MULTI-SCULPT: Multiplex Integration via Selective, CRISPR-Mediated, Ultralong Pathway Transformation in Yeast for Plant Natural Product Synthesis

清脆的 计算生物学 合成生物学 生物 酵母 基因 天然产物 多路复用 转化(遗传学) 基因组编辑 代谢工程 Cas9 遗传学 生物化学
作者
Franklin L. Gong,Jia-Ning Han,Sijin Li
出处
期刊:ACS Synthetic Biology [American Chemical Society]
卷期号:11 (7): 2484-2495 被引量:6
标识
DOI:10.1021/acssynbio.2c00135
摘要

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.
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