Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris

Fabrice Neiers,Loïc Briand,Christine Belloir,Nicolas Poirier,Michael Krohn,Christian Naumer

doi:10.3390/life11010046

Abstract

Brazzein is a small sweet-tasting protein found in the red berries of a West African evergreen shrub, Pentadiplandra brazzeana Baillon. Brazzein is highly soluble and stable over a large pH range and at high temperatures, which are characteristics that suggest its use as a natural sweetener. However, Pentadiplandra brazzeana culture is difficult at a large scale, limiting the natural source of brazzein. Heterologous expression of brazzein has been established in numerous systems, including bacteria, yeast, and transgenic plants. Brazzein requires four disulfide bonds to be active in eliciting an intense sweet taste, and the yeast Pichia pastoris appears to be one of the best options for obtaining functional brazzein in high quantities. Employing yeast secretion in the culture medium allows us to obtain fully active brazzein and facilitate purification later. To increase yeast secretion, we compared seven different signal peptides to successfully achieve brazzein secretion using the yeast P. pastoris. The brazzein proteins corresponding to these signal peptides elicited activation of the sweet taste receptor functionally expressed in a cellular assay. Among these tested signal peptides, three resulted in the secretion of brazzein at high levels.

Highlights

Brazzein is a 6.5-kDa sweet-tasting protein containing four disulfide bonds [1,2].Isolated from the pulp of the berries of a West African plant Pentadiplandra brazzeana Baillon, brazzein possesses a sweet taste close to that of sucrose with a high sweetness potency
P. pastorisBrazzein was previously To secreted into the buffered minimal medium of pastoris using the 85-amino-acid compare the production level and signal peptide processing of brazzein in the yeast secretion signal sequence from the Saccharomyces cerevisiae α-mating factor
Brazzein was natural signal peptide of brazzein is not known, the choice to select the best signal peptide secreted into the buffered minimal medium of P. pastoris using the 85-amino-acid secretion is mainly based on experimental findings

Summary

Introduction

Brazzein is a 6.5-kDa sweet-tasting protein containing four disulfide bonds [1,2]. Isolated from the pulp of the berries of a West African plant Pentadiplandra brazzeana Baillon, brazzein possesses a sweet taste close to that of sucrose with a high sweetness potency (from 500- to 2000-fold sweeter than sucrose solution on a weight basis). Brazzein presents exceptional thermostability and high solubility over a wide range of pH values, which are essential for food applications [2]. The high water stability, the extreme temperature and pH stability are highly promising for food application [1]. Among the sweet-tasting protein, brazzein appear as one of the most promising. Seven sweet-tasting proteins have been identified to date to brazzein: lysozyme, mabinlin, monellin, neoculin, pentadin and thaumatin

Methods

Results

Conclusion