文献类型：期刊文献

英文题名：Development and characterization of a Cre/loxP toolkit for genome engineering in Komagataella phaffii

作者：Song, Lili[1];Hua, Li[1];Wu, Yanyan[1];Jiang, Xinyu[1];Liu, Qi[1];Zheng, Yitao[1];Cai, Menghao[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2027

卷号：15

起止页码：113

外文期刊名：SYNTHETIC AND SYSTEMS BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:001826124500001)】；

基金：This work was supported by the Shanghai Explorer Program (24TS1411600) , National Key R&D Program of China (2022YFC2805102) , and Shanghai Agricultural Science and Technology Innovation Project (K2025017) . We thank Professor Shuli Liang from South China University of Technology for generously providing the pGACH plasmid used in this study.

语种：英文

外文关键词：Komagataella phaffii; Cre/loxP; Genome engineering; LoxP variant screening; Orthogonal loxP variants

摘要：The Cre/loxP system is a versatile tool for genome engineering, yet its application in the industrial yeast Komagataella phaffii has been limited primarily to marker recycling. This study reports a comprehensive Cre/loxP toolkit for K. phaffii. The N-terminal SV40 NLS fusion (NLS-Cre) was identified as the optimal expression strategy balancing high recombination efficiency (24.6%) with minimal cellular toxicity. Further, site-specific integration, inversion, and replacement were experimentally validated, demonstrating multiple recombination functionalities of the Cre/loxP systems in this host. High-throughput screening of 60 loxP variants revealed a structure-activity relationship, that is, spacer mutations frequently retained high recombination activity, whereas inverted repeat mutations uniformly abolished function. Therein, 12 variants with efficiency over 65% were identified, including five exceeding 80% (lox2271, loxm52, loxm71, lox514, and lox5171). Orthogonality assessment identified four variants (loxm72, loxm41, lox5272, loxm71) highly orthogonal to the wild-type loxP, offering potential for sequential genome engineering in strains with pre-existing sites. The obtained toolkit provides the first systematic validation of multiple recombination functions and loxP variants in K. phaffii, offering foundational components for genome engineering in this industrial strain.