Methods to mediate polyketide synthase module effectiveness

Details Methods to mediate polyketide synthase module effectiveness Methods to mediate polyketide synthase module effectiveness

2000-02-09

2004-06-22

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Reexamination Certificate

active

06753173

ABSTRACT:

TECHNICAL FIELD
The invention is directed to facilitating usage by polyketide synthase modules of nascent polyketide chains. More specifically, the invention concerns including intermodule and intramnodule linkers in constructions for synthesis of desired polyketides.
BACKGROUND OF THE INVENTION
Polyketides are a class of compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. Polyketides are biologically active molecules with a wide variety of structures, and the class encompasses numerous compounds with diverse activities. Tetracycline, erythromycin, epothilone, FK-506, FK-520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of polyketides. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds.
The biosynthetic diversity of polyketides is generated by repetitive condensations of simple monomers by polyketide synthase (PKS) enzymes that mimic fatty acid synthases. For instance, the deoxyerythronolide-B synthase catalyzes the chain extension of a primer with several methylmalonyl coenzyme A (MeMalCoA) extender units to produce the erythromycin core.
The cloning, analysis, and recombinant DNA technology of genes that encode PKS enzymes allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters. See, e.g., PCT publication Nos. WO 93/13663; 95/08548; 96/40968; 97/02358; 98/27203; and 98/49315; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146; 5,830,750; and 5,843,718; and Fu, et al., 1994
, Biochemistry
33: 9321-9326; McDaniel, et al., 1993
, Science
262: 1546-550; and Rohr, 1995
, Angew. Chem. Int. Ed. Engl
. 34(8): 881-888, each of which is incorporated herein by reference.
PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between acylthioester building blocks. The buildinhg blocks used to form complex polyketides are typically acylthioesters, such as acetyl, butyryl, propionyl, malonyl, hydroxymalonyl, methylmalonyl, and ethylmalonyl CoA. Two major types of PKS enzymes are known; these differ in their composition and mode of a synthesis of the polyketide synthesized. These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes.
The present invention concerns modular PKS. In the Type I or modular PKS enzyme group, a set of separate catalytic active sites (each active site is termed a “domain”, and a set thereof is termed a “module”exists for each cycle of carbon chain elongation and modification in the polyketide synthesis pathway. The typical modular PKS is composed of several large polypeptides, which can be segregated from amino to carboxy terminii into a loading module, multiple extender modules, and a releasing (or thioesterase) domain. The PKS enzyme known as 6-deoxyerythronolide B synthase (DEBS) is a typical Type I PKS. In DEBS, there is a loading module, six extender modules, and a thioesterase (TE) domain. The loading module, six extender modules, and TE of DEBS are present on three separate proteins (designated DEBS-1, DEBS-2, and DEBS-3, with two extender modules per protein). Each of the DEBS polypeptides is encoded by a separate open reading frame (ORF) or gene; these genes are known as eryAI, eryAII, and eryAIII. See FIG.
1
. There is considerable interest in the genetic and chemical reprogramming of modular PKSs (see, e.g., Khosla, 1997
, Chem. Rev
. 97:2577-2590, and Staunton, et al., 1997
, Chem. Rev
. 2611-2629, each of which is; incorporated herein by reference).
Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module. The loading module of DEBS consists of an acyltransferase (AT) domain and an acyl carrier protein (ACP) domain. Another type of loading module utilizes an inactivated KS, an AT, and an ACP. This inactivated KS is in some instances called KS
Q
, where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for ketosynthase activity. In other PKS enzymes, including the FK-520 PKS, the loading module incorporates an unusual starter unit and is composed of a CoA ligase activity domain. In any event, the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl) and transfers it as a thiol ester to the ACP of the loading module.
The AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and hydroxymalonyl) and transfers it to the ACP of that extender module to form a thioester. Each extender module is responsible for accepting a compound from a prior module, binding a building block, attaching the building block to the compound from the prior module, optionally performing one or more additional functions, and transferring the resulting compound to the next module. The transfer into a module is mediated by the KS domain which is upstream of the remaining catalytic domains. The additional functions are performed by enzymes which comprise a ketoreductase (Kg) which reduces the carbonyl group generated from the condensation to an alcohol, a dehydratase (DH) which converts the alcohol to a double bond, and an enoyl reductase (ER) which reduces the doublebond to a single bond. These catalytic domains appear to be immediately adjacent and not separated by any linking sequences. Collectively, they can be called “beta-carbonyl modifying” domains. Thus, a particular module may contain none of these activities, only KR, or KR+DH, or KR+DH+ER. Thus, the order of domains from the N-terminus of a particular module is KS, AT, beta-carbonyl modifying domains (if present), ACP. The order, N→C of the beta-carbonyl modifying enzymes is DH ER KR.
Thus, each extender module of a modular PKS contains zero, one, two, or three enzymes that modify the beta-carbon of the growing polyketide chain downstream of the AT catalytic domain. A typical (non-loading) minimal Type I PKS extender module is exemplified by extender module 3 of DEBS, which contains only a KS domain, an AT domain, and an ACP domain. The next extender module, module 4, contains all three 10 beta-carbonyl modifying enzymes. (The beta-carbonyl modifying enzymes effect such modification on the extender unit that has been added by the previous module.)
Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module one possesses an acyl-KS adjacent to a malonyl (or substituted malonyl) ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading building block (elongation or extension).
After traversing the final extender module, the polyketide encounters a releasing domain that cleaves the polyketide from the PKS and typically cyclizes the polyketide.
For example, final synthesis of 6-dEB is regulated by a TE domain located at the end of extender module six. In the synthesis of 6dEB, the TE domain catalyzes cyclization of the macrolide ring by formation of an ester linkage. In FK-

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