Biomedical Engineering Reference
In-Depth Information
discussed in the following sections. Variations of the canonical PKS or NRPS
assembly line described above are commonplace.
14.3. PRECURSOR-DIRECTED BIOSYNTHESIS
14.3.1. Introduction
Polyketide and nonribosomal peptide natural products have long been, and remain,
clinically important yet synthetically challenging classes of molecules. In many
cases, however, the complexity of these molecules belies the simplicity of the small
primary metabolites from which they arise. As shown in the above biosynthetic
systems, these simple precursor molecules are assembled into complex secondary
metabolites by a series of biosynthetic enzymes that act with a level of regio- and
chemoselectivity that is difficult to achieve in the chemical synthesis of large, highly
functionalized molecules.
Superficially, the two classes of molecules that will be discussed here, poly-
ketides and nonribosomal peptides, appear very different, but it is the similarities
in the logic of their construction, as illustrated in the previous section, that has led to
the successful application of precursor-directed biosynthesis as an approach to the
generation of analogues of each. Both biosynthetic pathways are initiated by the
priming of the system by a simple metabolite, which is then elongated through
iterative reactions with a series of simple extender molecules. During the course of
construction of the polyketide or peptide backbone, the molecule is regiospecifically
modified by enzymes such as reductases and epimerases.
Precursor-directed biosynthesis is a technique that exploits this assembly line
biosynthetic strategy by inserting unnatural precursors at targeted places along these
enzymatic cascades. The unnatural precursor can be inserted in any of the priming,
extending, or tailoring phases and allows all downstream biosynthetic enzymes to act
with fidelity, thereby yielding a predictable natural product analogue.
The strength of this approach lies in the simplicity of the precursor molecules
coupled with the ability of highly evolved biosynthetic pathways to introduce extreme
complexity in a highly efficient manner. As with any approach to the production of
complex molecules, there are several aspects of the design of precursor-directed
biosynthetic systems that need to be taken into account prior to attempting this type of
work. Among themost pressing of these concerns are the level of complexity to design
into the synthetic precursors, the substrate tolerance of the biosynthetic enzymes,
selection of the microbial host, and engineering of the biosynthetic system itself.
These issues will be discussed in detail later.
14.3.2. Precursor Complexity and Enzyme Tolerance
In the biosynthetic schemes shown previously, the nascent natural product grows in
complexity from a simple priming molecule to the final highly functionalized natural
product. Examples exist in which unnatural precursors are introduced at virtually all
points along this continuum of complexity, with the two extremes being unnatural
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