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Developing a RecT-assisted endogenous CRISPR/SzCas9 system for precise genome editing in Streptococcus zooepidemicus
Xuejing Zhang1, Feiyu Wang1, Xueping Guo2
1State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People's Republic of China.
International Journal of Biological Macromolecules
|December 14, 2024
Summary
Researchers developed a new CRISPR gene editing system for Streptococcus zooepidemicus, improving hyaluronic acid production. This system enables precise genetic modifications, leading to enhanced yields and safer products for industrial applications.
Area of Science:
- Microbiology
- Biotechnology
- Genetic Engineering
Background:
- Streptococcus zooepidemicus is key for industrial hyaluronic acid (HA) production.
- Limited genetic tools hinder S. zooepidemicus strain improvement for HA manufacturing.
Purpose of the Study:
- To develop an efficient, markerless gene editing system for industrial S. zooepidemicus strains.
- To engineer S. zooepidemicus for enhanced hyaluronic acid production and improved safety profiles.
Main Methods:
- Developed a RecT-assisted endogenous CRISPR/SzCas9 system for S. zooepidemicus.
- Performed markerless gene deletion (hylb), gene substitution (VHb), and stop codon insertion (sagA).
- Iterative editing was employed to achieve desired genetic modifications.
Main Results:
- Successfully engineered a non-hemolytic S. zooepidemicus strain (SD-3) with reduced toxicity.
- Achieved a 39.7% increase in hyaluronic acid production, reaching 10.8 g/L.
- Produced high molecular weight hyaluronic acid (up to 2.8 x 10^6 Da) with uniform characteristics.
Conclusions:
- The novel CRISPR/SzCas9 system provides efficient genetic manipulation for S. zooepidemicus.
- Engineered strain SD-3 demonstrates significant potential for industrial hyaluronic acid production with enhanced yield and safety.
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