PRECURSOR-DIRECTED BIOSYNTHESIS OF POLYKETIDE AND NONRIBOSOMAL PEPTIDE NATURAL PRODUCTS - Modern Tools for the Synthesis of Complex Bioactive Molecules - page 501

activity, but this modest increasewas contrasted by significant increase in cytotoxicity
in healthy cells [52].
The most recent work on using these types of approaches for the production of
unnatural beauvericins has moved beyond the native host to use an engineered strain
that is incapable of producing Hiv. The gene for ketoisovalerate reductase ( kivr ), the
gene responsible for the reduction of ketoisovalerate to Hiv, was identified and
knocked out, leaving a system that was capable of producing cleaner product profiles
through the elimination of competition from the wild-type substrate. When a 2-
hydroxy carboxylic acid was fed, the product in which it was incorporated at all three
positions was the sole product. Compounds

were produced from feeding of
2-hydroxybutyrate and 2-hydroxy-3-metylvalerate, respectively, as proof of principle
results (Scheme 14.9). This approach was then coupled with the feeding of 2- and 3-
fluorophenylalanine, leading to the production of

48 and 49

. These compounds were
tested in the same antihaptotactic assays as used previously and all were found to have
activity that was approximately equivalent to or diminished compared to wild
type [53].
To this point, precursor-directed biosynthesis has yet to yield a depsipeptide
analogue with improved bioactivity compared to beauvericin. Nonetheless, the work
discussed previously represents the development of a platform capable of the facile
combinatorial production of a large number of compounds from the feeding of simple
2-hydroxy carboxylates, a class of simple molecules easily accessible from
simple synthetic chemistry.

50 - 55

14.4.2.5. Pikromycin As with many of the polyketides discussed thus far, the
native producer of pikromycin 56 is a bacterium, S. venezuelae . As shown in
Scheme 14.9, the structure of pikromycin is very similar to that of erythromycin
and, unsurprisingly, the biosynthetic systems are very similar as well. What makes
pikromycin biosynthesis unique is that termination takes place after five or six rounds
of elongation, resulting in the production of both 12- and 14-membered ring
macrolides. This natural promiscuity of the thioesterase domain led researchers to
examine the ability of the TE for macrocylization of unnatural seco -pikromycin
analogues. While the in vitro cyclization of a linear precursor was successfully
employed for the formal total synthesis of thewild-type aglycone, it was unsuccessful
in the production of any unnatural compounds [54].
The first successful application of precursor-directed biosynthesis to the
production of pikromycin analogues, as illustrated in Scheme 14.10, involved an
approach very similar to the diketide feeding experiments described for erythro-
mycin. Examinations of the substrate specificity found that the upstream modules of
the pikromycin pathway had very little tolerance for any variation on their wild-type
substrates, leading researchers to construct a mutant in which the loading didomain as
well as the first two modules were removed. Synthetic triketides with slight variation
from the structure of the native substrate of module 3 were then fed to S. venezuelae
expressing this mutant pathway, where they were successfully loaded onto module 3
and past through the remainder of the biosynthetic system. These variations
were tolerated at two separate positions, leading to the production of compounds
57 - 60 [55].