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

Abstract

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.

Engineered biosynthesis of plant polyketides by type III polyketide synthases in microorganisms

“…CRISPR/Cas is another powerful tool that can enable pathway construction in short time by assembling multiple genetic parts/ genes simultaneously. For example, Gong et al (2022) has developed a CRISPR/Cas mediated method customized for complicated plant pathway reconstruction in yeast, which has succeeded in reconstructing a 12-gene, de novo isoflavone pathway in one pot. Apel et al (2017) developed a cloningfree toolkit for integration by employing CRISPR/Cas9 that can be applied to PNP synthesis too.…”

De novo biosynthesis of berberine and halogenated benzylisoquinoline alkaloids in Saccharomyces cerevisiae

“…The high accumulation of canadine and relatively low berberine production indicates that the canadine to berberine conversion catalyzed by STOX is another major bottleneck in the berberine pathway. We first tried to overexpress the enzyme STOX from B. wilsoniae (BwSTOX) 46 using high-copy plasmids, including pAG424 and pAG425 47 , in BBR4R to improve the conversion. However, no berberine accumulation was detected from the strains harboring the STOX-expressing plasmids.…”

“…PCR products were verified by agarose gel electrophoresis and purified with a gel DNA recovery kit following the manufacturer's instructions. Plasmids for yeast expression (pAG series 47 ) and plant transient expression (pCambia2300 series) were constructed using Gateway LR Clonase II Enzyme mix.…”