MULTI-SCULPT: Multiplex Integration via Selective, CRISPR-Mediated, Ultralong Pathway Transformation in Yeast for Plant Natural Product Synthesis.
MULTI-SCULPT: Multiplex Integration via Selective, CRISPR-Mediated, Ultralong Pathway Transformation in Yeast for Plant Natural Product Synthesis.
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DOI：
10.1021/acssynbio.2c00135
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发表时间：
2022-07-15
影响因子：
4.7
通讯作者：
Li, Sijin
中科院分区：
文献类型：
--
作者：
Gong, Franklin Leyang;Han, Jianing;Li, Sijin
酵母已成为通过异源生物合成途径重建复杂且有价值的天然产物生物合成的多功能模型宿主。天然产物途径阐明的最新进展揭示了许多大型而复杂的植物途径，其中包含 10-30 个基因，用于生物合成结构复杂、有价值的天然产物。然而，在酵母中有效重建超长途径的能力与对有价值的植物天然产物生物制造日益增长的需求不匹配。在这里，我们开发了一种酵母中的一锅多基因途径整合方法，名为 MULTI-SCULPT，通过选择性、CRISPR 介导的超长途径转化进行多重整合。利用通过 CRISPR/Cas9 进行的多位点基因组破坏、新开发的天然和合成遗传部分以及微调的基因整合和表征方法，我们成功地将包含 12 基因植物异黄酮生物合成途径的 21 个 DNA 插入片段整合到酵母中，并在 12 天内获得了 90-100% 的成功率。该方法能够实现快速高效的超长生物合成途径整合，并可以在未来实现更长途径的快速迭代整合。最终，该方法将加速已阐明的植物天然产物途径的组合优化，并加速异源假定途径的表征。
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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DOI：
10.1073/pnas.052545899
发表时间：
2002-03-19
影响因子：
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通讯作者：
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