pMOX: a new powerful promoter for recombinant protein production in yeast Pichia pastoris

Abstract AOX1 promoter (pAOX1) is a robust inducible promoter highly preferred for the production of recombinant proteins in Pichia pastoris (P. pastoris). However, repression by other carbon sources and induction by methanol, which is a fire hazard chemical and undesirable for industrial production, are remarkable drawbacks in large-scale use of this promoter. Hence, novel strong regulatory promoters are highly desired. In the present study, the promoter region of methanol oxidase gene (pMOX), from Hansenula polymorpha, was explored for the heterologous expression of foreign proteins in protease deficient and wild type P. pastoris strains. The promoter region of MOX was isolated and replaced with the pAOX1 in the pPINK-HC plasmid. The activity of pMOX and pAOX1 was compared using endoglucanase 3 (CMC3) and endoglucanase II (EgII) enzymes as the reporter proteins. Evaluation of carbon sources on pMOX activity showed complete inactivation in the presence of xylose and sorbitol and high activity by glycerol, glucose and methanol feeding. Furthermore, the results indicated that increasing the gene dosage and using protease deficient-trait significantly increased CMC3 and EgII expression under the control of pMOX. In conclusion, in this study, a new small powerful and methanol-free promoter is introduced for recombinant protein production in yeast P. pastoris.