Expression of a triple mutational des‐pGlu brazzein in transgenic mouse milk

Abstract

Brazzein has excellent potential for use as a sweetener because of its high level of sweetening potency and stability against extreme temperature and pH. It is extracted from the tropical and difficult—to‐cultivate African plant Pentadiplandra brazzeana , which hampers its commercial viability. Here we report the mammary‐specific expression of wildtype or triple mutational (H31R/E36D/E41A) des ‐pGlu brazzeins in the milk of transgenic mice. Using enzyme‐linked immunoassay (ELISA), western blot, and sweetness intensity testing, we confirmed that the triple mutation made the des ‐pGlu brazzein molecule 10,000 times sweeter than sucrose in a weight base, even after 10 min of incubation at 100 °C; in addition, the triple mutant was also significantly sweeter than the wildtype des ‐pGlu brazzein. This study provides new insights for producing brazzein or brazzein‐sweetened milk from animals for use in food and healthcare applications.

A sweeter future: Using protein language models for exploring sweeter brazzein homologs

“…Prior studies on WT brazzein have examined single or double mutations to uncover key areas vital for sweetness. The results indicate that the residues 29-33, 36, 39-43, and the N and C-termini are crucial for sweetness (Assadi-Porter, Aceti, & Markley, 2000; Ghanavatian et al, 2016; Jin, Danilova, Assadi-Porter, Aceti, et al, 2003; Jin, Danilova, Assadi-Porter, Markley, et al, 2003; Lee et al, 2013; Lim et al, 2016; Liu et al, 2021; R. Lu et al, 2022).…”

The sweet protein brazzein as a promising natural sweetener

“…To increase the level of sweetness of brazzein, mutant variants of this protein were created through site-directed mutagenesis, the sweetest of which is triple mutant H31R/E36D/E41A, which is 22,500 times sweeter than sucrose [2]. Since the content of brazzein in the fruits of the natural source (Pentadiplandra brazzeana) is extremely low (0.2%), various methods have been developed to obtain brazzein using heterologous expression systems, which used as producers: bacteria (Escherichia coli, Lactococcus lactis), yeast (Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum) and animals (Mus musculus) [3][4][5]. Despite the short peptide sequence, the industrial production of recombinant protein faced several problems, including low protein yield (e.g in mouse milk it was detectable on western blot analysis only) and loss of sweetness.…”