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Numerous important pharmaceuticals and nutraceuticals originate from plant specialized metabolites, most of which are synthesized via complex biosynthetic pathways. The elucidation of these pathways is critical for the applicable uses of these compounds. Although the rapid progress of the omics technology has revolutionized the identification of candidate genes involved in these pathways, the functional characterization of these genes remains a major bottleneck. Baker’s yeast (Saccharomyces cerevisiae) has been used as a microbial platform for characterizing newly discovered metabolic genes in plant specialized metabolism. Using yeast for the investigation of numerous plant enzymes is a streamlined process because of yeast’s efficient transformation, limited endogenous specialized metabolism, partially sharing its primary metabolism with plants, and its capability of post-translational modification. Despite these advantages, reconstructing complex plant biosynthetic pathways in yeast can be time intensive. Since its discovery, CRISPR/Cas9 has greatly stimulated metabolic engineering in yeast. Yeast is a popular system for genome editing due to its efficient homology-directed repair mechanism, which allows precise integration of heterologous genes into its genome. One practical use of CRISPR/Cas9 in yeast is multiplex genome editing aimed at reconstructing complex metabolic pathways. This system has the capability of integrating multiple genes of interest in a single transformation, simplifying the reconstruction of complex pathways. As plant specialized metabolites usually have complex multigene biosynthetic pathways, the multiplex CRISPR/Cas9 system in yeast is suited well for functional genomics research in plant specialized metabolism. Here, we review the most advanced methods to achieve efficient multiplex CRISPR/Cas9 editing in yeast. We will also discuss how this powerful tool has been applied to benefit the study of plant specialized metabolism.

The hallucinogenic plant, Salvia divinorum, synthesizes neoclerodane diterpenes, such as salvinorins, salvidivins, and salvinicins, which are agonistic or antagonistic to μor κ-opioid receptors. From S. divinorum trichomes, crotonolide G synthase (SdCS; CYP76AH39) was identified. It catalyzes the conversion of kolavenol to a dihydrofuran neoclerodane, crotonolide G. 18 O 2feeding studies confirmed that SdCS incorporates an aerobic oxygen into crotonolide G, rather than forming a cation at C16 that is trapped by the alcohol at C15. Structural modeling of SdCS accompanied by site-directed mutagenesis established the importance of V367 and F479 residues in substrate-binding. The dihydrofuran neoclerodane can serve as a unique lead structure for drug development.

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