Biochemical Characterization of the Phoslactomycin modular Polyketide Synthase
Polyketides are a class of natural products with a large structural diversity. They find use for example in infection-, cancer- and respiratory disease treatments. New discoveries, chemical modification and the engineering of polyketide biosynthetic pathways may lead to the identification of nove...
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|Polyketides are a class of natural products with a large structural diversity. They find use
for example in infection-, cancer- and respiratory disease treatments. New discoveries,
chemical modification and the engineering of polyketide biosynthetic pathways may lead
to the identification of novel products with altered, potentially improved properties.
The structural diversity of polyketides originates from the assembly line-like joining
of simple building blocks by polyketide synthases (PKS). The introduction of structural
features is guided by the choice of starter units and the incorporation of various extender
units, such as malonyl- or alkyl-malonyl-CoAs. Enoyl-CoA carboxylases/reductases
(ECRs) are the key-enzymes providing alkyl-malonyl-CoAs. ECR catalyzed carboxylation
of α,β-unsaturated enoyl-CoA thioesters, yields in the production of e.g. (2S)-ethylmalonyl-CoA. This work presents a route for the preparative scale chemo-enzymatic
synthesis of a versatile set of extender units. Combining ECR activity with the concept of
biocatalytic proof-reading for recycling of unwanted byproducts resulted in the efficient
production of various polyketide extender units.
This set of extender units enabled tackling the fundamental question, of how
site-specific incorporation of different extender units in PKS is conveyed. For that,
phoslactomycin PKS (Pn PKS) derived tetra-, penta- and hexaketide were produced in
vitro. Furthermore, challenging the Pn PKS assembly line, that naturally incorporates
malonyl- and ethylmalonyl-CoA, with seven different extender units, revealed a highly
promiscuous module. With detailed kinetic analysis of excised domains, it could be shown
that the transacylation reaction is the driving force determining the incorporation of
Furthermore, in this work, the Pn PKS associated type II thioesterase PnG could
be shown to possess an editing function and thereby increase the yield of native and
non-native phoslactomycin derived polyketides in vitro. This work underlines their
potential for improving the production yields of polyketides.
In summary, a detailed study of the phoslactomycin PKS, starting from the
production of extender units, over the in vitro reconstitution of Pn PKS, to the characterization of single domains and an accessory thioesterase, is presented. This dissertation
provides new insights in the molecular functions of this astonishing class of enzymes.