Dynamically Regulating Homologous Recombination Enables Precise Genome Editing in Ogataea polymorpha
Xin Ni1,2, Xiaoxin Zhai1,3, Wei Yu1
1Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China.
ACS Synthetic Biology
|September 4, 2024
Summary
This study enhances homologous recombination (HR) in *Ogataea polymorpha* yeast using a novel inducible system. This improves genetic engineering efficiency for producing valuable chemicals without impacting cell growth or fitness.
Area of Science:
- Synthetic biology
- Microbial biotechnology
- Molecular genetics
Background:
- *Ogataea polymorpha* is a promising yeast cell factory for chemical production.
- Low homologous recombination (HR) efficiency limits its metabolic engineering potential.
- Constitutive overexpression of HR genes causes cellular stress and reduces productivity.
Purpose of the Study:
- To engineer a dynamically regulated HR repair pathway in *O. polymorpha*.
- To improve genetic manipulation efficiency without compromising cellular fitness.
- To enhance bioproduction titers using the engineered system.
Main Methods:
- Constructed a CRISPR-Cas9 system in *O. polymorpha*.
- Engineered an HR pathway using *ScRAD51* under an l-rhamnose-inducible promoter (PLRA3).
- Evaluated HR rates, cell growth, and fatty alcohol titers under inducible conditions.
Main Results:
- Achieved up to 60% HR rates, a 10-fold increase over wild-type.
- Demonstrated no detectable influence on cell growth in methanol.
- The dynamic system yielded 50% higher fatty alcohol titers compared to static regulation.
Conclusions:
- Developed a feasible platform for convenient genetic manipulation in *O. polymorpha*.
- The inducible HR system enhances efficiency without perturbing cellular fitness.
- This approach facilitates advanced metabolic engineering for industrial bioproduction.
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