Hac1p homologues from higher eukaryotes can improve the secretion of heterologous proteins in the yeast Pichia pastoris.

Objectives: To systematically explore the effects of overexpressing Hac1p homologues from different sources on protein secretion in Pichia pastoris system.

Results: Effects of Hac1p homologues encompassing P. pastoris (PpHac1p), S. cerevisiae (ScHac1p), Trichoderma reesei (TrHac1p) and Homo sapiens (HsXbp1), on secretion of three reporter proteins-β-galactosidase, β-mannanase and glucose oxidase were investigated. No individual Hac1p was optimal for all the enzymes. Rather, by testing a set of Hac1p, the secretory expression of each of the enzymes was improved. Notably, HsXbp1 overexpression improved β-mannanase production from 73 to 108.5 U β-mannanase mL while PpHac1p had no impact in shake flask culture. Moreover, HsXbp1 led to 41 and 67% increases in β-mannanase production in the single- and four-copy strain, respectively in 1-L laboratory fermenter. Transcription analysis of indicative chaperones suggested that HsXbp1 may cause a stronger and prolonged activation of the UPR target chaperone genes.

Conclusion: Mammalian HsXbp1 worked better than yeast Hac1p in terms of improving β-mannanase secretion in P. pastoris, and Hac1p screening may offer an effective strategy to engineer the secretion pathway of eukaryotic expression systems.