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

Dr. Bryan W Berger, PhD
Dr. Bryan W Berger, PhD
Lehigh University
Assistant Professor
Bethlehem, PA | United States

Biotechnol Bioeng 2019 Apr 1. Epub 2019 Apr 1.

Department of Biomedical Engineering, University of Virginia, Thornton Hall, Charlottesville, Virginia.

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.

Download full-text PDF

http://dx.doi.org/10.1002/bit.26982DOI Listing
April 2019
3 Reads
4.126 Impact Factor

Publication Analysis

Top Keywords

hfbi secretion
copy number
increasing hfbi
pichia pastoris
recombinant yields
hydrophobin hfbi
chaperone strain
gene copy
1-copy-hfbi/no chaperone
number increasing
reaction increasing
chain reaction
hfbi copy
chaperone overexpression
increased hfbi


(Supplied by CrossRef)
Real‐time PCR‐based determination of gene copy numbers in Pichia pastoris
Abad S. et al.
Biotechnology Journal: Healthcare Nutrition Technology 2010
Protein expression in Pichia pastoris: Recent achievements and perspectives for heterologous protein production
Ahmad M. et al.
Applied Microbiology and Biotechnology 2014
Surface hydrophobin prevents immune recognition of airborne fungal spores
Aimanianda V. et al.
Nature 2009
Can too many copies spoil the broth?
Aw R. et al.
Microbial Cell Factories 2013
Hydrophobins—Unique fungal proteins
Bayry J. et al.
PLOS Pathogens 2012
Expression of a modified dutch elm disease toxin
Bolyard M. G. et al.
Molecular Plant‐Microbe Interactions 1992
Heterologous protein expression in the methylotrophic yeast Pichia pastoris
Cereghino J. L. et al.
FEMS Microbiology Reviews 2000
Enhancement of the gene targeting efficiency of non‐conventional yeasts by increasing genetic redundancy
Chen Z. et al.
PLOS One 2013
Production of mouse epidermal growth factor in yeast: High‐level secretion using Pichia pastoris strains containing multiple gene copies
Clare J. J. et al.
Gene 1991
Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production
Daly R. et al.
Journal of Molecular Recognition 2004
Cooverexpression of chaperones for enhanced secretion of a single‐chain antibody fragment in Pichia pastoris
Damasceno L. M. et al.
Applied Microbiology and Biotechnology 2007

Similar Publications