Abstract
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−1 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.
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Acknowledgements
This work was supported by the Key International Cooperation Project of the Chinese Academy of Sciences (155112KYSB20160010) and the CAS-TWAS President’s Fellowship Program 2014/2018.
Supporting information
Supplementary Table 1: Primers used in this study.
Supplementary Table 2: Strains and plasmids used in the study.
Supplementary Fig. 1: Multiple sequence alignments of Hac1p homologues.
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Bankefa, O.E., Wang, M., Zhu, T. et al. Hac1p homologues from higher eukaryotes can improve the secretion of heterologous proteins in the yeast Pichia pastoris. Biotechnol Lett 40, 1149–1156 (2018). https://doi.org/10.1007/s10529-018-2571-y
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DOI: https://doi.org/10.1007/s10529-018-2571-y