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

08:00 EDT 1st April 2019 | BioPortfolio

Summary of "Effect of gene copy number and chaperone co-expression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris."

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. 1-copy, 2-copy, 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 qPCR. 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-copy 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. This article is protected by copyright. All rights reserved.

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Name: Biotechnology and bioengineering
ISSN: 1097-0290
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Medical and Biotech [MESH] Definitions

Stretches of genomic DNA that exist in different multiples between individuals. Many copy number variations have been associated with susceptibility or resistance to disease.

The in vitro fusion of GENES by RECOMBINANT DNA techniques to analyze protein behavior or GENE EXPRESSION REGULATION, or to merge protein functions for specific medical or industrial uses.

The use of techniques that produce a functional MUTATION or an effect on GENE EXPRESSION of a specific gene of interest in order to identify the role or activity of the gene product of that gene.

A copy number variation that results in reduced GENE DOSAGE due to any loss-of-function mutation. The loss of heterozygosity is associated with abnormal phenotypes or diseased states because the remaining gene is insufficient.

The number of copies of a given gene present in the cell of an organism. An increase in gene dosage (by GENE DUPLICATION for example) can result in higher levels of gene product formation. GENE DOSAGE COMPENSATION mechanisms result in adjustments to the level GENE EXPRESSION when there are changes or differences in gene dosage.

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