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Published on: January 11, 2011
Development and characterization of a Cre/loxP toolkit for genome engineering in Komagataella phaffii
Lili Song1, Li Hua1, Yanyan Wu1
1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
None:
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.
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