Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris

Abstract

Hydrophobins are small highly surface-active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression host Pichia pastoris. One-, 2-, and 3-copy-HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction.Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression of PDI1 or ERO1 caused insignificant changes in HFBI secretion in 1-and 2-copy strains, but a statistically significant HFBI secretion increase in 3-copy strain. KAR2 overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3-copy-HFBI secreting 22±1.6 fold more than the 1-copy-HFBI/no chaperone strain. The highest increase was seen in 3-copy-HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1-copy-HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small-scale format used in this study.

CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris

“…Multicopy strains are often constructed and screened for production enhancement of compounds and proteins [49, 50]. However, the identical expression cassette may be lost or misplaced when multicopy integration is performed by single-crossover in P. pastoris [43].…”

A Combinational Strategy for Effective Heterologous Production of Functional Human Lysozyme in Pichia pastoris

“…Therefore, co-expressing three molecular chaperones, Bip, Pdi1, and Ero1, to enhance the post-translational efficiency was applied in our studies. These three molecular chaperones were reported to function on prompting on protein folding and disulfide bond formation (Rose et al, 1989;Farquhar et al, 1991;Hudson et al, 2015) and applied to successfully increase the heterologous expression of hydrophobin HFBI (Sallada et al, 2019), Candida rugosa lipase Lip1 (Li et al, 2016), and human albumin (HSA) fusion protein IL2-HSA in P. pastoris (Guan et al, 2016). In our FIGURE 6 | High-density cultivation of hLYZ in 5-L fermenter.…”

Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins

“…Nevertheless, upregulation of the UPR is beneficial only in the cases where protein folding, rather than its passage through the secretory pathway, becomes rate-limiting (Love et al 2012 ). In P. pastoris , increased expression or secretion of many different recombinant proteins resulted from co-expression of the following: the ER-chaperone Kar2p or protein disulfide isomerase Pdi1 (Inan et al 2006 ; Damasceno et al 2007 ; Sallada et al 2019 ), enzymes involved in the ER redox control and oxidative stress such as Ero1, Gpx1, Aha1, or Ypt6 (Sha et al 2013c ; Ben Azoun et al 2016a ; Huangfu et al 2016 ; Sallada et al 2019 ), the UPR transcription factor Hac1p (Guerfal et al 2010 ; Vogl et al 2014 ; Li et al 2015 ; Krainer et al 2016 ; Huang et al 2017 ; Han et al 2020 ; Liu et al 2020 ), the kinase/RNase Ire1p (Yu et al 2020 ), or new co-chaperones (Huangfu et al 2016 ) (Table 2 ). Glycosylation activity was also increased (Moon et al 2015 ) or product homogeneity and processing of the secretion α-factor were improved (Guerfal et al 2010 ).…”