Combinatorial biosynthesis modification

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. 

Modif/ing the ClYcosylation Pattern in Actinomycetes by Combinatorial Biosynthesis... 

The biosynthesis of many hydroxylated natural products proceeds through regio- and enantioselective modification of polyketides, which are assembled through chain elongation via acetate or propionate units [2]. The enzymes responsible for the chain elongation and subsequent reduction, elimination, aromatiza-tion, and further modifications are classified as polyketide synthases [3]. These multifunctional enzymes have been used for whole-cell biotransformation toward unnatural metabolites that are within the scope of combinatorial biosynthesis... 

Rix U, Fischer C, Remsing LL et al (2002) Modification of post-PKS tailoring steps through combinatorial biosynthesis. Nat Prod Rep 19 542-580... 

Combinatorial biosynthesis is an approach aimed at the modification of the cellular machinery involved in the biosynthetic pathways that produce natural products. There are several groups active in the field, and an example reported by McDaniel et al. (60)... 

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... 

Natural product research is at its highest level now due to unmet needs, remarkable diversity of structures and activities, utility as biochemical probes, novel and sensitive assay methods, improvements in isolation, purification and characterization, and new production methods." Many new products have been made by genetic methods involving modification or exchange of genes between organisms to create hybrid molecules the technique is known as combinatorial biosynthesis. ... 

Manipulation of the DEBS system has led to the most impressive demonstration of combinatorial biosynthesis to date. McDaniel and coworkers have utilized specific module-swapping strategies to access a variety of 6-deoxyerythronolide B analogs with modifications at each carbon of the macrolide backbone [26]. Modules 1-6 of DEBS were systematically replaced with individual rapamycin synthase components to alter oxidation state and methylation in the final polyketide product. The study produced 60 unique structures at yields ranging from 1 to 70% of that of 6-deoxyerythronolide B (Fig. 9.2-5). However, each new compound required independent synthase engineering, which made library construction quite tedious. 

Combinatorial biosynthesis is a contemporary approach with the ability not only to produce new natural product analogues but also to afford new drug candidates per se. This methodology involves the engineering of biosynthetic gene clusters in microorganisms. For example, the modification of bacterial polyketide synthases has led to production of some 200 new polyketides that do not occur naturally (43). 

R is initially hydrogen in steffimycin this position is modified with a -OCH3 moiety. Although the steffimycin biosynthetic cluster has been cloned and sequenced [23], the genes responsible for this modification have not been unequivocally identified, and this modification has not been used in combinatorial biosynthesis. 

Combinatorial biosynthesis can be practically realized by (i) chemical modification by biocatalysts (biotransformation), (ii) mutasynthesis, (iii) combinatorial metabolism in hybrids, and (iv) activation of silent metabolism (Figure 2.11). 

Another noteworthy approach is combinatorial biosynthesis, that is, the deliberate generation of novel natural products via supplementation of unnatural precursors or modification of enzymes [153]. 

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