posted on 2019-01-24, 19:39authored byEdward Kalkreuter, Jared M. CroweTipton, Andrew N. Lowell, David H. Sherman, Gavin J. Williams
There
is significant interest in diversifying the structures of
polyketides to create new analogues of these bioactive molecules.
This has traditionally been done by focusing on engineering the acyltransferase
(AT) domains of polyketide synthases (PKSs) responsible for the incorporation
of malonyl-CoA extender units. Non-natural extender units have been
utilized by engineered PKSs previously; however, most of the work
to date has been accomplished with ATs that are either naturally promiscuous
and/or located in terminal modules lacking downstream bottlenecks.
These limitations have prevented the engineering of ATs with low native
promiscuity and the study of any potential gatekeeping effects by
domains downstream of an engineered AT. In an effort to address this
gap in PKS engineering knowledge, the substrate preferences of the
final two modules of the pikromycin PKS were compared for several
non-natural extender units and through active site mutagenesis. This
led to engineering of the methylmalonyl-CoA specificity of both modules
and inversion of their selectivity to prefer consecutive non-natural
derivatives. Analysis of the product distributions of these bimodular
reactions revealed unexpected metabolites resulting from gatekeeping
by the downstream ketoreductase and ketosynthase domains. Despite
these new bottlenecks, AT engineering provided the first full-length
polyketide products incorporating two non-natural extender units.
Together, this combination of tandem AT engineering and the identification
of previously poorly characterized bottlenecks provides a platform
for future advancements in the field.