Department of Organic Chemistry, São Paulo State University (UNESP), Rua Prof. Francisco Degni, 55, Quitandinha, Araraquara 14800-060, Brazil.
Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, Gothenburg 41296, Sweden.
FEMS Yeast Res. 2018 Aug 1;18(5). doi: 10.1093/femsyr/foy036.
A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.
与原核生物相比，酵母中途径表达的复杂性在于需要为每个表达的基因分别使用启动子和终止子。通过使用 2A 病毒肽可以实现单个转录物的表达和分离的蛋白质生产，但需要进行详细的特征分析以评估其适用性和应用。本工作旨在对多种 2A 肽序列进行表征，以确定其在酿酒酵母代谢工程应用中的适用性。我们筛选了放置在荧光蛋白序列之间的 22 个肽。通过对应于报告蛋白的切割和未切割形式的条带的 Western blot 强度计算切割效率。在 22 个序列中有 3 个显示出高切割效率：马鼻炎 B 病毒（91%）、猪口蹄疫病毒-1（85%）和 Operophtera brumata cypovirus-18（83%）的 2A 肽。此外，释放的蛋白质的表达与其单顺反子表达相当。作为概念验证，通过用 ERBV-1 的截断形式连接 HMG1 的形式表达三萜类化合物 friedelin 的合酶，在相同的酵母菌株中成功生产了 friedelin，产量与单顺反子表达相当（通过单独的启动子）。这些结果表明，这些肽可适用于酿酒酵母代谢工程应用中多个蛋白质的表达和翻译。
