Abstract: This study attempted to improve the expression level of recombinant human lysozyme in Pichia pastoris by co-expression of the chaperone protein disulfide isomerase (PDI) and the transcriptional activator of ferrous transport-1 (Aft1) from P. pastoris. The constructed expression plasmids, pG418-PDI and pG418-Aft1, were linearized and integrated into the genome of P. pastoris KM71-HLY through homologous recombination by electroporation. The positive transformants were selected based on G418 resistance, yielding two recombinant strains, KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1. Shake flask fermentation was performed to analyze the expression level of HLY in the engineered yeasts. Experimental results showed that KM71-HLY, KM71-HLY-pG418-PDI and KM71-HLY-pG418-Aft1 all produced human lysozyme (14.7 kDa) with inhibitory effects on Micrococcus lysodeik under the induction of methanol. These recombinant strains exhibited only a slight difference in the cell growth at the early stage of induction. However, the biomasses of KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1 were lower than that of KM71-HLY after 70 h and their optical density values (OD600 nm) at the end of fermentation were 92.8% and 84.1% as compared to KM71-HLY, respectively. When induced by methanol for 168 h, the yields of total secretory protein of KM71-HLY, KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1 were 324.02, 350.87 and 474.8 mg/L, and the lysozyme activities of the fermentation supernatants were 34 880, 45 600 and 50 180 U/mL, respectively. As compared to KM71-HLY, KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1 increased total secretory protein production by 8.3% and 46.5%, and extracellular lysozyme activity by 30.7% and 43.9%, respectively.

Key words: human lysozyme, chaperone, transcriptional factor, co-expression, Pichia pastoris