Understanding substrate translocation in polyketide synthase (PKS) assembly lines

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The term "assembly line" usually evokes associations with car production facilities: the vehicle passes through several stations at which work steps are carried out. Nature uses a similar synthetic principle in the synthesis of polyketide natural products. Like car assembly lines, modular polyketide synthases (PKSs) consist of stations, named modules, at which an initially simple precursor molecule, often activated carboxylic acid, is processed. While the precursor molecule is simple, the products are complex bioactive compounds with wide applications in medical therapy. As clear as the mechanism of macroscopic car production lines is - cars are transported on belts or tracks, and robots or people carry out, e.g., screwing, welding or gluing work - the molecular basis of PKS production lines remains until today largely elusive and controversially discussed. How is the growing polyketide channeled through the assembly line? How does a module recognize that the polyketide has been processed and should now be forwarded to the next module? And linked to this, how does a module prevent repeated processing of a polyketide? There are only vague attempts to explain these questions. The project presented in this grant proposal seeks to provide clear answers to the molecular basis of the vectorial synthesis of PKS assembly lines. It comprises basically two axes: (i) The biochemical part of the project aims at the functional understanding of the vectorial synthesis of PKS production lines, following an “understanding by remodeling” approach. In order to understand which units are responsible for vectorial synthesis, PKS assembly lines will be remodeled by explicitly non-vectorial units. Specifically, we transplant units of non-vectorial homologous proteins into the PKS assembly line, and monitor whether the ability for vectorial synthesis is retained. For this approach, we use the non-vectorial (but prototypically iterative) fatty acid synthase (FAS) and two well-characterized iterative PKSs as donor proteins. (ii) In the structural biology part of the project, we aim at the structural characterization of vectorial synthesis. On the basis of carefully selected subunits of PKS production lines and using cryogenic electron microscopy (cryo-EM), we particularly seek to structurally describe the passage of the growing polyketide from one module to the other, the so-called translocation step. Here, intelligent protein design aims to lock key conformations of vectorial synthesis. State-of-the-art single-particle cryo-EM can handle heterogeneity of complex protein samples, and is able to extract structural information from ensembles of conformations at near-atomic resolution. The structural biology part is designed as collaborative project with Prof. Timm Maier (Biozentrum Basel). His laboratory has outstanding expertise in the structural analysis of PKSs and related multidomain proteins.