Facultad de Ciencias Biológicas, Instituto de Biotecnología, Universidad Autónoma de Nuevo León UANL, Av. Universidad S/N Col. Ciudad Universitaria, 66455, San Nicolás de los Garza, Nuevo León, Mexico.
World J Microbiol Biotechnol. 2023 Jul 8;39(9):246. doi: 10.1007/s11274-023-03682-5.
The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P) and transcriptional terminator (T) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P-T and P-T pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.
甲醇营养型酵母毕赤酵母（同义词：巴斯德毕赤酵母）是一种广泛用于通过整合到酵母基因组中的表达盒来胞外生产异源蛋白的宿主。表达盒中的强启动子并不总是异源蛋白生产的最佳选择，特别是如果蛋白质的正确折叠和/或翻译后加工是限制步骤。转录终止子是表达盒中的另一个调节元件，可改变异源基因的表达水平。在这项工作中，我们鉴定并功能表征了一个组成型基因（即 1033 基因）的启动子（P）和转录终止子（T），其具有较弱的非甲醇依赖转录活性。我们构建了两个毕赤酵母菌株，它们具有来自 1033 和 AOX1 基因的调节 DNA 元件的两种组合（即 P-T 和 P-T 对），并评估了调节元件组合对异源基因和内源性 1033 和 GAPDH 基因在葡萄糖或甘油中生长的细胞中的转录水平，以及对细胞外产物/生物量产量的影响。结果表明，P 具有 GAP 启动子 2-3%的转录活性，并且可通过细胞生长和碳源进行调节。调节元件的组合使异源和内源性基因具有不同的转录活性，这取决于碳源。启动子-终止子对和碳源影响异源基因的翻译和/或蛋白质分泌途径。此外，低水平的异源基因转录水平与甘油培养一起增加了翻译和/或蛋白质分泌。
