This research characterized a novel Komagataella phaffii strain generated through intraspecific crossing between a wild isolate and a laboratory strain. This segregant, called S467, expressed 2.2-fold more secreted recombinant β-glucosidase than its parental strains in microtiter scale, which suggested that S467 could be an attractive host for bioprocess optimization. S467 was grown alongside the laboratory strain CBS7435 expressing β-glucosidase (CBS_BGL9), as a control, in a 1.5 L bioreactor to determine kinetics parameters, and similar cell growth rate (0.12 h⁻¹) but higher recombinant protein activity, measured as enzymatic activity, was observed in S467. The effect of specific cell growth rate was studied using continuous cultures (chemostat) at different dilution rates, identifying conditions that provided up to a twofold increase in enzymatic activity in S467. RT-qPCR was conducted on key genes associated with the genetic background of S467, in order to clarify differences at the transcriptomic level that render S467 as a potential superior host for recombinant protein production. Overall, this study provides quantitative evidence of the positive effect of the natural isolate IRA1 allele for the generation of recombinant β-glucosidase and highlights the usability of natural genetic diversity in K. phaffii.