Efficient Biosynthesis of Rare Ginsenoside Compound K Using Engineered <i>Pichia pastoris</i> with Surface-Anchored <i>Sulfolobus solfataricus</i> β-Glucosidase

<i>Sulfolobus solfataricus</i> β-glucosidase (SS-bgly) is vital for converting rare ginsenoside compound K (CK), but suffers from stability and cost issues in practical applications. To tackle these, we developed a whole-cell immobilization method by genetically anchoring SS-bgly on <i>Pichia pastoris</i> GS115, using GPI-anchored GCW61 protein. The enhanced strain, Cbg61, underwent ARTP mutagenesis, yielding the improved Cbg61-T37 strain. This strain was further engineered to coexpress N-acetyltransferase MPR1 for oxidative stress resistance, resulting in Cbg61-T37-MPR1. Following fermentation optimization, the β-glucosidase activity of strain Cbg61-T37-MPR1 increased by 58.9% over that of strain Cbg61, with the process also resulting in a 93.79% conversion rate and a CK yield of 6.42 mg/mL. The strain exhibited high stability, retaining 63.17% efficiency after five cycles. This strategy of enzyme surface display, directed evolution, and reaction optimization significantly enhances CK production's practicality and economic feasibility.