Mariya Ryabushkina
Engineering of Enzymatic Domains for Synthesis of new Polyketides
Polyketides have been known to display a wide array of functions such as anticancer, antifungal, and antiparasitic activity. The molecule of interest, laulimalide, has been isolated from a marine sponge inhabited by hundreds of symbiotic bacteria. Due to the inability to target the host producer of laulimalide, isolation and characterization of the native laulimalide pathway has proved to be impossible. Proposed biosynthetic pathways for the production of laulimalide incorporating the rearrangement of known modules from pimaricin, rifamycin, amphotericin, and deoxyerythronolide are a possiblity. Modular polyketide synthase genes can be rearranged via intramodular linker regions and intermodular linker regions to facilitate communication between heterologous modules. Restriction sites can be introduced to allow interchange of modules from various pathways. To test the efficiency of the proposed protein-protein interactions, three biosynethic pathways for portions of the core of laulimalide have been proposed. The pathway under study, C13 – C19, contains a linker region created from the module 2 and module 3 interface in the 6-deoxyerythronolide B pathway for the interpolypeptide interface of module 2 of pimaricin and module 12 of amphotericin. A NdeI restriction site has been added at the start of pimaricin module 1 and an EcoRI restriction site has been added after the stop codon of pimaricin module 2. Module 1 and 2 of pimaricin have compatible XbaI and SpeI overlaps. The interpolypeptide linker contains an SpeI site for compatibility with the XbaI site of module 2. Site directed mutagenesis of pUC19 has removed the inherent XbaI site for proper installment of pimaricin module 1, pimaricin module 2, and the interpolypeptide linker.
Aim 1: Install pimaricin module 1 and pimaricin module 2 inserts into pUC 19 vector. Express and check for activity. Continue with biosynthetic pathway for C13 – C19 of the laulimalide core.
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