Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeastPichia pastoris

This study attempted to fully explore the expression potentials of Pichia pastoris for producing porcine insulin precursor (PIP) through PIP copy number optimization.Multi-copy strains were screened employing a highly efficient improved in vivo method and their copy numbers were quantified by real-time qPCR. A range of Mut(+)P. pastoris strains harbouring 0, 1, 3, 6, 12, 18, 29, 52 copies of PIP were obtained. After 96 h methanol induction, a bell-shaped correlation curve was observed between gene dosage and protein yield, and the maximum PIP expression level of 181 mg l(-1) was achieved by a 12-copy strain. Specific growth rate and methanol utilization capacity were found to decrease remarkably for high copy strains (>12 copies). Transcriptional analysis of KAR2 suggested higher copy strains were suffering more from ER stress.A copy number around 12 is optimal for secretory expression of PIP in P. pastoris. Excess PIP gene dosage (>12 copies) significantly impaired the growth of P. pastoris hosts.The methods developed and the discoveries made by this systematical investigation will be helpful to the application and understanding of Pichia pastoris expression system for heterologous overexpression.