Media optimisation for enhanced β-glucosidase production in Komagataella pastoris

Abstract

Cost-effective protein production requires maximal target protein expression levels in affordable cultivation media. The Komagataella pastoris pGAPZαA expression vector was adapted for expression of the Phanerochaete chrysosporium β-glucosidase reporter gene in Komagataella pastoris and Saccharomyces cerevisiae resulting in volumetric activities of 0.26 and 0.52 U/mL, respectively. The K. pastoris[pccbgl1] strain was subsequently cultivated in modified BMGY with different nitrogen source substitutions for YNB. Soy whey yielded an extracellular β-glucosidase activity of 0.60 U/mL, 2.07-fold greater than obtained with standard BMGY. Hereafter, novel simple media (SM) combinations were designed, which resulted in a 2.71-fold increase in β-glucosidase activity relative to BMGY in shake flask cultivations. The simple media compared well with the routinely used basal salt medium (BSM) in 1-L batch fermentation studies. The BSM, SMb (SM with brewer's spent yeast) and SMs (SM with soy whey) resulted in volumetric activities of 2.26, 2.79 and 1.29 U/mL and productivities of 84, 120 and 49 U/g DCW, respectively after 30 h of aerobic cultivation [2.8, 4.0 and 1.63 U/g DCW/h]. The novel SMb compares well with the BSM and has potential use for the economical production of recombinant proteins on an industrial scale. A key consideration when using the current waste streams as a nitrogen source is the presence of additional proteins, which may impact the downstream purification process when a highly pure product is required.