Controlled Access. Restriction lift date: 2028-05-31
The development of a platform for novel sweet taste receptor ligand discovery & an optimisation method for heterologous protein secretion in Saccharomyces cerevisiae
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Date
2024
Authors
O'Riordan, Nicola
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
Excess sugar and artificial sweetener consumption is associated with many modern-day diseases which impose a major burden on global healthcare systems (Malik et al., 2019). Sweet-tasting proteins represent a promising alternative and could help control the incidence of diabetes, obesity, hyperlipemia, and many other metabolic-related diseases (Zhao et al., 2021). Unfortunately, the cultivation of sweet proteins from their natural sources is technically challenging and commercially unfeasible (Bilal et al., 2022). Sweet taste is initiated by the interaction of an appropriate ligand with the sweet taste receptor which functions as an obligate heterodimer, composed of two subunits; TAS1R2 and TAS1R3 (taste receptor type 1 member 2 and 3) (Ahmad and Dalziel, 2020). Widespread issues with heterologous protein expression of the sweet taste receptor and its ligand binding domains (LBDs) have been reported (Smith et al., 2021, Nango et al., 2016). Therefore, expression of the sweet taste receptors LBDs across insect, bacterial, and mammalian cells was compared, and protein expression was optimised to facilitate screening for a novel sweet protein. A stable cell line expressing the full-length versions of the receptors was characterised to assess the interaction of the anticipated novel sweet protein candidates with the sweet taste receptor. An affibody-A phage display library and a nanobody yeast display library were screened and binding to TAS1R2 and TAS1R3 was assessed. Unfortunately, no suitable ligand was identified therefore, a novel method to optimise protein secretion in Saccharomyces cerevisiae (S. cerevisiae) was established to facilitate simplified downstream processing of commercially significant proteins. The expansion involved modification of the yeast modular cloning (MoClo) toolkit (Lee et al., 2015) to include a panel of secretion-promoting sequences and translational fusion partners (TFPs) selected from the literature. The utility of this expansion was validated using 5 unique proteins of commercial interest including the naturally occurring sweet proteins, brazzein and monellin. This work represents the first reliable documentation of brazzein secretion in S. cerevisiae close to its expected molecular weight but its utility extends to the production of many heterologous proteins of commercial interest. It is anticipated that this will help to overcome the barriers associated with sweet protein production and presents a viable commercial alternative to sugar and artificial sweeteners.
Description
Controlled Access
Keywords
Sweet taste receptor , Protein secretion , Saccharomyces cerevisiae , TAS1R2 , TAS1R3 , Secretion signal peptide , Translational fusion partner
Citation
O'Riordan, N. 2024. The development of a platform for novel sweet taste receptor ligand discovery & an optimisation method for heterologous protein secretion in Saccharomyces cerevisiae. PhD Thesis, University College Cork.