Improved Secretory Production of the Sweet-Tasting Protein, Brazzein, in Kluyveromyces lactis

Abstract

Brazzein is an intensely sweet protein with high stability over a wide range of pH values and temperatures, due to its four disulfide bridges. Recombinant brazzein production through secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect disulfide formation, which is crucial for achieving the final protein structure and stability. Protein disulfide bond formation requires protein disulfide isomerase (PDI) and Ero1p. Here, we overexpressed KlPDI in K. lactis or treated the cells with dithiothreitol to overexpress KlERO1 and improve brazzein secretion. KlPDI and KlERO1 overexpression independently increased brazzein secretion in K. lactis by 1.7-2.2- and 1.3-1.6-fold, respectively. Simultaneous overexpression of KlPDI and KlERO1 accelerated des-pE1M-brazzein secretion by approximately 2.6-fold compared to the previous system. Moreover, intracellular misfolded/unfolded recombinant des-pE1M-brazzein was significantly decreased. In conclusion, increased KlPDI and KlERO1 expression favors brazzein secretion, suggesting that correct protein folding may be crucial to brazzein secretion in K. lactis.

3M-Brazzein as a Natural Sugar Substitute Attenuates Obesity, Metabolic Disorder, and Inflammation

“…Our recombinant wild-type brazzein showed 1800 times higher sweetness than sucrose; moreover, the brazzein mutants with critical residue mutation had sweeter molecules than those of wild-type brazzein. Among the mutants, the brazzein protein with triple mutations (H31R/E36D/E41A) (3M-brazzein, 3M-Brz) was 22,500 times sweeter than sucrose on a weight basis, representing an 18-fold sweetness compared to that of wild-type brazzein. − Furthermore, brazzein secreted by the yeast K. lactis exhibited antioxidant, anti-inflammatory, and antiallergic activities without any antibacterial and antifungal effects, making it more suitable for use in industrial food processes.…”

Bioprospecting and biotechnological insights into sweet-tasting proteins by microbial hosts―a review

“…Its derivatives with mutation of critical residues were even sweeter than the native brazzein. Amongst the mutants, brazzein with three mutations (H31R/E36D/E41A) was 22,500-times sweeter than sucrose and represented 18-fold sweetness than wild-type brazzein [ 74–77 ]. Moreover, brazzein produced by the K. lactis displayed anti-inflammatory, antiallergic, and antioxidant potentialities rendering it alluring for utilization in food processes [ 75 ].…”

Customized yeast cell factories for biopharmaceuticals: from cell engineering to process scale up

“…If the protein is misfolded prior to secretion, it will be degraded and ends in enhanced stress in endoplasmic reticulum. Hence for the proper secretion of heterologous proteins, the protein folding chaperones and redox enzymes in S. cerevisiae [ 63 , 64 ], K. lactis [ 65 , 66 ] and P. pastoris [ 67 ] are overexpressed. The folding and secretion of proteins is controlled by Hsp70 and Hsp40 families of chaperones.…”