Synthetic Operon Assembly and CRISPR Genome Editing

The assembly of synthetic operon in YEp352 was performed according to the BioBrick principles^{23 (link)} described by Zelcbuch et al.^{24 (link)} To further simplify the cloning process, a seamless, recombination-based cloning strategy was applied for the synthetic operon construction in YEplac181 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The knockout of ROX1 and knockdown of ERG9 were achieved via CRISPR/Cas9 technologies.^{25 (link)} For the CRISPR genome editing, yeast cells were pre-transformed with p414-TEF1p-Cas9-CYC1t plasmid (Addgene). All the PCR products were analyzed on 1% agarose gels and positive colonies were grown overnight for plasmid extraction using the Tianprep Rapid Mini Plasmid Kit (Tiangen, Beijing China) according to the manufacture's instructions. All clones were verified through the expression cassette sequencing (Sangon, China). The constructed plasmids containing the whole expression operon were chemically transformed into the yeast by S. cerevisiae EasyComp™ Transformation Kit (Invitrogen, CA, United States) and aliquots were plated on the corresponding auxotrophic minimal media. Table S4† lists the primers used to construct these plasmids and strains.