Combinatorial Biosynthesis of Natural Products

Natural products (NPs) have long been a source of biologically active compounds, and their extraction and synthetic modification, especially for pharmaceutical purposes (238, 239), have been well-smdied. Their stmcmres exhibit a wide degree of diversity and levels of complexity that are seldom attained in totally synthetic structures, and this is reflected in the wide range of biological properties they display. 

Recently, combinatorial technologies and libraries have somewhat replaced NPs as a source of diversity in pharmaceutical research. A comparison of combinatorial technologies with the fermentation of an NP-producing organism show that they both produce a library of compounds. While the former library is structurally determined a priori either by the selected synthetic scheme or by recombinant genetic information in the case of biosynthetic peptide and ON libraries, the latter is the result of the metabolic complexity of the producing strain and has to be deconvoluted in order to 

Polyketides (PKs) are a typical example of a large and diverse class of NPs that derive from several related biosynthetic pathways. Their stmctures contain repeating units iteratively assembled into a range of diverse chemical structures (Fig. 10.43). PKs can be taken as an example of the application of combinatorial biosynthesis as both the 

Modular PKS enzymes are responsible for the synthesis of a wide diversity of structures and seem to have more relaxed specificities in several of the enzymatic steps. Their enormous appeal for combinatorial purposes, though, derives from the presence of multiple modules that can be manipulated independently, allowing the production of rings of different sizes and with potential stereochemical variation at each PK carbon. The higher complexity of these pathways has somewhat hindered their exploitation, but recently, several have been fully characterized. Among them, by far the most studied modular multienzyme complex is 6-deoxyerythronolide B synthase (DEBS 240,266,267), which produces the 14-member macrolide 6-deoxyerythronolide B (10.70, Fig. 10.45). DEBS contains three large subunits each of which contains two PKS enzyme modules. Each module contains the minimal PKS enzyme vide supra) and either none (M3), one (ketoreductase KR Ml, M2, MS, and M6), or three (dehydratase DH-enoyl reductase ER-ketoreductase KR, M4) catalytic activities that produce a keto (M3), an hydroxy (Ml, M2, MS and M6), or an unsubstituted methylene (M4) on the last monomeric unit of the growing chain (Fig. 10.45). A final thioesterase (TE) activity catalyzes lactone formation with concomitant release of 10.70 from the multienzyme complex. Introduction of TE activity after an upstream module allows various reduced-size macrolides (10.71-10.73, Eig. 10.45) to be obtained. 

The specificity of many components of DEBS has been thoroughly studied to allow its careful manipulation (268-272), and its potential for combinatorial biosynthesis including several combinatorial applications via rational modifications of DEBS has been thoroughly reviewed (251, 252, 273-279). Other large modular PKS enzymes have also been characterized (280-288) and will eventually be used to design and 

---

Zhang W, Tang Y (2008) Combinatorial biosynthesis of natural products. J Med Chem 51 2629-2633... 

Examples of Combinatorial Biosynthesis of Pharmaceutical Natural Products... 

Since the first description was only two decades ago, combinatorial biosynthesis has advanced from a limited set of proof-of-principle experiments into a more mature scientific discipline. To reach the maximal potential of natural product structural diversity, the combination of this approach with other established and emerging technologies will ultimately provide access to a rich variety of unnatural natural products with improved properties or new biological activities for future drug discovery and development. 

Thomas, M.G., Bixby, K.A. and Shen, B. (2005) Combinatorial biosynthesis of anticancer natural products, in Anticancer Agents from Natural Products (eds G.M. Cragg, D.G.I. Kingston and D.J. Newman), CRC Press, Boca Raton, FL, pp. 519-551. 

In this chapter, we will introduce an exciting class of natural product biosynthetic enzymes, the modular synthases, as well as their associated enzyme partners. We will discuss the use of metabolic engineering as a tool for small-molecule discovery and development, both through directed fermentation and combinatorial biosynthesis. In addition, we will review six classes of partner enzymes involved in the modification of polyketide (PK) and nonribosomal peptide (NRP) natural products. We believe that these enzymatic transformations hold great opportunities for synthetic chemists and will serve as the foundation for a new trend in both discovery and process chemistry. 

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

The power of combinatorial biosynthesis has been best demonstrated through the engineered biosynthesis of polyketides." Polyketides consist of a structurally diverse family of natural products and are mostly biosynthesized by soil-bome actinomyces as secondary metabolites. Fungi and plants have also been sources of polyketides. 

The three types of PKSs described here, the enediyne PKS, the C-0 bondforming PKS, and the AT-less PKS, are only representive examples that reside outside the archetypical PKS paradigms. Continued exploration on the mechanism of polyketide biosynthesis will undoubtly uncover more unusual PKSs. These novel PKSs, in combination with the archetypical ones, will ultimately enhance the toolbox available to facilitate combinatorial biosynthesis and production of iinnatural natural products. The full realisition of the potential embodied by combinatorial biosynthesis of PKSs for natural product structural diversity, however, depends critically on the fundamental characterization of PKS structure, mechanism, and catalysis. 

---