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 IIV80A achieved a maximum protein concentration and activity in the secreted supernatant of 2.3g L-1 and 41.4 kU L-1, respectively. The preparative biosynthesis of salidroside by ASG IIV80A exhibited a high space-time yield of 33.1g 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.