The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and 6 extension modules which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the S. venezuelae mutant were 50% of that of an S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. 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. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products.
The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the Saccharopolyspora erythraea 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products. A series of radiolabeling studies of the loading module has shown that transfer from the AT to the ACP occurs much faster for propionate than for isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP is the kinetic product, while isobutyryl-ACP is the thermodynamic product. A slowed transfer from the loading domain ACP to first-extension module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored equilibrium to be realized and likely accounts for the observed shift in triketide lactone products.
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 Cterminus 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 ii 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 Cterminal (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 derive...
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