Efficient Production and Characterization of the Sweet-Tasting Brazzein Secreted by the Yeast Pichia pastoris

Abstract

Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and (1)H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms.

Refined single‐interval adjustment matrix yes‐no task for estimating the absolute thresholds of sweet‐tasting molecules

“…The estimated threshold values of sucrose and brazzein obtained with the SIAM yes‐no task were approximately 3.2 mg/ml and 4.4 × 10 −3 mg/ml, respectively. These values were significantly different from those reported by Assadi‐Porter et al (), but similar to those reported by Poirier et al (). Other studies using a variety of methods estimated the threshold of sucrose to be 2.7–4.3 mg/mL, which is 2.3‐ to 3.7‐fold lower than the value estimated by Assadi‐Porter et al (), Fukunaga, Uematsu, and Sugimoto (), James, Laing, and Oram (), Kunka, Doty, and Settle (), and Shepherd, Quek, and Pathirana ().…”

“…Other studies using a variety of methods estimated the threshold of sucrose to be 2.7–4.3 mg/mL, which is 2.3‐ to 3.7‐fold lower than the value estimated by Assadi‐Porter et al (), Fukunaga, Uematsu, and Sugimoto (), James, Laing, and Oram (), Kunka, Doty, and Settle (), and Shepherd, Quek, and Pathirana (). Poirier et al () suggested that these differences in threshold were related to differences in the methods used to estimate the thresholds. Therefore, the sweetness potency (or ratio) between the control substance sucrose and brazzein should be analyzed for comparison.…”

“…However, the threshold values of these methods cannot be compared because they were obtained using different methods and sample aliquots for tasters. For example, the minor‐type brazzein was determined to be approximately 800‐fold sweeter than sucrose by weight using a four‐alternative forced‐choice procedure (Poirier et al, ), but was found to be approximately 500‐fold sweeter using another method scoring the sweetness of brazzein and sucrose (Assadi‐Porter, Aceti, & Markley, ). Therefore, a standard sensory analysis is needed to determine the absolute threshold, sweet potencies, and sensitive differences of sweet‐tasting molecules and proteins.…”

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis

“…The yeasts Saccharomyces cerevisiae, Pichia pastoris, Hansulae polymopha, and Kluyveromyces lactis have been widely used as a host to produce heterologous proteins [11][12][13]. These yeast expression systems have advantages that include eukaryotic quality-control machinery, fast growth rate, high cell density, facile genetic manipulation, and inexpensive growth medium.…”

“…Most of the recombinant proteins produced in yeast have been expressed by S. cerevisiae strains [11,12]. Recently, Poirier et al [13] reported the heterologous expression in the yeast P. pastoris of two natural forms of brazzein, and a mutant called Q1-bra. Recombinant proteins were induced by adding 1% methanol twice a day for 6 days, and approximately 30-90 mg/L was obtained.…”