Shen Qi, Yu Zhuang, Zhou Xiao-Ting, Zhang Shi-Jia, Zou Shu-Ping, Xiong Neng, Xue Ya-Ping, Liu Zhi-Qiang, Zheng Yu-Guo
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Appl Microbiol Biotechnol. 2021 Apr;105(8):3211-3223. doi: 10.1007/s00253-021-11195-0. Epub 2021 Apr 5.
Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (P). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (P) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the P has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• P was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by P was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the P for driving antibiotic-resistant genes.
在毕赤酵母中高效表达异源蛋白的常规方法包括使用强甲醇调控的乙醇氧化酶（AOX1）启动子和多个表达盒插入片段。为了筛选含有多个整合片段的转化子，将抗抗生素基因（如印度链霉菌博来霉素基因）构建到表达载体中，因为表达载体上抗抗生素基因的插入数量越多，转化子对更高浓度抗生素的抗性就越强。抗抗生素基因通常由强组成型翻译延伸因子1a启动子（P）驱动。然而，抗抗生素蛋白仅在筛选过程中是必需的。它们在异源蛋白表达过程中的产生可能会增加细胞负担，特别是对于含有多个抗抗生素基因表达盒拷贝的高拷贝菌株。此外，由于TEF1启动子对抗抗生素基因的有效表达，选择多个插入片段需要高浓度的昂贵抗生素。为了解决这些限制，我们用一个较弱的启动子（P）取代了TEF1启动子，该启动子来源于2-脱氧葡萄糖-6-磷酸磷酸酶基因的潜在启动子区域，用于驱动抗抗生素基因的表达。重要的是，与通常用于筛选过程的葡萄糖相比，在基于AOX1启动子生产重组蛋白所使用的碳源（甘油和甲醇）下，P的活性更低。通过使用含有100μg/ml博来霉素的平板，该策略已成功筛选出含有超过3个目的基因拷贝的转化子。同时，在这些多拷贝菌株的异源蛋白表达过程中，通过免疫印迹检测不到博来霉素抗性蛋白的水平。
关键点
• P被鉴定为一种新型葡萄糖调控启动子。
• P驱动的抗抗生素基因表达在重组蛋白表达过程中受到抑制，从而减轻了代谢负担。
• 使用P驱动抗抗生素基因，经济高效地筛选出了含有多个整合片段的转化子。
