Engineering Saccharomyces cerevisiae for the production of natural osmolyte glucosyl glycerol from sucrose and glycerol through Ccw12-based surface display of sucrose phosphorylase
Abstract Background Yeast Saccharomyces cerevisiae is widely recognised as a versatile chassis for constructing microbial cell factories. However, producing chemicals from toxic, highly concentrated, or cell-impermeable substrates, or chemicals dependent on enzymatic reactions incompatible with the...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2024-11-01
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| Series: | Journal of Biological Engineering |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13036-024-00468-7 |
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| Summary: | Abstract Background Yeast Saccharomyces cerevisiae is widely recognised as a versatile chassis for constructing microbial cell factories. However, producing chemicals from toxic, highly concentrated, or cell-impermeable substrates, or chemicals dependent on enzymatic reactions incompatible with the yeast’s intracellular environment, remains challenging. One such chemical is 2-O-(α-D-glucopyranosyl)-sn-glycerol (glucosyl glycerol, αGG), a natural osmolyte used in the cosmetics and healthcare industries. This compound can be synthesised in a one-enzyme reaction from sucrose and glycerol by Leuconostoc mesenteroides sucrose phosphorylase (SucP), an enzyme which, in a low-water, glycerol-rich, phosphate-free environment, transfers the glucosyl moiety from sucrose to glycerol. Results In this study, we engineered a yeast microbial cell factory for αGG production. For this purpose, we first focused on the abundant yeast GPI-anchored cell wall protein Ccw12 and used our insights to develop a miniature Ccw12-tag, which adds only 1.1 kDa to the enzyme of interest while enabling its covalent attachment to the cell wall. Next, we Ccw12-tagged SucP and expressed it in an invertase-negative strain of yeast S. cerevisiae from the PHO5 promoter, i.e., promoter strongly induced under phosphate-free conditions. Such SucP isoform, covalently C-terminally anchored to the outer cell surface, produced extracellularly 37.3 g l− 1 (146 mM) of αGG in five days, while the underlying chassis metabolised reaction by-products, thereby simplifying downstream processing. Conclusions The here-described S. cerevisiae strain, displaying C-terminally anchored sucrose phosphorylase on its cell surface, is the first eukaryotic microbial cell factory capable of a one-step αGG production from the readily available substrates sucrose and glycerol. |
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| ISSN: | 1754-1611 |