Prototyping Yarrowia lipolytica for industrial production of hyperthermophilic enzymes- a case of β-glucosidase (CelB) from Pyrococcus furiosus

Abstract

Hyperthermozymes have recently gained significant recognition as prospective industrial biocatalysts. However, the necessity to ease their large scale synthesis in a cost-effective manner often challenges the viability of industrial bioprocesses. In the present study, Yarrowia lipolytica, because of its high secretion capacity and robust growth, was assessed for its efficacy to express and secrete one of the widely used hyperthermozyme, CelB (β-glucosidase) of Pyrococcus furiosus. Preliminary expression studies using preproLIP2 and preXPR2 as signal peptides indicated that preproLIP2 was more efficient in targeting CelB outside the cell with enzyme yields of 0.42 ± 0.016 U/mL. To further improve the extracellular CelB yields, the preproLIP2-CelB expression cassette was integrated in one, two or three copies at different genomic loci of Y. lipolytica. Though variances in CelB expression were observed across the integration sites, its constitutive expression did not hamper the cell fitness. Also, with increasing CelB copy number linear increase in expression was observed reaching the highest enzyme yields of ∼5 U/mL for Po1f_LP3 strain, 12 fold higher than the base strain (Po1g_LP). Interestingly, comparable enzyme yields were achieved when rich YPD medium for cultivating Y. lipolytica was replaced with a low-cost SPD medium. Such high titres observed in shake-flask cultivation demonstrates the potential of Y. lipolytica for fermentative production of hyperthermozymes.