(449e) MULTI-Sculpt – Multiplex Integration Via Selective, CRISPR-Mediated, Ultra Long Pathway Transformation in Yeast for Natural Product Synthesis

Yeast has been a versatile model host for complex and valuable natural product biosynthesis via the reconstruction of heterologous biosynthetic pathways. Recent advances in natural product pathway elucidation have uncovered many large and complicated pathways that contain 10-30 genes catalyzing the biosynthesis of structurally complex, valuable natural products in plants and microorganisms. However, the ability to reconstruct ultra-long pathways efficiently in yeast does not match the increasing demand for valuable natural product biomanufacturing. Here, we developed a one-pot, multi-gene pathway integration method in yeast, named MULTI-SCULPT for MULTiplex Integration via Selective, CRISPR-mediated, Ultra Long Pathway Transformation. Leveraging multi-locus genomic disruption via CRISPR/Cas9, newly developed native and synthetic genetic parts, and fine- tuned gene integration and characterization methods, we managed to integrate 21 DNA inserts that contain a 12-gene plant isoflavone biosynthetic pathway into yeast with a 100% success rate in 12 days, with all tested cells yielding the correct, engineered phenotype. This method enables fast and efficient ultra-long biosynthetic pathway integration and can allow for the fast iterative integration of even longer pathways in the future. Ultimately, this method will accelerate the combinatorial optimization of elucidated natural product pathways and accelerate putative natural product pathway characterization heterologously.