Construction of Constitutive Saccharomyces Cerevisiae Engineered Strain of β-glucosidase

Saccharomyces cerevisiae engineered strain of β-glucosidase was constructed constitutive in this study. The complete PαBC expression cassette was constructed by overlap- extension PCR technique; then the complete PαBC expression cassette was connected with the pYES2 expression vector and rDNA core sequence to construct the pYES2-PαBC-rDNA vector, the new vector was transferred into Saccharomyces cerevisiaeINVSc1 by r-DNA integration method subsequently. A total of 23 positive mutants were obtained by SC-U nutrient deficient medium and strain solution PCR screening. Digital droplet PCR (dd-PCR) technique was used to identify the copy numbers of positive transformants. Copy numbers of the β-glucoside gene sequence were: 1, 2, 3, 5, 6, 9, 10, 12, 13, 15, analyzing the relationship between the number of copies and the expression of β-glucoside enzyme activity, the result showed that β-glucoside have the highest activity at 10 copies (29.8 IU/mL). The β-glucoside enzyme activities of transformants were verified stable inheritance. The molecular weight of β- glucosidase is 60 kDa, and it is relatively stable at 40–55 °C and pH5.0–6.0 slightly acidic environment. Mn²⁺ has a significant effect on the promotion of β-glucosidase activity (138%), while Ag⁺ and Cu²⁺ had an inhibitory effect on the activity of β-glucosidase, only 13.3% and 39% of pre-addition.