Functional Separation of Multimodular Type I PKS Polypeptides by Utilizing Matched Docking Domains From a Heterologous PKS System

摘要

Bacterial type I modular polyketide synthases (PKS) are large multifunctional enzyme systems responsible for the biosynthesis of complex polyketide natural products such as erythromycin, pikromycin, and borrelidin. Type I systems are comprised of a loading module which generally selects an appropriate acyl group starter unit, and multiple discrete extension modules, responsible for each single round of acyl group incorporation into the final polyketide core structure. These modules can exist naturally as either single discrete polypeptides, such as modules 5 and 6 from the pikromycin PKS (PikA3 and PikA4 respectively), or as multimodular polypeptides fused together by short intrapolypeptide linkers such as the loading module and the first and second extension modules of the erythromycin and pikromycin PKSs (DEBS1 and PikAI respectively). While short peptide linkers between modules on the same polypeptide facilitate the transfer of polyketide intermediates from one module to the next via their close proximity to one another, docking domains found at the C-terminus of one module and the N-terminus of the next subsequent module facilitate the needed protein-protein interactions for the passage of biosynthetic intermediates between modules on separate polypeptides. The ability to utilize docking domains in place of intrapolypeptide linkers was explored in the pikromycin and erythromycin PKSs by dissecting the tri-modular PikAI and DEBS1 polypeptides with matched docking domains. It has been shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin PKS are used in place of the intrapolypeptide linker. In both cases the yields of pikromycin produced by the S. venezuelae host mutant, which is a PikAI deletion strain were 50% of that of an S. venezuelae strain expressing the native trimodular PikAI. Additionally, expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a 10-fold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. The utilization of docking domains to separate linked modules was also demonstrated in the erythromycin PKS. Expression of the first protein involved in erythromycin biosynthesis (DEBS1) with the DEBS thioesterase fused to the C-terminal (DEBS1-TE) in S. venezuelae results in the production of triketide lactone products. Separation of DEBS1-TE resulted in 50% triketide lactone production, consistent with the observations in the pikromycin system. Published work has shown that the DEBS loading module has relaxed substrate specificity, and is capable of incorporating acetate, butyrate and isobutyrate in addition to the normally observed propionate starter unit, which typically predominates. However, in the current study when the DEBS loading module is separated from module 1 with matched docking domains, a dramatic shift in the starter unit, favoring the isobutyrate derived tri-ketide lactone is observed. This apparent shift in starter unit preference for a dissected PKS system has resulted in insights into the kinetics of acyl group loading, off loading, as well as the hydrolysis and transfer from the AT to ACP domains. In addition to the separation of multimodular PKS polypeptides with docking domains, it has also been shown that the individual catalytic domains of single discrete module, BorA5 from the borrelidin PKS can be expressed as stand alone proteins while retaining catalytic functionality in vitro. This work has provided a basis for future studies of this module, which has been proposed to function iteratively, catalyzing three rounds of chain elongation.