RESUMO
As a plant-derived pentacyclic triterpenoid, ß-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, ß-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of ß-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic ß-cyclodextrin (ß-CD) has been previously employed to aid the efflux of ß-amyrin out of the cells. Nevertheless, the supplemented ß-CD in the medium is not consistent with ß-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of ß-CD was developed in this study to enhance the biosynthesis of ß-amyrin and its efflux. The in situ synthesis of ß-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized ß-CD could capture 16% of the intracellular ß-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of ß-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular ß-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.
