| Popis: |
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 plant pathways that contain 10-30 genes for the biosynthesis of structurally complex, valuable natural products. However, the ability to reconstruct ultralong pathways efficiently in yeast does not match the increasing demand for valuable plant natural product biomanufacturing. Here, we developed a one-pot, multigene pathway integration method in yeast, named MULTI-SCULPT for multiplex integration via selective, CRISPR-mediated, ultralong pathway transformation. Leveraging multilocus 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 90-100% success rate in 12 days. This method enables fast and efficient ultralong biosynthetic pathway integration and can allow for the fast iterative integration of even longer pathways in the future. Ultimately, this method will accelerate combinatorial optimization of elucidated plant natural product pathways and accelerate putative pathway characterization heterologously. |
