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In order to improve the extracellular endo-1,4-β-mannosidase (MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides (W1, MF4I, INU1A, αpre, HFBI) were chosen to be analyzed by SignalP 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector pGAPZαA 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, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-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 α-factor with MF4I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Pichia pastoris pGAP (glyceraldehyde dehydrogenase promoter) expression system was widely used for the expression and production of heterologous proteins. Screening multi-copy recombinants was an effective strategy to improve the heterologous protein production in P. pastoris. Because multiple gene insertion events occurred with a low frequency, hundreds to thousands of antibiotic-resistance recombinants need to be screened. The common way of improving screening efficiency was to increase antibiotic concentration in screening plates. Here we developed a screening method by selecting small colonies from low-concentration antibiotic screening plates. This strategy greatly improved the probability of obtaining multi-copy mannanase gene (man) recombinants and it could replace the common strategy by increasing antibiotic concentration in screening plates. The further study in liquid shake flask cultures revealed that cell concentrations, growth rates and substrate consumption rates of recombinants gradually decreased with the increase in man copy number. This indicated that such a screening strategy was effective to screen multi-copy recombinants based on colony size.

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