[Construction of a Kluyveromyces lactis strain with multi-copy integration for enhanced bovine chymosin production by CRISPR/Cas9 and UV mutagenesis].
Sheng Wu Gong Cheng Xue Bao
; 40(9): 2983-2997, 2024 Sep 25.
Article
in Zh
| MEDLINE
| ID: mdl-39319719
ABSTRACT
Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcymD, and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcymD-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Ultraviolet Rays
/
Kluyveromyces
/
Mutagenesis
/
Chymosin
/
CRISPR-Cas Systems
Limits:
Animals
Language:
Zh
Journal:
Sheng Wu Gong Cheng Xue Bao
Year:
2024
Document type:
Article