Assessing and harnessing updated polyketide synthase modules through combinatorial engineering

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase. Every combinatorial possibility of modules 2-6 inserted between the first and last modules of the native synthase was constructed and assayed. Anticipated products were observed from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. Ketosynthase gatekeeping and module-skipping were determined to be the principal impediments to obtaining functional synthases. The platform was also used to create functional hybrid synthases through the incorporation of modules from the Erythromycin, Spinosyn, and Rapamycin assembly lines. The relaxed gatekeeping observed from a ketosynthase in the Rapamycin synthase is especially encouraging in the quest to produce designer polyketides.