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 Erolp. Here, we overexpressed KIPDI in K. lactis or treated the cells with dithiothreitol to overexpress KIEROI and improve brazzein secretion. KIPDI and KIERO1 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 KIEROI expression favors brazzein secretion, suggesting that correct protein folding may be crucial to brazzein secretion in K. lactis.