Biomedical Engineering Reference
In-Depth Information
cluster must first be cloned into expression vectors that are compatible with the
heterologous host. Conditions must then be identified that lead to the successful
expression and folding of the biosynthetic enzymes. Frequently, coexpression of
accessory proteins such as posttranslational modification enzymes is required in the
heterologous host. However, once these hurdles are overcome, the engineering
potential can be greatly increased.
Precursor-directed biosynthesis involves the loading of unnatural substrates
onto biosynthetic enzymes. A frequently encountered problem is the competitive
loading of both unnatural and natural substrates onto the PKS or NRPS assembly line,
whichmost often favors the natural substrate. To eliminate this competition, either the
domain responsible for loading the natural precursor is genetically deleted or
inactivated or biosynthesis of the natural precursor is disrupted. Such approaches
lead to vastly increased titers of the desired natural product analogues and also much
cleaner product profiles and are sometimes referred to as mutasynthesis or chemo-
biosynthesis. The term precursor-directed biosynthesis, as it is used throughout this
chapter, encompasses both these approaches.
14.4. APPLICATIONS OF PRECURSOR-DIRECTED
BIOSYNTHESIS
Several of the most important issues that one must take into consideration when
establishing a system for the production of natural product analogues through
precursor-directed biosynthesis are presented above. As is the case for the total
synthesis of natural product analogues, this initial setup is a rather arduous exercise (in
this case, in molecular biology). There exist, however, many examples of the
successful application of this type of approach, which will be discussed later.
Examples are organized into sections depending on the synthetic complexity of
the precursors introduced. We begin with systems where unnatural primer or extender
units are used. Oftentimes, these precursors are commercially available and require
little or no synthetic manipulation. In the systems with precursors of intermediate
complexity, most often the precursor is not commercial and must be obtained through
simple synthetic schemes. These applications utilize multiple rounds of chain
elongation and tailoring performed by the target biosynthetic enzymes. We conclude
with a few examples in which the researchers have synthesized highly complex
precursor molecules and exploited only biosynthetic enzymes for the performance of
one or two reactions.
The examples presented here were chosen as they are each representative of a
slight variation on the theme of precursor-directed biosynthesis. More in-depth
discussions of each example, as well as many not included here, have been undertaken
in several excellent review articles [4-8].
14.4.1. Simple Precursors
Arguably themost powerful exploitations of precursor-directed biosynthesis are those
in which simple molecules are incorporated as unnatural primer and extender units.
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