College of Life Sciences, Jilin Agricultural University, Changchun, China.
Key Laboratory of Straw Biology and Utilization, The Ministry of Education, Changchun, China.
Sci Rep. 2020 Jul 15;10(1):11686. doi: 10.1038/s41598-020-68570-6.
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.
基于酿酒酵母的表达系统依赖于安全的、食品级的菌株，成本低、操作简单，可用于大规模发酵。然而，酿酒酵母对外源蛋白表达水平低限制了其广泛应用。内质网（ER）对外源蛋白的折叠和加工能力是限制外源蛋白表达的重要因素。本研究考察了参与未折叠蛋白反应途径的转录因子 Hac1p 对黑曲霉木聚糖酶基因 xynB 在酿酒酵母中的表达的影响。重叠延伸聚合酶链反应（PCR）、rDNA 整合和液滴数字 PCR 技术用于产生含有 8 个 xynB 拷贝的酿酒酵母菌株（S8），允许木聚糖酶的高效分泌表达。然后使用 GeXP 系统检查随后在菌株 S8 中过表达 HAC1 对 ER 中与蛋白折叠相关基因表达的影响。结果证实，通过 rDNA 整合表达盒，xynB 的多个拷贝可实现组成型分泌表达，木聚糖酶的最大产量为 325 U/mL。然而，HAC1 的过表达进一步提高了 S8 菌株的木聚糖酶产量，使产量达到 381 U/mL。
