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

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.