Metabolic engineering of <i>Saccharomyces cerevisiae</i> for <i>de novo</i> production of dihydrochalcones with known antioxidant, antidiabetic, and sweet tasting properties

Dihydrochalcones are plant secondary metabolites comprising molecules of significant commercial interest as antioxidants, antidiabetics, or sweeteners. To date, their heterologous biosynthesis in microorganisms has been achieved only by precursor feeding or as minor by-products in strains engineered for flavonoid production. Here, the native ScTSC13 was overexpressed in Saccharomyces cerevisiae to increase its side activity in reducing p-coumaroyl-CoA to p-dihydrocoumaroyl-CoA. De novo production of phloretin, the first committed dihydrochalcone, was achieved by co-expression of additional relevant pathway enzymes. Naringenin, a major by-product of the initial pathway, was practically eliminated by using a chalcone synthase from barley with unexpected substrate specificity. By further extension of the pathway from phloretin with decorating enzymes with known specificities for dihydrochalcones, and by exploiting substrate flexibility of enzymes involved in flavonoid biosynthesis, de novo production of the antioxidant molecule nothofagin, the antidiabetic molecule phlorizin, the sweet molecule naringin dihydrochalcone, and 3-hydroxyphloretin was achieve

SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae

Additional file 1: Figure S1. The SWITCH recombination event triggering substitution of cas9 (human codon optimized) for dcas9 (yeast codon optimized). Figure S2. Confirmation of cas9 integration into the X-3 locus by diagnostic PCR. Figure S3. Control screening in strain S-0 of the three gRNAs tested for swapping cas9. Figure S4. Exploiting SWITCH for marker-free integration into a specific locus. Figure S5. The three assembler fragments used with SWITCH for marker-free integration of the complete naringenin pathway into locus XI-2. Figure S6. Naringenin producers created by SWITCH and assembler. Figure S7. Gene regulation by SWITCH. Figure S8. Implementing SWITCH for TSC13 down regulation. Table S1. The main primers used in this study. Table S2. Plasmid list