ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Microbiotechnology
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1605229
Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production
Provisionally accepted
- Wuhan Polytechnic University, Wuhan, China
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, primarily sourced from calf abomasum, serves as a 删除了: employed 27 删除了: enhance 28 删除了: production 29 删除了: preparation 30 删除了:conventional coagulant in milk curdling during cheese production. To improve the enzymatic properties and enhance the expression of calf chymosin (BtChy) in Kluyveromyces lactis to meet the demands of the cheese industry, the in silico engineering via hotspot scanning and molecular dynamics analysis was adopted. This approach improved the activity of BtChy on milk curdling and increased its sensitivity at 65°C. Multiple strategies were utilized to develop an environmentally friendly method for chymosin production. These included screening for constitutive promoters and signal peptides, as well as in vitro construction of a concatemer of the BtChy gene.The optimal combination, comprising the PTDH3 promoter, invertase signal peptide, and a four-copy BtChy gene integrated into the yeast genome, was identified. After highdensity cultivation in a 5-L bioreactor, the recombinant yeast achieved an activity of 42,000 SU/mL, a 52.5-fold increase over the original wild-type chymosin gene.
Keywords: Calf chymosin, in silico engineering, signal peptide, Promoter, Gene Dosage
Received: 03 Apr 2025; Accepted: 14 May 2025.
Copyright: © 2025 Han, Zhang, Rao, Lei and Yang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Jiang-Ke Yang, Wuhan Polytechnic University, Wuhan, China
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