Facilitating secretory expression of apple seed β‐glucosidase in Komagataella phaffii for the efficient preparation of salidroside

Abstract Plant‐derived β‐glucosidases hold promise for glycoside biosynthesis via reverse hydrolysis because of their excellent glucose tolerance and robust stability. However, their poor heterologous expression hinders the development of large‐scale production and applications. In this study, we overexpressed apple seed β‐glucosidase (ASG II) in Komagataella phaffii and enhanced its production from 289 to 4322 U L −1 through expression cassette engineering and protein engineering. Upon scaling up to a 5‐L high cell‐density fermentation, the resultant mutant ASG II V80A achieved a maximum protein concentration and activity in the secreted supernatant of 2.3 g L −1 and 41.4 kU L −1 , respectively. The preparative biosynthesis of salidroside by ASG II V80A exhibited a high space‐time yield of 33.1 g L −1 d −1 , which is so far the highest level by plant‐derived β‐glucosidase. Our work addresses the long‐standing challenge of the heterologous expression of plant‐derived β‐glucosidase in microorganisms and presents new avenues for the efficient production of salidroside and other natural glycosides.