Intraspecific protoplast fusion of Brettanomyces anomalus for improved production of an extracellular β-glucosidase

Improvement of production of an extracellular β-glucosidase with high activity by Brettanomyces anomalus PSY-001 was performed by using recursive protoplast fusion in a genome-shuffling format. The initial population was generated by ultraviolet irradiation, ultrasonic mutagenesis and, then, subjected to recursive protoplast fusion. Mutant strains exhibiting significantly higher β-glucosidase activities in liquid media were isolated. The best mutant strain showed increased cell growth in a flask culture, as well as increased β-glucosidase production. A recombinant strain, F3-25, was obtained after three rounds of genome shuffling and its production of β-glucosidase activity reached 4790 U L-1, which was a nearly eightfold increase compared to the original strain B. anomalus PSY-001. The subculture experiments indicated that F3-25 was genetically stable.