Aided‐efflux and high production of β‐amyrin realized by β‐cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae. Issue 4 (17th January 2023)

• Record Type: Journal Article
• Authors: Zhu, Ying; Zhou, Chen; Liu, Xia; Li, Xinying; Shi, Caifang; Zhang, Yapeng; Wang, Ying; Li, Chun
• Is Part Of: Biotechnology and bioengineering. Volume 120:Issue 4(2023)
• Page Start: 1147
• Page End: 1158
• Publication Date: 2023-01-17
• Subjects: β‐amyrin -- β‐cyclodextrin -- aided‐efflux -- in situ synthesis -- Saccharomyces cerevisiae; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.28327
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: As a plant‐derived pentacyclic triterpenoid, β‐amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae . However, β‐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 β‐amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β‐cyclodextrin (β‐CD) has been previously employed to aid the efflux of β‐amyrin out of the cells. Nevertheless, the supplemented β‐CD in the medium is not consistent with β‐amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided‐efflux system based on in situ synthesis of β‐CD was developed in this study to enhance the biosynthesis of β‐amyrin and its efflux. The in situ synthesis of β‐CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β‐CD could capture 16% of the intracellular β‐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 β‐amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β‐amyrin. Overall, this work provides novel strategies for the aided‐efflux of natural products with high hydrophobicity in engineered S. cerevisiae . Abstract : β‐Amyrin was accumulated intracellularly when heterogeneously synthesized in Saccharomyces. cerevisiae, which limits the industrial application of its microbial production. In this study, we provided a novel strategy to realize its efflux via β‐cyclodextrin in situ synthesized on cell surface. The β‐cyclodextrin‐aided efflux of β‐amyrin was further improved by two‐phase fermentation as well as the regulation of lipid synthesis. As a result, the production of β‐amyrin was increased to 73 mg/L in shake flask, with 31 mg/L β‐amyrin was transported to the outside of the cell.