The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University, No. 94 Weijin Road, Nankai District, Tianjin, China.
Tianjin Academy of Environmental Sciences, No. 17 Fukang Road, Nankai District, Tianjin, China.
FEMS Yeast Res. 2017 Sep 1;17(6). doi: 10.1093/femsyr/fox064.
In biotechnological industry, increased expression cassette stability and copy number serve as important means of maintaining consistently high production levels of heterologous proteins in Saccharomyces cerevisiae. In this study, we combined δ sequences for site-specific integration with TPI1 gene from Schizosaccharomyces pombe (POT1) as a selection marker to realize high-copy integration and stable expression of heterologous proteins in S. cerevisiae. With the newly developed POT1 platform, a 32-copy integration of enhanced green fluorescent protein (EGFP) expression cassette was obtained in a single round and was stably maintained after 100 generations of growth in a rich complex medium. Talaromyces emersonii cellobiohydrolase I gene was synthesized with S. cerevisiae codon bias and expressed under the control of TPI1 promoter and terminator via POT1-mediated δ-integration; the highest specific activity yielded 238 mU g-1 of dry cell weight when p-nitrophenyl-β-D-cellobioside was used as substrate, whereas the highest activity in cellulose hydrolysis reached 67% Avicel conversion. POT1-mediated δ-integration produces high protein levels over a wide dynamic range and enables broad applications in metabolic engineering and synthetic biology.
在生物技术产业中，提高表达盒的稳定性和拷贝数是维持酿酒酵母中异源蛋白持续高产的重要手段。在本研究中，我们将δ序列与来自裂殖酵母的 TPI1 基因（POT1）结合，作为选择标记，以实现酿酒酵母中异源蛋白的高拷贝整合和稳定表达。利用新开发的 POT1 平台，在一轮中获得了增强型绿色荧光蛋白（EGFP）表达盒的 32 拷贝整合，并在丰富的复杂培养基中生长 100 代后仍能稳定维持。通过 POT1 介导的δ整合，根据酿酒酵母密码子偏好性合成了塔宾曲霉纤维二糖水解酶 I 基因，并在 TPI1 启动子和终止子的控制下表达；当使用对硝基苯基-β-D-纤维二糖苷作为底物时，最高比活达到 238 mU g-1 干细胞重量，而在纤维素水解中最高活性达到 67% Avicel 转化率。POT1 介导的 δ 整合可在较宽的动态范围内产生高蛋白质水平，可广泛应用于代谢工程和合成生物学。
