Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-beta-mannosidase (MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides (W1, MF4I, INU1A, alpha pre, HFBI) were chosen to be analyzed by SignalP 4.0, among which W1 was designed. Then, the widely used signal peptide alpha-factor in expression vector pGAPZ alpha A was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4I, INU1A, apre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with alpha-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by alpha-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/mL, while those mediated by MF4I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/mL, respectively. The maximum MAN activity reached 347.5 U/mL with 4 gene copies mediated by MF4I. These results indicate that replacing the signal peptide alpha-factor with MF4I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.