Heterologous expression of wild-type and Lys99Ala mutant L-asparaginase from Lachancea thermotolerans in Pichia pastoris: culture optimization and laboratory-scale bioreactor production

Abstract L-Asparaginase (L-ASNase) is an enzyme of significant therapeutic value within the pharmaceutical industry, primarily due to its clinical efficacy in treating acute lymphoblastic leukemia (ALL). Despite its benefits, the clinical application of commercially available bacterial-derived L-ASNases is frequently impeded by severe hypersensitivity reactions and immunogenicity. This limitation has necessitated the exploration of novel L-ASNase variants and the development of alternative heterologous expression systems that minimize adverse immune responses. As unicellular eukaryotic models, yeast-based expression platforms represent a robust alternative for the production of therapeutic proteins. In the present study, the asnase gene from Lachancea thermotolerans ( L. thermotolerans , Lt ASNase), along with its Lys99Ala mutant variant was heterologously expressed in Pichia pastoris ( P. pastoris ). To maximize yield, cultivation parameters were systematically optimized, followed by scale-up in a bioreactor under stabilized conditions. Initial shake-flask cultivations of the wild-type and mutant Lt ASNase yielded maximum whole-cell activities of 191 U g DCW −1 (628 U L − 1 ) and 57 U gdcw − 1 (174 U L⁻¹) over induction periods of three and two days, respectively. Process optimization identified the ideal conditions as pH 6.0 and 20 °C for the wild-type enzyme, and pH 7.5 and 16 °C for the mutant. Notably, bioreactor-scale expressions facilitated 4- and 7-fold increase in volumetric whole-cell activity for the wild-type and mutant enzymes, compared to shake-flask results. These findings suggest that P. pastoris is a viable host for microbial Lt ASNase production, achieving yields higher than established Escherichia coli ( E. coli ) systems; however, further investigation into secretory expression pathways is required to provide cost-effective production.