Covering: mid 1990s to 2018 Over the last two decades, diverse approaches have been explored to generate new polyketides by engineering polyketide synthases (PKSs). Although it has been proven possible to produce new compounds by designed PKSs, engineering strategies failed to make polyketides available via widely applicable rules and protocols. Still, organic synthetic routes have to be employed whenever new polyketides are needed for applications in medicine, agriculture, and industry. In light of the rising demand for commodity products from feedstock and for fast and cheap access to pharmaceutical compounds, the need for harnessing PKSs to produce such molecules is more urgent than ever before. In this review, we focus on a multitude of approaches to engineer modular PKSs by swapping and replacing PKS modules and domains, which we analyze in the light of recent structural and biochemical data. We conclude with an outlook on possible strategies on how to increase success rates of PKS engineering in future.

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzymesubstrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.The assembly line architecture of multimodular polyketide synthases (PKSs) 5 represents a promising catalytic framework for combinatorial biosynthesis (1). A particularly well studied example of this family of multienzyme systems is the 6-deoxyerythronolide B synthase (DEBS), which produces 6-deoxyerythronolide B (Fig. 1), the parent aglycone of the macrolide antibiotic erythromycin (2). DEBS is comprised of three distinct homodimeric proteins: DEBS1, DEBS2, and DEBS3, each containing two PKS modules, with each module being responsible for a distinct round of polyketide elongation and modification. DEBS uses propionyl-CoA to prime the loading didomain (LDD) of module 1 and six methylmalonyl-CoA-derived extender units in catalysis of the six cycles of polyketide chain elongation, followed by terminal release of the mature polyketide chain by thioesterase (TE)-catalyzed macrolactone formation.Since the original genetic characterization of DEBS over two decades ago (3, 4), extensive in vivo and in vitro analysis has revealed that specific protein-protein interactions play a critical role in the proper vectorial channeling of biosynthetic intermediates from one PKS module to the next (5). A particularly effective mini-assembly line for mechanistic analysis has been a simple bimodular derivative of DEBS (Fig. 2) harboring modules 1 and 2 with a fused TE domain. This bimodular PKS has served as a prototype for many analogous constructs.The potential for engineering chimeric PKSs by recombining modules from paralogous polyketide biosynthetic pathways has been explored for nearly two decades. Early studies revealed the importance of ACP-KS interactions (6, 7), as well as the utility of intermodul...

Engineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain-domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a noncovalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced noncovalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.

Modular polyketide synthases (PKSs) produce complex, bioactive secondary metabolites in assembly line-like multistep reactions. Longstanding efforts to produce novel, biologically active compounds by recombining intact modules to new modular PKSs have mostly resulted in poorly active chimeras and decreased product yields. Recent findings demonstrate that the low efficiencies of modular chimeric PKSs also result from rate limitations in the transfer of the growing polyketide chain across the non-cognate module:module interface and further processing of the non-native polyketide substrate by the ketosynthase (KS) domain. In this study, we aim at disclosing and understanding the low efficiency of chimeric modular PKSs and at establishing guidelines for modular PKSs engineering. To do so, we work with a bimodular PKS testbed and systematically vary substrate specificity, substrate identity, and domain:domain interfaces of the KS involved reactions. We observe that KS domains employed in our chimeric bimodular PKSs are bottlenecks with regards to both substrate specificity as well as interaction with the ACP. Overall, our systematic study can explain in quantitative terms why early oversimplified engineering strategies based on the plain shuffling of modules mostly failed and why more recent approaches show improved success rates. We moreover identify two mutations of the KS domain that significantly increased turnover rates in chimeric systems and interpret this finding in mechanistic detail..