Type I Polyketide Synthases:Methodology, Biocatalysis, and Evaluation of Substrate Promiscuity.

[摘要] The gram-positive prokaryotes of the Streptomyces genus are prolific producers of secondary metabolites including a plethora of complex polyketide compounds.These natural products are constructed through decarboxylative Claisen condensations of simple malonic acids from primary metabolism by multidomain, modular enzymes called polyketide synthases (PKS) in a manner analogous to an industrial assembly line.A prominent example of one such pathway is the pikromycin (Pik) cluster from S. venezuelae ATCC 15439, which biosynthesizes a suite of 12- and 14-membered macrolide antibiotics.This pathway has been a workhorse in the Sherman lab for in vivo work, in vitro biochemistry, and more recently, biocatalysis and in depth structural analysis.This dissertation describes synthetic chemistry, in vitro biochemistry, and in vitro biocatalysis focused on the final two PKS modules from the Pik cluster, PikAIII and PikAIV.First, the native pentaketide from the Pik pathway was chemically synthesized and employed to optimize in vitro biochemistry/biocatalysis with PikAIII-TE and PikAIII/PikAIV, culminating in a biocatalytic platform for macrolide production in 13 linear steps.Next, the native hexaketide from the Pik pathway was synthesized from fermentation derived 10-deoxymethynolide and employed to optimize in vitro biochemistry of PikAIV and excised Pik thioesterase (TE) domain, revealing the ability to control the catalytic cycle of PikAIV and gain entry to 12- or 14-membered macrolactones with greater than 10:1 selectivity for either ring size.Finally, we simulated ;;combinatorial biosynthesis” in a controlled in vitro environment with PikAIII-TE and PikAIII/PikAIV to identify catalytic bottlenecks using unnatural pentaketide substrates that mimic engineering early in the pathway. Analyses of results generated to date indict the TE domain as the bottleneck in combinatorial biosynthesis, and a crucial target for protein engineering.

[发布机构] University of Michigan

[效力级别] Chemistry

[关键词] polyketide synthases;Chemistry;Science;Medicinal Chemistry