Expression and function of an HAC1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae

Abstract

To investigate the effects of transcription factor HAC1, which is involved in the unfolded protein response pathway, on the expression of xynB in Saccharomyces cerevisiae , we used overlap extension polymerase chain reaction (PCR), rDNA integration, and droplet digital PCR technology to generate a S . cerevisiae strain (S8) containing eight copies of the xylanase gene, allowing high-yield secretory expression of xylanase. HAC1 was then overexpressed in the S8 strain, and the GeXP system was used to study the effects of HAC1 overexpression on the expression of genes associated with protein folding in the endoplasmic reticulum (ER). Results confirmed the constitutive secretory expression of the multiple copies of the xylanase gene in S . cerevisiae following rDNA-based integration of the expression cassette. Specifically, recombinant S . cerevisiae strain S8, containing eight copies of the xylanase gene, showed maximum xylanase expression, with a yield of 325 U/mL. However, overexpression of HAC1 further improved xylanase production by strain S8, resulting in a yield of 381 U/mL. These results confirmed that overexpression of HAC1 improves the expression of genes associated with protein folding in the ER, enhancing the protein folding and assembly functions of the ER and increasing xylanase expression.