The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and has been engineered for consolidated bioprocessing (CBP) of lignocellulosic biomass through heterologous cellulase expression. However, S. cerevisiae produces low titers of cellulases and one suspected reason for this is that heterologous proteins induce the unfolded protein response (UPR). Current methods of measuring the UPR are RNA based and can be inconsistent and cumbersome. We developed vector-based biosensors that will detect and quantify UPR activation. The vector consisted of either the Trichoderma reesei xylanase 2 or codon optimized green fluorescent protein (eGFP) reporter genes under the control of the S. cerevisiae PHAC1 or PKAR2 promoters. The eGFP reporter under control of PKAR2 was identified as the preferred combination due to its superior dynamic range and its greater sensitivity when measuring UPR induction in cellulase producing strains. To our knowledge, we show for the first time that significant UPR activation differences could consistently be observed for different cellulase candidate genes unlike previous RNA-based tests, which were unable to detect these differences. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them favorable for use in CBP yeasts.

中文翻译：

---

体内检测和测量产生重组纤维素酶的酿酒酵母菌株中未折叠的蛋白质反应。

酵母酿酒酵母具有工业上所需的乙醇生产特性，并且经过工程改造，可通过异源纤维素酶表达进行木质纤维素生物质的合并生物处理（CBP）。但是，酿酒酵母产生的纤维素酶滴度低，一个可疑的原因是异源蛋白质诱导了未折叠的蛋白质反应（UPR）。当前测量UPR的方法是基于RNA的，并且可能不一致且麻烦。我们开发了基于矢量的生物传感器，可以检测和量化UPR激活。该载体由里氏木霉木聚糖酶2或在酿酒酵母PHAC1或PKAR2启动子控制下的经密码子优化的绿色荧光蛋白（eGFP）报告基因组成。PKAR2控制下的eGFP报告基因被鉴定为首选组合，因为它在测量纤维素酶生产菌株中的UPR诱导时具有出色的动态范围和更高的灵敏度。据我们所知，我们首次展示了不同的纤维素酶候选基因可以一致地观察到明显的UPR激活差异，这与以前的基于RNA的测试不同，后者无法检测到这些差异。量化UPR诱导的能力将有助于鉴定不会大大诱导UPR的候选纤维素酶基因，使其适合用于CBP酵母。我们首次表明，与以往基于RNA的测试不同，无法针对这些纤维素酶候选基因连续观察到显着的UPR激活差异，而后者无法检测到这些差异。量化UPR诱导的能力将有助于鉴定不会大大诱导UPR的候选纤维素酶基因，使其适合用于CBP酵母。我们首次表明，与以往基于RNA的测试无法检测到这些差异相比，对于不同的纤维素酶候选基因，可以一致地观察到显着的UPR激活差异。量化UPR诱导的能力将有助于鉴定不会大大诱导UPR的候选纤维素酶基因，使其适合用于CBP酵母。