title: Engineering the Synthetic Capabilities of a Modular Type I Polyketide Synthase
title: Entwicklung der Synthesefähigkeiten einer modularen Typ I Polyketidsynthase
titleType: AlternativeTitle
Creators: name: Gerlinger, Patrick Dennis
Publisher: name: Philipps-Universität Marburg
Year: 2022
Subjects: synthetic biology
PKS
Life sciences
Biowissenschaften, Biologie
polyketide synthases
Polyketidsynthase
synthetische Biologie

description: Polyketide are a vast class of natural products, with biological activities such as antibacterial, antiviral or antitumor activity. The elucidation of the biosynthetic machinery behind them, Polyketide Synthases (PKS), revealed polyketides to be derived from simple acyl- and malonyl-Coenzyme A thioester building blocks in a highly predictable fashion regarding substrate specificity and stereoconfiguration. The prospect of harnessing and modifying these enzymes in order to create designer polyketides has thus attracted the attention of research for multiple decades. The availability of a broad panel of substrates if a fundamental prerequisite to study and engineer the substrates specificity of PKSs. To meet these requirements, multiple chemo-enzymatic strategies for their in vitro synthesis have been developed. Each of these routes has, however, inherent drawbacks and yields either the incorrect stereoisomer or requires chemical precursor synthesis. This work describes a nature-inspired, fully enzymatic one-pot synthesis route to generate polyketide extender units from carboxylic acids via enzymatic ligation, oxidation and carboxylation. Avoiding chemical precursor synthesis, this method showed to be highly efficient for aliphatic substrates, allowing for a high-yield preparative scale synthesis of their corresponding extender units. Successful engineering efforts to facilitate the incorporation of non-native extender units into PKS assembly lines have focused largely on the specificity-conferring acyltransferase (AT) domain, and were mostly carried out in terminal or standalone modules. By expanding the exploration space for site-directed engineering to other domains than the AT (i.e. ketoreductase and ketosynthase), this work describes their effects on substrate incorporation in a fully reconstituted PKS assembly line in vitro. Mutagenesis of those domains can greatly influence the processing of non-native intermediates through enhanced promiscuity. Additionally, a single amino acid substitution in the ketosynthase domain was sufficient to completely invert substrate specificity by creating a concomitant shunt mechanism. These findings further led to the development of an alternative route to achieve specificity for extender unit supply through orthogonally added acyl-carrier protein bound thioesters. Despite the multitude of PKS engineering approaches in various contexts, only few overlaps to broader research fields of synthetic biology, such as the development of artificial pathways, exist. In order to bridge this gap, this work shows the successful combination of a polyketide synthase with an artificial CO2-fixation cycle. Intermediates of this cycle would serve as substrates for polyketide production, and drainage thus stall the continuous CO2-fixation. This was avoided by implementing anaplerotic feedback pathways into the metabolic network, catalyzing the conversion of the cycle’s output molecule back into intermediates. The resulting metabolic in vitro network, consisting of more than 20 enzymes and catalyzing up to 50 reactions, was able to produce polyketide yields from starting substrate and CO2 at comparable yields to the isolated PKS supplied with an excess of substrates.