Biomedical Engineering Reference
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of its DEBS analogue. This hybrid system allowed a series of simple carboxylic acids
to be loaded in the place of the wild-type propionate [40,41].
Most recently, with the heterologous expression of the DEBS pathway in
E.
coli
[42], a truncated derivative of DEBS has also been constructed in which the
loading didomain and module 1 were removed entirely [43,44]. The need for the
purification of the aglycone intermediate has also been removed by the reconsti-
tution of the entire biosynthetic pathway of 6-deoxyerythromycin D in
E. coli
[45].
This system has the potential for greatly enhancing the pace at which synthetic
precursors can be screened for their ability to be converted into superior erythro-
mycin analogues.
14.4.2.3. Calcium-Dependent Antibiotic The calcium-dependent antibiotics
(CDAs) are a group of nonribosomal peptides produced natively in
S. coelicolor
. They
belong to a class of molecules termed the lipopeptides, the best known of which is
daptomycin. CDAs are so named due the dependence of their biological activity on the
binding of a calcium ion. This chelate is then postulated to insert itself into the bacterial
cell wall of Gram-positive bacteria, producing a pore that results in membrane
depolarization through the release of potassium ions.
The CDAs are undecapeptides that, like balhimycin, contain several nonpro-
teogenic amino acids. Of pertinence for the precursor-directed approaches that have
been applied to this molecule are D-4-hydroxyphenylglycine, D-3-phosphohydrox-
yasparagine, and L-3-methylglutamic acid (Scheme 14.8). Another defining feature
of the CDAs is the presence of the 2,3-epoxyhexanoyl fatty acid moiety that has
proven essential for bioactivity.
In initial efforts toward precursor-directed production of a CDA, a mutant of
S.
coelicolor
was produced in which the biosynthesis of D-4-hydroxyphenylglycine was
blocked. The authors examined replacement of the hydroxyl group with various
functionalities and discovered that the A domain responsible for the incorporation of
this amino acid was rather discriminating as the only permissible alterations were
replacement with hydrogen or fluorine [46].
In a subsequent study, L-3-trifluoromethylglutamic acid and L-3-ethylglutamic
acid were fed to cultures of
S. coelicolor
and the product profiles were analyzed. The
desired analogues were found to be produced despite competition from their natural
counterparts [47,48].
CDA contains tryptophan, or amino acids derived therefrom, at positions C3
and C11. In a recent study, the authors used a strain of
S. coelicolor
that was
auxotrophic for tryptophan and supplemented the media with several tryptophan
analogues. Low-level incorporation of 5-hydroxytryptophan was found at both these
positions, while 4-fluorotryptophan was incorporated at considerably higher
levels [49].
The final portion of the CDA scaffold to be altered by precursor-directed
biosynthesis was the short fatty acid side chain. This work was performed in a manner
analogous to that described for erythromycin. The carrier protein domain of module 1
was inactivated by an alanine mutation of the active-site serine. A series of
N
-acyl-
serine-SNAC thioesters were then fed to the mutant strain. When acylated with fatty
chains of six or fewer carbons, the desired CDA analogues were produced in modest
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