Polyketides are a class of compounds synthesized by the enzymatic polymerization of acetyl, proprionyl, butyryl and methoxyacetyl moieties (extender units) into a polyketide backbone through a series of decarboxylative condensation and reduction reactions and subsequent modifications. Polyketide natural products biosynthesized by polyketide synthases have diverse biological activities such as antibacterial, antifungal, anticancer, and immunosuppressant activities. Many of these products play important roles in the treatment of a variety of human diseases.
The Type I polyketide synthases are highly modular proteins. Each Type I PKS module consists of several domains with defined functions, separated by short spacer regions. Type I PKSs 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 (Donadio et al., 1991, Science 252: 675-679; MacNeil et al., 1992, Gene 115: 119-125). The active sites required for one cycle of condensation and reduction are typically clustered as “modules”. For example, 6-deoxyerythronolide B synthase (DEBS) consists of the three multifunctional proteins, DEBS 1, DEBS 2, and DEBS 3, each of which possesses two modules that incorporate an extender unit into a polyketide chain (Caffrey et al., 1992, FEBS Lett. 304: 225-228).
The diverse activities of polyketides are partly due to their extensive structural diversity available via Type I PKS enzymology (FIG. 1). However, their use for medicinal purposes is being challenged by the development of resistance to these molecules by microorganisms and by cancer cells. Additionally, use of a particular polyketide for the treatment of a patient can be inhibited by unwanted side effects caused by the molecule, or the failure of the molecule to have the desired level of activity. To combat these issues, new derivatives of polyketides are needed. One approach to generate new derivatives is to modify known polyketides through synthetic or semisynthetic chemistry. While successful in many cases, the more complicated the polyketide structure, the less efficient a synthetic or semisynthetic approach is. A complementary approach is to use metabolic engineering of the polyketide biosynthetic pathway to generate new structural derivatives. Although this approach has been successful in generating new polyketides, one of the restrictions in its use has been the limited number of precursors, or extender units, which can be incorporated into a polyketide backbone by the Type I PKSs.
The flexibility of Type I PKSs has been exploited for the generation of metabolically engineered “natural” products through combinatorial biosynthesis. One example is to replace a catalytic domain from one Type I PKS with an alternative domain from a different Type I PKS, resulting in a hybrid enzyme that generates a hybrid product. This approach was shown to generate a library of nearly 60 erythromycin derivatives by exchanging catalytic domains from the erythromycin Type I PKS with catalytic domains from other Type I PKSs (McDaniel et al., 1999, Proc. Natl. Acad. Sci. USA 96: 1846-1851). Thus, combinatorial biosynthesis complements the more traditional approaches of using total or semisynthetic chemistry to generate structural diversity.
Changing the extender unit(s) incorporated into a polyketide can be used to vary the moiety that extends away from the backbone of the polyketide, which can have effects on its interaction with its biological target. Changes available using this approach are limited because of the limited number of known Type I PKS extender units—only four: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl (MM)-ACP (FIG. 2). These extender units result in the incorporation of acetyl, propionyl, butyryl, or methoxyacetyl moieties into the polyketide backbone, respectively. The chemical attributes of these extender units are similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. However, for all of these extender units, the moieties on the α-carbons lack simple chemical reactivity for further downstream modification by semisynthetic chemistry. Due to these limitations, there is an interest in identifying or generating new extender units with different chemical attributes to enhance structural diversification by combinatorial biosynthesis and increase the opportunities for downstream modification by semisynthetic chemistry.
The inventors of the present patent application previously published a paper proposing the existence of previously unknown hydroxymalonyl-ACP (HM-ACP), and aminomalonyl-ACP (AM-ACP) extender units (Emmert et al., 2004, Appl. Environ. Microbiol. 70: 104-113). In that paper, the mechanism of AM-ACP formation was proposed and has been subsequently confirmed. Although Emmert et al. proposed a mechanism for HM-ACP formation, the proposed mechanism has since been determined to be incorrect. The precursor is not glycerate, a glyceryl-CoA intermediate is not formed, and Orf2 (ZmaF) does not play a role in HM-ACP formation. Emmert et al. further proposed the minimal biosynthetic machinery for zwittermicin A assembly involving the incorporation of AM-ACP and HM-ACP, shown in FIG. 4; however, the identity of the necessary acyltransferase (AT) domains for incorporation of these extender units was not disclosed. These AT domains are the essential components needed for AM-ACP and HM-ACP recognition and incorporation. At the time, there were many potential mechanisms for AM-ACP and HM-ACP incorporation, but it was not known which mechanism was correct. In accordance with the present invention, novel enzymes responsible for the biosynthesis of zwittermicin A have been discovered.