Modular biosynthetic systems

The natural diversity in molecular structure found within these groups points to the evolutionary logic behind modular systems for the synthesis of these types of secondary metabolites. The theoretical simplicity of this modular organization coupled with the near infinite permutations of possible molecular structures has fueled enormous academic and industrial efforts to understand and modify modular biosynthetic systems, and new pathways continue to be revealed in the pursuit of this objective [7-10, 17]. 

Conceptually, the modular biosynthetic systems with their assembly line structure are, perhaps, the most appealing for the purpose of rational biosynthetic engineering. If they could be properly harnessed, one can reasonably envision the construction of large, macrocyclic structures comprised of hydrocarbons of varying length, branches and oxidation via PKS modules, as well as nearly any amino acid imaginable via NRPS modules. Indeed, so many... 

For biosynthetic systems other than modular PKSs and NRPSs, the relationship between the structures of biosynthetic enzymes and the structure of the product(s) formed is often much less clear. Consequently, predicting the structures or structural features of metabolic products of novel biosynthetic systems uncovered by genomics can be challenging. Nevertheless, some structural parameters of such compounds can often be inferred. Indeed, sequence analyses of some classes of biosynthetic enzyme can lead to predictions of substrate specificity. 

Figure 1 Hypothetical pentaketide biosynthetic system, which illustrates the enzymatic logic of type I modular polyketide synthases (PKSs) and the catalytic role of acyl transferase (AT) domains. Each AT domain selects substrates from the cellular pool and tethers them as thioesters to acyl carrier protein (ACP) domains. In a typical PKS module, the AT and ACP domains are present in all modules. The ketosynthase (KS) domain is present in all chain extension modules. The dehydratase (DH), enoyl reductase (ER), and ketoreductase (KR) domains are optional domains. The final thioesterase (TE) domain catalyzes the release of the product from the PKS.

Typically, the last domain in the final module of modular PKS and NRPS systems is a thioesterase (TE) domain. This domain catalyzes the release of the assembled polyketide or peptide chain from carrier protein domain within the last module of the PKS or NRPS. Separately encoded, stand-alone TE enzymes are also found in some systems, such as the coelichelin biosynthetic system. TE domains catalyze two related types of chain release reactions. The first type is the hydrolysis or intermolecular condensation with a soluble amine and the second is intramolecular amide or ester bond formation. These chain release reactions result in distinct metabolic products. The intermolecular reactions lead to linear products with a carboxyl-terminus, whereas the intramolecular reactions lead to cyclic products. Sequence comparisons of TE domains from various modular PKSs that assemble known metabolic products have established a correlation between the phylogenetic relatedness of the domains and the type of chain release reaction catalyzed. " However, this predictive tool, which has been developed by Ecopia BioSciences, is not yet publicly accessible. 

Particularly important to the pathways of modular synthases is the incorporation of novel precursors, including nonproteinogenic amino acids in NRP systems [17] and unique CoA thioesters in PK and fatty acid synthases [18]. These building blocks expand the primary metabolism and offer practically unlimited variability applied to natural products. Noteworthy within this context is the contiguous placement of biosynthetic genes for novel precursors within the biosynthetic gene cluster in prokaryotes. Such placement has allowed relatively facile elucidation of biosynthetic pathways and rapid discovery of novel enzyme mechanisms to create such unique building blocks. These new pathways offer a continued expansion of the enzymatic toolbox available for chemical catalysis. 

According to this modular analysis, each protein catalyses two cycles of chain extension. The term cassette has been proposed for the giant proteins [34]. All three cassettes in the erythromycin cluster are bimodular, but in other systems, such as the rapamycin [35] and tylosin [36] PKSs, the size of a cassette can vary from one to six chain extension modules. The three cassettes co-operate in some way to form an extraordinarily complex molecular assembly hne. The biosynthetic intermediates remain PKS-bound throughout the whole synthetic sequence via thioester links. A challenging feature of this organisation is the mechanism which controls the ordering of the cassettes in the assembly Hne so that the transfer of the growing chain from one cassette to the next is correctly controlled. 

NRPS-independent siderophore (NTS) synthetases constitute another class of biosynthetic enzymes that can be divided into three types according to their amino acid sequence.These types are proposed to be specific for different substrates. Thus, type A enzymes are specific for citric acid, type B enzymes are proposed to be specific for a-ketoglutaric acid, and type C enzymes are specific for derivatives of citric or succinic acid. The type C enzymes are further divided into modular and iterative subtypes depending on whether they catalyze one or multiple condensation reactions. Thus for novel NTS synthetase systems uncovered by genome sequencing, structural features of their metabolic products can often be predicted. 

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