Molecular study on the role of vacuolar transporters in glycyrrhetinic acid production in engineered Saccharomyces cerevisiae

Glycyrrhetinic acid (GA) is one of the major bioactive components of the leguminous plant, Glycyrrhiza spp. (Chinese licorice). Owing to GA's complicated chemical structure, its production by chemical synthesis is challenging and requires other efficient strategies such as microbial synthesis. Earlier investigations employed numerous approaches to improve GA yield by refining the synthetic pathway and improving the metabolic flux. Nevertheless, the metabolic role of transporters in GA biosynthesis in microbial cell factories has not been studied so far. In this study, we investigated the role of yeast ATP binding cassette (ABC) vacuolar transporters in GA production. Molecular docking of GA and its precursors, β-Amyrin and 11-oxo-β-amyrin, was performed with five vacuolar ABC transporters (Bpt1p, Vmr1p, Ybt1p, Ycf1p and Nft1p). Based on docking scores, two top scoring transporters were selected (Bpt1p and Vmr1p) to investigate transporters' functions on GA production via overexpression and knockout experiments in one GA-producing yeast strain (GA166). Results revealed that GA and its precursors exhibited the highest predicted binding affinity towards BPT1 (ΔG = −10.9, −10.6, −10.9 kcal/mol for GA, β-amyrin and 11-oxo-β-amyrin, respectively). Experimental results showed that the overexpression of BPT1 and VMR1 restored the intracellular as well as extracellular GA production level under limited nutritional conditions, whereas knockout of BPT1 resulted in a total loss of GA production. These results suggest that the activity of BPT1 is required for GA production in engineered Saccharomyces cerevisiae.