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Engineering complex phenotypes in Halomonas bluephagenesis TD01 via large-fragment manipulation and multiplex base
Yu-Hang Zhang1, Yuan Yuan2, Bai-Tao Chen2
1State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, 361102, China; School of Advanced Interdisciplinary Biomedical Sciences, Xiamen University, Xiamen, Fujian, 361102, China.
New genetic tools for Halomonas bluephagenesis enable easier strain development for industrial biotechnology. These advancements in genome engineering facilitate the creation of novel polyhydroxyalkanoate (PHA) copolymers and improve cellular functions.
Area of Science:
- Industrial Biotechnology
- Synthetic Biology
- Microbial Engineering
Background:
- Halomonas bluephagenesis is a key industrial strain for open fermentation due to its tolerance to high salinity and alkalinity.
- Limited genome engineering tools hinder the development of H. bluephagenesis for advanced applications.
Purpose of the Study:
- To develop novel genetic tools for efficient genome engineering in H. bluephagenesis.
- To enable large-fragment manipulation and multiplex base editing for strain improvement.
Main Methods:
- Developed a single-plasmid system (pHaloFM) for sequential insertion/deletion of large DNA fragments using homologous recombination.
- Adapted a CRISPR/nCas9-assisted cytidine base editor (pHaloBE) for multiplex base editing of multiple target sites.
- Applied these tools for engineering cellular morphology and constructing polyhydroxyalkanoate (PHA) biosynthetic pathways.
Main Results:
- The pHaloFM system allows insertion of fragments up to 8 kb and deletion of regions up to 50 kb.
- The pHaloBE system achieved multiplex editing at nine target sites.
- Successfully engineered cellular morphology and constructed P34HB and PHBV PHA copolymer pathways in H. bluephagenesis.
Conclusions:
- The developed toolkit overcomes significant genetic manipulation bottlenecks in H. bluephagenesis.
- Provides a systematic framework for engineering complex phenotypes in non-model organisms.
- Facilitates the advancement of next generation industrial biotechnology (NGIB) through enhanced strain development.
