Abstract
Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation. However, low levels of foreign protein expression by S. cerevisiae have limited their widespread application. The ability of the endoplasmic reticulum (ER) to fold and process foreign proteins is an important factor restricting the expression of foreign proteins. In the current study, the effects of transcription factor Hac1p, which is involved in the unfolded protein response pathway, on S. cerevisiae-based expression of xylanase gene xynB from Aspergillus niger were examined. Overlap extension polymerase chain reaction (PCR), rDNA integration and droplet digital PCR technology were used to generate a S. cerevisiae strain (S8) containing eight copies of xynB, allowing high-yield secretory expression of xylanase. The effects of subsequent overexpression of HAC1 in strain S8 on the expression of genes associated with protein folding in the ER were then examined using the GeXP system. Results confirmed the constitutive secretory expression of the multiple copies of xynB following rDNA-based integration of the expression cassette, with a maximum xylanase yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL.
Highlights
Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation
High-yield xylanase-producing S. cerevisiae strains were generated via promoter modification, the introduction of secretory signal peptides and increasing the gene copy number
Analysis showed that the relationship between xylanase gene copy number and enzyme activity was not linear, with more than eight copies of xynB shown to be detrimental to protein expression
Summary
Saccharomyces cerevisiae-based expression systems, which rely on safe, food-grade strains, are low cost, simple to operate, and can be used for large-scale fermentation. The effects of transcription factor Hac1p, which is involved in the unfolded protein response pathway, on S. cerevisiae-based expression of xylanase gene xynB from Aspergillus niger were examined. Overlap extension polymerase chain reaction (PCR), rDNA integration and droplet digital PCR technology were used to generate a S. cerevisiae strain (S8) containing eight copies of xynB, allowing high-yield secretory expression of xylanase. The effects of subsequent overexpression of HAC1 in strain S8 on the expression of genes associated with protein folding in the ER were examined using the GeXP system. Results confirmed the constitutive secretory expression of the multiple copies of xynB following rDNA-based integration of the expression cassette, with a maximum xylanase yield of 325 U/mL. Multiple proteins are involved in the signalling pathway, including Hac1p, Cpr5p, Cne1p, Ero1p, Kar2p, Pdi1p and Sec53p. The genes coding for each of these proteins are likely to be affected by Hac1p-mediated activation of the UPR pathway, resulting in both short- and long-term physiological e ffects[9,10]
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