases (PKSs) are multifunctional enzymes which catalyse the condensation of simple carboxylic acids, activated as their coenzyme A (CoA) esters, via decar-

The cloning and sequence analysis of the clustered biosynthetic genes governing polyketide biosynthesis has revealed that aromatic PKS genes are organised very differently from the PKS genes that govern the synthesis of complex or reduced polyketides (reviewed in [l]). Typically, aromatic PKS genes encode a single set of small proteins, each carrying a single type of active site, together with a discrete acyl carrier protein (ACP) to which the growing polyketide chain is covalently attached during each round of chain extension. At least some of the constituent enzymes act in every successive cycle, so these have been called iterative PKSs. Specialised enzymes responsible for chain initiation, chain termination, and loading of extender units onto the ACP are not readily identifiable in the PKS gene sequence but are presumably also present in bacterial cells. In contrast to aromatic PKSs, the PKSs for synthesis of reduced polyketides consist of giant polypeptides housing sets or ‘modules’ of enzymatic activities, each module containing the appropriate enzymes required to carry out one particular cycle of chain extension and (where appropriate) reduction. The gene sequencing reveals that additional modules are present which are required for specific chain initiation and release, so that this type of PKS appears completely self-sufficient. Interestingly, fungal PKS multienzymes appear to be organised as giant multienzyme polypeptides, but apparently use a single set of active sites iteratively. The very different structural organisation of iterative aromatic PKSs and modular PKSs

Combinatorial approaches to polyketide biosynthesis Leadlay

has largely

determined

have been

used

the

to analyse

experimental and manipulate

approaches these

This review will focus on the current prospects neered biosynthesis of combinatorial polyketide Previous reviews [Z-4] have emphasised the diversity of such polyketide libraries, and the the engineering approach. It is argued here that

that

generated.

If the

modules

catalysing

all these

163

outcomes

systems.

are available to be grafted together, then potential hybrid PKSs containing six such

for engilibraries. potential logic of an even

million. Although not all of these molecular assembly lines may produce a compound, this approach offers a facile way of generating molecular diversity.

better fundamental understanding of polyketide synthase structure and function is required for a truly rational approach to construction of such combinatorial polyketide libraries.

Polyketides as targets for combinatorial biosynthesis Natural products continue to be the source of structural novelty and they form the basis for several important therapeutic agents. Nevertheless there is a perception, which I share, that the rewards of screening compounds from microbial and fungal isolates are diminishing, and meanwhile combinatorial chemistry has succeeded in generating highly diverse libraries of compounds that can be rapidly screened for their therapeutic potential. Recent advances in the development and exploitation of oligonucleotide (reviewed in [5,6]) and peptide (reviewed in [7]) libraries have highlighted some crucial advantages of biopolymers in accessing molecular diversity: very large libraries can easily be made, stored, amplified, and screened; and candidate molecules obtained from such libraries can be iteratively modified and reselected. Unnatural polymer libraries [8] have already been developed to mimic the structures of peptides, nucleic acids and oligosaccharides, but they must be synthesised and screened using the conventional techniques of combinatorial chemistry This has focused attention on the possibilities of combinatorial biochemistry, in which access is gained to the genes encoding the enzymes that synthesise naturally occurring polymeric small molecules, such as terpenoids, oligosaccharides and polyketides. Using standard methods of genetic engineering, it may then be possible to alter the molecules produced by a metabolic pathway by deleting enzymes or adding new ones, or by altering the structure of one or more enzymes to alter the substrate specificity. Depending on the number of ways in which this can be done, the number of molecules potentially accessible by this route could be very large indeed. For example, the modular PKS for erythromycin contains six modules or sets of enzymes, and each module catalyses a single round of chain extension, with a range of specific chemical and stereochemical outcomes. How many such specific outcomes are conceivable! Consideration of the mechanism of fatty acid and polyketide biosynthesis suggests at least ten alternative outcomes are possible, in terms of the degree of reduction, the type of extender unit used, and the alternative stereoisomers that might be

Polyketides

share

the advantages

of other

the number of modules is one

small

molecule

drugs, including oral availability and relative stability in biological systems. They are conformationally restricted, because of the formation of a range of aromatic, polycyclic or macrocyclic ring systems; they offer a range of chemical functionality for further elaboration; and they show a desirable and adjustable range of mixed hydrophobic and hydrophilic character. Each individual clone in a library of recombinant strains would produce one or a few polyketide molecules so that the fermentation products of the whole collection would provide a library of novel polyketide structures for screening. The array of clones growing on an agar dish would serve as a spatially addressable library so that any clone producing a ‘hit’ in a screening procedure could be immediately identified and used in further work. Using existing technology, the daily preparation of thousands of such clones would be straightforward. Even if the products are not themselves bioactive, combinatorial polyketide biosynthesis might make available a wide range of novel, chiral scaffolds for use in combinatorial chemistry.

Hybrid aromatic polyketide

synthases

The first experiments to construct hybrid aromatic PKSs were carried out by Strohl and co-workers [9] by transferring actinorhodin PKS genes from Streptomyces coelicolor, where they normally govern the synthesis of the isochromanequinone actinorhodin, to the anthracyclineproducing organism S. g&‘/ells. The enzymes expressed by the introduced genes apparently collaborated with some of the resident PKS proteins, leading to the synthesis of altered aromatic polyketides. Since then, such ‘mix and match’ experiments have been carried out on a systematic basis, using an increasing number of natural aromatic PKS gene sets. Particularly important insights were obtained using an ingenious plasmid-based system for rapid engineering and controlled expression of the actinorhodin PKS genes and other hybrid genes in S. coelicolor are favoured

[lo]. The actinorhodin for such work because

PKS and S coelicolor they are respectively

one of the best-characterized PKS gene sets and the genetically best-defined actinomycete species. From the results, a set of ‘design rules’ has been deduced for the rational production of aromatic polyketides [ll]. According to these rules, there is a ‘minimal PKS’ consisting of a ketosynthase (KS) required for condensation, an ACP to carry the growing chain. and a ‘chain length factor’ (CLF) to specify chain length. These are proposed to be sufficient tc form a polyketone chain of appropriate length and to carry out one cyclisation to form an aromatic ring at a specified point in the chain [l 11.

164

Next

generation therapeutics

An alternative

expression

system

has allowed

analysis

of

hybrid PKS genes in S. glaucescens using as the starting point the tetracenomycin PKS genes that govern the

assessed, however, is whether the ‘degrees represented by utilisation of individual components in a hybrid PKS are truly

synthesis

of one

of the decaketide

antibiotic

the intermediate tcmF2 [12]. Recently, the products obtained from an in vitro production has shown conclusively that and not the minima1 PKS, dictates the the first cyclisation of the ACP-bound intermediate [13’].

tetracenomycin

via

careful analysis of system for tcmF2 a specific cyclase, regiospecificity of linear polyketide

Another important recent development was the demonstration [ 14’1 that when the frenolicin minimal PKS, which in its native context synthesises both Cl6 and Cl8 chains, was combined with the tetracenomycin cyclase, a novel nonaketide product was obtained whose structure was in complete agreement with the ‘design rules’. In the absence of the TcmN cyclase, however, only octaketides were produced. It can therefore now be concluded, after a period of some uncertainty in the literature, that the chain length of an aromatic polyketide is not dictated solely by the intrinsic specificity of the KS and CLF subunits within the minimal PKS (see also [ 12,15]. Protein-protein interactions within a well-defined aromatic PKS multienzyme complex might also exert a profound influence on the nature of the products. This question could be addressed through further in vitro study of aromatic polyketide biosynthesis [13’,16].

Towards combinatorial polyketides

another.

The

most

recent

results

At present, the number of well-characterised novel compounds remains modest, each successive publication normally reporting on only one or two such structures. McDaniel et a/. [ll] point out, however, that typically each major hybrid product is accompanied by S-10 minor products of unknown structure, presumably reflecting the chemical possibilities and constraints operating on the highly reactive poly-p-0x0 intermediates. Figure 1

(a)

OH

OH

$y Ho$ HO

0 SEKlS

SEK43

In their key paper [ll], Khosla, Hopwood and their co-workers have argued that the individual enzyme components of aromatic PKSs can be deployed in heterologous combinations in a rational manner to make ‘designer’ polyketides. Figure 1 illustrates two examples of the success of this approach. After consideration of the pathway to the known metabolite SEK1.5, which is generated in the S coelicolor expression system by the action of the tetracenomycin minimal PKS, a route to SEK43 was designed. SEK43 was obtained by combining

0

HO

minimal PKS (to the actinorhodin

ketoreductase (to ensure reduction of the keto function at C-9); and the griseusin aromatase (to secure aromatisation of the ring) [ll]. The second example is of the design of the nonaketide PK8, which was successfully obtained by examining the synthesis path to the decaketide RMOb, and deducing that PK8 required a combination of the frenolicin minima1 PKS with the tetracenomycin cyclase [14*]. It is clearly now practicable to devise suitable combinations of PKS components in order to achieve certain modifications of the basic set of observed product scaffolds. The extension of the mix and match approach, to allow the efficient production of large numbers of aromatic polyketides, certainly requires the recruitment of further natural PKS gene sets, which is going on apace [17]. A more serious limitation which cannot yet be confidently

[12,13*,14’,15]

strongly suggest that they are not independent, that the ‘design rules’ [l l] are inadequate, and that optimistic estimates [3,4] of the potential diversity of such libraries of aromatic polyketides should be regarded with caution.

libraries of aromatic

three components: the tetracenomycin ensure a decaketide was produced);

of freedom’ heterologous independent

RM8Ob 0 1997 Current O~m~on in Chemical B~oloor Examples of successful application of the ‘design rules’ [l 11 for hybrid aromatic polyketide synthases. (a) A pathway to SEK43 was designed by reference to the known pathway to SEK15. to the nonaketide PK8 was designed in a similar fashion to the known

pathway

to the decaketide

Hybrid modular polyketide

(b) A route by reference

RM8Ob.

synthases

In principle, the mode of assembly of complex polyketides by modular PKSs lends itself readily to the ideas of combinatorial biosynthesis, because there is apparently a separate enzyme for each catalysed step. When targeted alterations are made in the structural genes a clear prediction can be made for the precise point at which chain assembly will be affected, assuming that all the constituent

Combinatorial

Figure

approaches

to polyketide

biosynthesis

Leadlay

165

2

DEBS 3

DEBS 1

Intermediates in Chain Extension Cycles

2

0 1997 Current Op~mon m Chemtcal Bwlogy The organisation of the three multienzymes of the 6-deoxyerythronolide B synthase (DEBS). Each circle represents a discrete active ketosynthase (KS); an acyltransferase (AT); an acyl carrier protein (ACP); a dehydratase (DH), ketoreductase (KR) or enoylreductase multienzyme contains two complete modules each catalysing a particular cycle of polyketide chain extension.

enzymes act autonomously. Deletion, substitution, or addition of activities can all be contemplated. The use of the high-level expression system pRhl5 in S. roe&o/or strain CH999 [lo] has been extremely important for work aimed at exploring the creation of hybrid modular PKSs for two reasons: the speed with which mutations can be made to the genes, and the enhanced and controllable levels of PKS expression. Additionally, detailed information has been published on the three constituent multienzymes of one modular PKS, the 6-deoxyerythronolide B synthase (DEBS) from SauharopoJ~~spora erythmea. DEBS governs the synthesis of the macrolactone (14-carbon ring) of erythromycin A [l&19]. The domain structure of these multienzymes (DEBSl, DEBSZ and DEBS3: Figure 2) has been determined using limited proteolysis, and has revealed surface-accessible linker regions [20,21’]. These linker regions are obvious points in the multienzyme at which to graft in new enzymatic domains, with minimal disturbance to the structure. In recent engineering experiments reported using the DEBS genes, the sites of fusion in the hybrid PKSs are often chosen to be within an amino acid residue or two of the proteolytic cleavage sites previously reported for the purified multienzymes. Another decisive advantage of using the DEBS genes as a model for creation of hybrid modular PKSs is that in contrast to most other modular PKS gene clusters, the gene sequences have been published [19,20]. Furthermore, it has been shown that a convenient, smaller modular PKS called DEBSl-TE can be created by relocation of the

site, for a (ER). Each

chain-terminating thioesterase (TE) from the carboxyl terminus of DEBS3 to the carboxyl terminus of DEBSl, where it specifically accelerates release of the chain after two cycles to form a triketide lactone [22]. Use of the DEBSl-TE system obviously facilitates the required genetic manipulation, and the system has been rapidly adopted by the groups working in this area (see, for example, [23-26,27”]. A DEBSl-TE construct has been reported in which the DEBS module 2 reductive activities have been replaced by the defunct reductive activities of DEBS module 3 (Figure 3), and the resulting hybrid PKS produces the predicted 3-ketolactones [24]. Another reason for the recent rapid increase in interest in hybrid PKS genes is the recent publication and detailed analysis of the entire DNA sequence for the PKS governing the biosynthesis of the immunosuppressant rapamycin [28,29’]. This effort was undertaken both to investigate the possibility of generating novel rapamycins by genetic engineering, and to provide a plentiful and diverse supply

of well

characterised

combinatorial PKS construction, (and particularly the DEBSl-TE)

‘spare

part’

PKS

genes

for

using the DEBS genes as the starting point.

The first erythromycin/rdpdmycin hybrid to be reported [30**] involved the substitution of the methylmalonyl-CoAspecific acyltransferase (AT) domain from module 2 of DEBSl-TE by its malonyl-CoA-specific counterpart from module 2 of the rap PKS (Figure 4). As predicted, the substitution led to the production of novel triketide lactones lacking the methyl branch at C-4 but otherwise

166

Next generation therapeutics

Figure 3

DEBS 1

IllOdUW+lE

DEBS1

module3+TB

0 1997 Current Opinion in Chemical Biology

DEBSl, reductive

/

containing modules 1 and 2, and a truncated loop (in module

on a truncated

DEBS

2 of DEBS)

multienzyme.

DEBSP,

containing

by a loop from the rap PKS module

Circles

represent

discrete

active

3, fused to the TE domain

4, containing

sites as described

unchanged. This is significant because the AT domains are core domains in a modular PKS [21*] and their replacement might have been expected to cause problems through unfavourable protein :protein interactions. Furthering this idea is the very recent substitution of a larger reductive loop, obtained from rap module 4, in place of the original reductive loop of DEBSl module 2 [27”], as part of a trimodular DEBS-derived tetraketide synthase [25,26]. The hybrid PKS efficiently produces altered products containing a specific double bond rather than the original B-hydroxy group. The generation of hybrid PKSs is a seminal advance because these examples show that domains can be transferred not only within PKS, but also between different PKSs. In a given indicates strong structural homology between different PKS systems, which is essential for maximum diversity to be attainable in combinatorial biosynthesis.

Towards combinatorial synthases

module

modular polyketide

There are two other relevant examples of hybrid PKS multienzymes, in which alterations in the loading module

a dehydratase, in Figure

are shown,

leads to synthesis

Substitution

of one

of an enoylthioester

2.

of a macrolide PKS have been been made in order to achieve a change in the specificity of the PKS for its starter unit. In an elegant experiment [31”], an Eli Lilly group deleted the first multienzyme of the PKS for the 16-membered macrolide platenolide (which has an acetate starter unit) and then replaced it with an altered version in which the amino-terminal loading module was substituted with the closely-related PKS for the 16-membered macrolide tylonolide (where the starter unit is propionate). Tylonolide and platenolide are the respective precursors of the valuable antibiotics tylosin and spiramycin. The hybrid PKS efficiently produced the predicted homologue of platenolide, showing that the altered chain was readily accommodated by all the activities

of the PKS

[31**].

The same idea, of moving a loading module, has been exemplified in a recent experiment [32”] carried out to convert the rather narrowly specific loading module of DEBS into one which would accept branched-chain starter units. This is a major change in substrate specificity, aimed at future combinatorial feeding of alternative starter units.

Combinatorial

Figure

approaches

to polyketide

biosynthesis

Leadlay

167

4

DEBS I-TE

RAPS1

f

~

---1

\ PK-

P”

R=Me R = Et Hybrid Products

Q 1997

Current

Opinion

4n Chemical

Eboloqy

Alteration of a modular PKS in order to change the type of extender unit used. Substitution of an acetate-specific AT domain from module 4 of the rapamycin PKS gene for the propionate-specific AT in DEBS module 2 leads to synthesis of novel lactones lacking a specific methyl group. Ctrcles represent discrete active sites as described from the rapamycin PKS.

in Figure 2. The shaded

The source of the promiscuous loading module was the avermectin PKS, which normally synthesises a pentacyclic polyketide. The altered DEBSl-TE containing the avermectin loading module was indeed found to synthesise authentic C-Z branched derived from endogenous Z-methylbutyryl-CoA.

triketide lactones, presumably intracellular isobutyryland

exchanges

of

the

library construction that the predicted

kind

acetate-specific

discussed

AT domain derived

here,

and

enable

to proceed with a fair expectation chemical products will be formed.

Meanwhile, in a development nicely complementary to the biosynthetic effort, a promising report has appeared discussing iterative chemical combinatorial synthesis of polyketides on solid-phase support [33]. It remains to be seen whether the chemical or biosynthetic route is better able to deliver large and diverse polyketide libraries.

Conclusions The question now is not whether combinatorial libraries can be constructed, but whether be diverse enough to be really useful. The

circle denotes the transplanted

polyketide they will remaining

problems in constructing libraries of aromatic PKSs are concerned with the extent to which the novel products of the recombinant PKSs represent genuine biosynthetic diversity, and with trying to increase the size of the libraries that can be constructed. In contrast, there is apparently a great deal of untapped variation within naturally occurring modular PKSs, and the problem is that we do not yet know exactly which subunits or groups of domains can be swapped without compromising the workings of the integrated complex. Such information should, with luck, emerge from further systematic domain

References

and recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . l

*

of special interest of outstanding interest

1.

Katz L, Donadio S: Polyketide synthesis: prospects antibiotics. Annu Rev Microbial 1993, 47:875-912.

for hybrid

2.

Rohr J: Combinatorial biosynthesis - an approach future. Angew Chem Int Ed 1995, 34:681-085.

in the near

3.

Tsoi CJ, Khosla C: Combinatorial biosynthesis of unnatural natural products - the polyketide example. Chem Biol 1995, 2:355-362.

166

Next generation therapeutics

4.

Khosla C, Zawada RJX: Generation of polyketide libraries via combinatorial biosynthesis. Trends Biotechnol 1996, 14:335341.

The authors present evidence for a functionally dimeric model for modular polyketide synthases, using data from limited proteolysis, cross-linking, and ultracentrifugation.

5.

Breaker RR: DNA aptamers B/o/ 1997, 1:26-31.

22.

6.

Pan T: Novel and variant ribozymes obtained through in vitro selection. Curr Opin Chem Biol 1997, I:1 7-25.

Cortbs J, Wiesmann KEH, Roberts GA, Brown MJB, Staunton J, Leadlay PF: Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage. Science 1995, 268:1487-l 489.

23.

7.

Hruby VJ, Ahn J-M, Liao S: Synthesis of oligopeptide and peptidomimetic libraries. Curr Opin Chem No/ 1997, 1:114-l

Pieper R, Luo GL, Cane DE, Khosla C: Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases. Nature 1995, 378:263-266.

24.

Bedford D, Jacobsen JR, Luo G, Cane DE, Khosla C: A functional chimeric modular polyketide synthase generated via domain replacement Chem Biol 1996, 3:827-831.

25.

Kao CM, Luo GL, Katz L, Cane DE, Khosla C: Engineered biosynthesis of structurally diverse tetraketides by a trimodular polyketide synthase. J Am Chem Sot 1996, 118:9184-9185.

26.

Pieper R, Gokhale RS, Luo GL, Cane DE, Khosla C: Purification and characterisation of bimodular and trimodular derivatives of the erythromycin polyketide synthase. Biochemistry 1997, 36:1846-1851.

and DNA enzymes.

Curr Opin Chem

19.

8.

Soth MJ, Nowick JS: Unnatural oligomers and unnatural oligomer libraries. Curr Opin Chem Biol 1997, 1 :I 20-l 29.

9.

Bartel PL, Zhu CB, Lampel JS, Dosch DC, Connors NC, Strohl WR, Beale JM, Floss HG: Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthetic genes in Streptomycetes - clarification of actinorhodin gene functions. J Bacterial 1990, 172:4816-4826.

10.

McDaniel R, Ebeti-Khosla S, Hopwood DA, Khosla C: Engineered biosynthesis of novel polyketides. Science 1993, 262:15461550.

11.

McDaniel R, Ebert-Khosla S, Hopwood DA, Khosla C: Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature 1995, 375:549-554.

12.

Shen B, Summers RG, Wendt-Pienkowski E, Hutchinson CR: The Streptomyces glwcescens TcmKL polyketide synthase and TcmN polyketide cyclase genes govern the size and shape of aromatic polyketides. J Am Chem Sot 1995, 117:681 l-6821.

Shen B, Hutchinson CR: Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase. Proc Nat/ Acad Sci USA 1996, 93:6600-6604. A well executed study using a cell-free enzyme system to reveal the central role of the cyclase TcmN in determining the fate of the linear polyketide chain.

27. ..

McDaniel R, Kao CM, Fu H, Hevezi P, Gustafsson C, Betlach M, Ashley G, Cane DE, Khosla C: Gain-of-function mutagenesis of a modular polyketide synthase. J Am Chem Sot 1997, 119:4309-4310. The authors present another important example of the utility of the rapamycin polyketide synthase gene cluster for spare part surgery, replacing a ketoreductase domain in a trimodular DEBS-derived multienzyme by a ketoreductase plus a dehydratase didomain from rapamycin PKS module 4. 28.

13. .

14. .

Kramer PJ, Zawada RJX, McDaniel R, Hutchinson CR, Hopwood DA, Khosla C: Rational design and engineered biosynthesis of a novel 1 &carbon aromatic polyketide. J Am Chem Sot 1997, 119:635-639. An important study showing how the design rules for recombinant aromatic polyketide synthases should be revised.

Schwecke T, Aparicio JF, Molnar I, Koenig A, Khaw LE, Haydock SF, Oliynyk M, Caffrey P, Co&s J, Lester JB, et al.: The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proc Nat/ Acad Sci USA 1995, 92:7839-7843.

29. .

Aparicio JF, Molnar I, Schwecke, T, Koenig, A, Haydock SF, Khaw LE, Staunton J, Leadlay PF: Organisation of the biosynthetic gene cluster for rapamycin in Stiepfomyces hygroscopicus - analysis of the enzymatic domains in the modular polyketide synthase. Gene 1996, 169:9-l 6. Detailed information on the domain of organisation of the rapamycin polyketide synthase (PKS), essential for using PKS genes as spare parts in domain and module swaps.

30.

15.

Rajgarhia VB, Strohl WR: Minimal Streptomyces strain C5 daunorubicin polyketide biosynthetic genes required for aklanonic acid biosynthesis. J Bacterial 1997, 178:2690-2696.

16.

Carreras CW, Pieper R, Khosla C: Efficient synthesis of aromatic polyketides in vitro by the actinorhodin polyketide synthase. J Am Chem Sot 1996,118:5158-5 159.

1 7.

Alvarez MA, Fu H, Khosla C, Hopwood DA, Bailey JE: Engineered biosynthesis of novel polyketides - properties of the WhiE aromatase/cyclase. Nat Biotechnol 1996, 14:336-338.

18.

Cort&s J, Haydock SF, Roberts GA, Bevitt DJ, Leadlay PF: An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopo/yspor8 8rythr8ea. Nature 1990, 348:176-l 78.

19.

Donadio S, Staver MJ, McAlpine JB, Swanson SL, Katz L: Modular organisation of genes required for complex polyketide biosynthesis. Science 1991, 252:675-679.

20.

Aparicio JF, Caffrey P, Marsden AFA, Staunton J, Leadlay PF: Limited proteolysis and active-site studies of the first component of the etythromycin-producing polyketide synthase. J Biol Chem 1994, 269:8524-8528.

21. .

Staunton J, Caffrey P, Aparicio JF, Roberts GA, Bethell SS, Leadlay PF: Evidence for a double-helical structure for modular polyketide synthases. Nat Struct Biol 1996, 3:188-192.

Olivnvk M. Brown MJB. Coti& J. Staunton J. Leadlav PF: A hvbrid poiyietide synthase obtained by domain swappiig. Chem-8iol 1996, 3:833-839. The authors demonstrate the creation of a hybrid modular polyketide synthase multienzyme using an acetate-specific acyltransferase domain from the rap gene cluster to substitute for a propionate-specific acyltransferase domain in the DEBSl -TE, and its use to obtain a specifically altered triketide lactone product.

..

31. ..

Kuhstoss S, Huber M, Turner JR, Paschal JW, Rao RN: Production of a novel polyketide synthase through the construction of a hybrid polyketide synthase. Gene 1996, 183:231-236. Another excellent example of the creation of a hybrid modular polyketide synthase multienzyme, containing a propionate-specific loading module in place of an acetate-specific one, and its use to complement other polyketide synthase multienzymes in trans to create a 16-membered macrolide. Marred only by the lack of supporting experimental evidence. 32. ..

Leadlay PF, Staunton J, Marsden AFA, Wilkinson B, Dunster NJ, Co&s J, Oliynyk M, Hanefeld U, Brown MJB: Rational engineering of complex polyketide biosynthesis - where do we begin? In Generics and Molecular Biology of Industrial Microorganisms., Edited by Hegeman GD, Baltz RH, Skatrud PL. Washington: American Society for Microbiology; 1997: in press. Significant, as the first module swap to create a more promiscuous polyketide synthase for possible combinatorial studies. 33.

Reggelin M, Brenig V: Towards polyketide libraries - iterative, asymmetric aldol reactions on a solid support Tetrahedron Lett 1996, 37:6851-6852.