Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washington Luis km 235, 13565-905 São Carlos, SP, Brazil.
N Biotechnol. 2014 Jan 25;31(1):90-7. doi: 10.1016/j.nbt.2013.08.005. Epub 2013 Sep 4.
Saccharomyces cerevisiae is the most important microorganism used in the ethanol fermentation process. The PE-2 strain of this yeast is widely used to produce alcohol in Brazil due to its high fermentation capacity. The aim of the present study was to develop an expression system for recombinant proteins using the industrial PE-2 strain of S. cerevisiae during the alcoholic fermentation process. The protein chosen as a model for this system was CaneCPI-1, a cysteine peptidase inhibitor. A plasmid containing the CaneCPI-1 gene was constructed and yeast cells were transformed with the pYADE4_CaneCPI-1 construct. To evaluate the effect on fermentation ability, the transformed strain was used in the fermentation process with cell recycling. During the nine-hour fermentative cycles the transformed strain did not have its viability and fermentation ability affected. In the last cycle, when the fermentation lasted longer, the protein was expressed probably at the expense of ethanol once the sugars were exhausted. The recombinant protein was expressed in yeast cells, purified and submitted to assays of activity that demonstrated its functionality. Thus, the industrial PE-2 strain of S. cerevisiae can be used as a viable system for protein expression and to produce alcohol simultaneously. The findings of the present study demonstrate the possibility of producing recombinant proteins with biotechnological applications during the ethanol fermentation process.
酿酒酵母是乙醇发酵过程中最重要的微生物。由于其高发酵能力，该酵母的 PE-2 菌株在巴西被广泛用于生产酒精。本研究旨在开发一种使用工业酿酒酵母 PE-2 菌株在酒精发酵过程中表达重组蛋白的表达系统。该系统选择 caneCPI-1 作为模型蛋白，caneCPI-1 是一种半胱氨酸蛋白酶抑制剂。构建了含有 caneCPI-1 基因的质粒，并将 pYADE4_caneCPI-1 构建体转化到酵母细胞中。为了评估对发酵能力的影响，使用具有细胞回收的转化菌株进行发酵过程。在九个小时的发酵周期中，转化菌株的活力和发酵能力没有受到影响。在最后一个周期中，当发酵时间延长时，由于糖耗尽，可能会以牺牲乙醇的代价表达蛋白质。重组蛋白在酵母细胞中表达、纯化并进行活性测定，证明其具有功能。因此，工业酿酒酵母 PE-2 菌株可同时用于表达蛋白和生产酒精。本研究的结果表明，在乙醇发酵过程中生产具有生物技术应用的重组蛋白是可行的。
