TREHALULOSE SINTASE PRODUCTION BY RECOMBINANT YEAST FOR SUGAR CANE MOLASSES BIOREFINERY

FRESCURA, JULIETA M; ZAPATA, ARLEY D; ROJAS, NATALIA L

Buzios

Simposio; Simposio Nacional de Fermentaciones; 2022

The excessive sucrose intake is related to health disorders such as cardiovascular disease, diabetes, obesity, cancer, osteoporosis, and dental infections. Trehalulose is a disaccharide, sucrose isomer, naturally found in small amounts in honey, pollen, and sugar cane. It is a promising substance to replace sucrose as sweetener because of its greater sweetening power compared to other sucrose isomers of sucrose (70% of sucrose sweetness) and its structural similarity which does not generate residual flavors. On the other hand, due to its slow metabolism, trehalulose contributes to the prevention of dental caries, to mitigate type-2 diabetes, to weight control, and also it has prebiotic effects. These characteristics suggest a wide range of potential applications in the food, cosmetic and pharmaceutical industries. The objective of this study is the development of strategies of genetic engineering and bioprocess intended for an enzymatic process for the production of trehalulose from sucrose. Three microorganisms were selected as sources of enzymes for the potential production of trehalulose from sucrose, based on previous reports and the bioinformatic analysis purposed.Those genes were modified and arranged for their expression in the P. pastoris yeast heterologous system. Enzyme production was analyzed in shake flasks. Among them, after recovering the supernatant of P. pastoris PC1 (carrying P. carotovorum gene), SDS-PAGE analysis revealed the expression of a protein which molecular weight corresponds to the expected for trehalulose sintase (76kDa). In a preliminary enzyme activity analysis, using sucrose as substrate, trehalulose sintase activity resulted in 200 mU/mL, thus constituting a promising recombinant system to produce trehalulose. These advances could be applied in the sugar cane molasses biorefinery.