4.6 Article

Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast

Journal

BIOTECHNOLOGY AND BIOENGINEERING
Volume 119, Issue 5, Pages 1314-1326

Publisher

WILEY
DOI: 10.1002/bit.28040

Keywords

ajmalicine; alkaloids; CRISPR; Cas9; multiplex genome integration; plant natural products; sanguinarine

Funding

  1. National Key Research and Development Program of China [2018YFA0901800, 2021YFC2103200]
  2. Natural Science Foundation of China [21808199]
  3. China Postdoctoral Science Foundation [2020M681903]
  4. Natural Science Foundation of Zhejiang Province [LR20B060003]

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Yeast cell factories have been widely used for the production of plant-derived natural products. However, the stability of genes involved in plant natural product biosynthesis remains a challenge. In this study, a genomic approach was used to identify stable integration sites for the successful construction of multigene biosynthetic pathways in yeast. Additionally, stable yeast cell factories were established, enabling the production of the highest titer of monoterpene indole alkaloids (MIAs) ever reported, with potential applications in the biotechnology production of other high-value MIAs.
Yeast cell factories have been increasingly employed for producing plant-derived natural products. Unfortunately, the stability of plant natural product biosynthetic pathway genes, particularly when driven by the same sets of promoters and terminators, remains one of the biggest concerns for synthetic biology. Here we profile genomic loci flanked by essential genes as stable integration sites in a genome-wide manner, for stable maintenance of multigene biosynthetic pathways in yeast. We demonstrate the application of our yeast integration platform in the construction of sanguinarine (24 expression cassettes) and ajmalicine (29 expression cassettes) de novo biosynthetic pathways for the first time. Moreover, we establish stable yeast cell factories that can produce 119.2 mg L-1 heteroyohimbine alkaloids (containing 61.4 mg L-1 ajmalicine) in shake flasks, representing the highest titer of monoterpene indole alkaloids (MIAs) ever reported and promising the complete biosynthesis of other high-value MIAs (such as vinblastine) for biotechnological applications.

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