life Article Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris Fabrice Neiers 1, Christine Belloir 1, Nicolas Poirier 1, Christian Naumer 2, Michael Krohn 2 and Loïc Briand 1,* 1 Centre des Sciences du Goût et de l’Alimentation, CNRS, INRAE, Université Bourgogne Franche-Comté, F-21000 Dijon, France; [email protected] (F.N.); [email protected] (C.B.); [email protected] (N.P.) 2 BRAIN AG, Darmstaedter Str. 34-36, 64673 Zwingenberg, Germany; [email protected] (C.N.); [email protected] (M.K.) * Correspondence: [email protected]; Tel.: +33-380-681615 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 Citation: Neiers, F.; Belloir, C.; in the secretion of brazzein at high levels. Poirier, N.; Naumer, C.; Krohn, M.; Briand, L. Comparison of Different Signal Peptides for the Efficient Keywords: brazzein; signal peptide; sweet taste receptor; sweet-tasting protein; Pichia pastoris Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris. Life 2021, 11, 46. https://doi.org/ 10.3390/life11010046 1. Introduction Brazzein is a 6.5-kDa sweet-tasting protein containing four disulfide bonds [1,2]. Received: 9 December 2020 Isolated from the pulp of the berries of a West African plant Pentadiplandra brazzeana Accepted: 11 January 2021 Baillon, brazzein possesses a sweet taste close to that of sucrose with a high sweetness Published: 13 January 2021 potency (from 500- to 2000-fold sweeter than sucrose solution on a weight basis). In addition, brazzein presents exceptional thermostability and high solubility over a wide Publisher’s Note: MDPI stays neu- range of pH values, which are essential for food applications [2]. Its long history of human tral with regard to jurisdictional clai- consumption combined with its absence of bitterness makes brazzein a good alternative ms in published maps and institutio- to natural sweeteners [1]. Moreover, the high water stability, the extreme temperature nal affiliations. 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 additionally to brazzein: lysozyme, mabinlin, monellin, neoculin, Copyright: © 2021 by the authors. Li- pentadin and thaumatin. censee MDPI, Basel, Switzerland. Natural brazzein has been found to occur in the fruit in two isoforms. The major This article is an open access article isoform (54 amino acids, ~80%), called pyrE-bra, contains an N-terminal pyroglutamic distributed under the terms and con- acid, whereas the minor isoform (53 amino acids, ~20%), called des-pyrE-bra, lacks the N- ditions of the Creative Commons At- terminal pyroglutamate residue [2]. Sensory analyses have revealed that the minor isoform tribution (CC BY) license (https:// is twice as sweet as the major form [3,4]. The three-dimensional structure of brazzein creativecommons.org/licenses/by/ has been solved by NMR spectroscopy and X-ray crystallography [5,6]. It is composed of 4.0/). Life 2021, 11, 46. https://doi.org/10.3390/life11010046 https://www.mdpi.com/journal/life Life 2021, 11, 46 2 of 12 three antiparallel beta-strands held together by four disulfide bridges and a short alpha- helix [5,6]. Site-directed mutagenesis experiments combined with cellular assays and sensory analysis have been performed to elucidate the structure-function relationships of brazzein [7,8]. Key amino acid residues responsible for the interaction of brazzein with the TAS1R2/TAS1R3 heterodimeric sweet taste receptor have been identified [7,9]. Although some models of receptor activation by brazzein have been proposed, the exact mechanism is not known. Interestingly, substitutions of some amino acid residues have been shown to significantly increase the sweetness of brazzein [7,10,11]. Because brazzein is difficult to obtain from its natural source, it has been expressed using various recombinant expression systems, including bacteria (Escherichia coli and Lactococcus lactis)[7,12,13], yeasts (Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis)[4,14–16] and transgenic plants [17]. The methylotrophic yeast Pichia pastoris (also called Komagataella phaffi) is known to be safe and is widely used for recombinant protein expression suitable for industrial production. This system allows high-level expression, proper folding, and posttranslational modifications such as phosphorylation and glycosyla- tion under the control of strong and tightly regulated promoters. In addition, proteins can be secreted by the yeast P. pastoris at high levels in minimal medium, facilitating purification from the culture supernatant and reducing downstream costs. The natural secretion of proteins is also advantageous in that the protein possesses an N-terminus identical to the natural protein if the signal peptide is properly cleaved. The yeast P. pastoris has already been successfully used to express the sweet-tasting proteins thaumatin, monellin, lysozyme and the sweet-taste-suppressing protein gurmarin [18–23]. In our previous study, we found that brazzein can be efficiently secreted by the yeast P. pastoris under the control of the methanol-inducible alcohol oxidase promoter [4]. Using the yeast prepropeptide signal from the Saccharomyces cerevisiae α-mating factor without the Glu-Ala-Glu-Ala spacer peptide, we secreted the two natural isoforms of brazzein, pyrE- bra and des-pyrE-bra, at a level of approximately 90 mg/L. Both recombinant isoforms were purified and biochemically characterized. Mass spectrometry analysis revealed that the signal peptide of both brazzein isoforms was properly cleaved, whereas 1H NMR spectroscopy indicated that both proteins were properly folded. In addition, we found that both recombinant brazzein isoforms were functional and able to activate the human sweet taste TAS1R2/TAS1R3 receptor using a cellular assay in agreement with their sensory properties, revealing that these proteins are identical to the brazzein isoforms naturally occurring in the fruit. An interesting property of yeast P. pastoris is its ability to efficiently secrete recombinant proteins in its culture medium. A secreted protein requires the presence of a signal peptide to target the secreted protein to the secretion pathway. The N-terminal part of the mature protein and/or the protein fold are also probably important for an efficient cleaving. For example, the signal peptide cleaving is not occurring properly for lysozyme and thaumatin when expressed in P. pastoris [21,22]. It is known that signal peptide selection can influence the amounts of proteins that are secreted in yeast culture medium [24,25]. Unfortunately, incorrect signal peptide processing can occur, generating extra N-terminal residues, which could affect protein functionality [26]. In this study, we compared 6 signal peptides to the previously tested α-factor mating prepropeptide for des-pyrE-bra secretion in P. pastoris. The corresponding proteins were purified, characterized and compared for their abilities to activate the sweet taste receptor using a cell-based assay. 2. Materials and Methods 2.1. Construction of the Expression Vectors The pPpT4_Alpha_S vector [27] was used for brazzein expression experiments. Syn- thetic DNA encoding des-pyrE-Bra (mature minor form of brazzein, 53 amino acid residues) fused with the different N-terminal signal peptides was codon optimized for P. pastoris expression using GeneDesigner (DNA 2.0, Menlo Park, CA, USA) [28]. The signal peptide cleavage was analyzed using the SignalP 4.0 Server (http://www.cbs.dtu.dk/services/ Life 2021, 11, 46 3 of 12 SignalP/). Plasmid names and signal peptide sequences are provided in Table1. DNA sequences were subcloned in the pPpT4_Alpha_S plasmid between the EcoRI and NotI restriction sites. Escherichia coli strain DH5α was used for amplification of the expression plasmids. The integrity of the constructs was checked by DNA sequencing. Table 1. Sequences of the signal peptides. For each signal peptide, the organism at the origin of the peptide sequence is indicated as well as the protein from which it originates. The amino acid sequences of the signal peptides and the vector name are also indicated. Construct Sequence: Signal Signal Peptide Organism References Name Peptide Saccharomyces