Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production
Chymosin (EC3.4.23.4), primarily sourced from calf abomasum, serves as a 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 i...
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| Format: | Article |
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Frontiers Media S.A.
2025-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1605229/full |
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| author | Ying Han Liu-Qun Zhang De-Ming Rao Lei Lei Jiang-Ke Yang |
| author_facet | Ying Han Liu-Qun Zhang De-Ming Rao Lei Lei Jiang-Ke Yang |
| author_sort | Ying Han |
| collection | DOAJ |
| description | Chymosin (EC3.4.23.4), primarily sourced from calf abomasum, serves as a 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 high-density 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. |
| format | Article |
| id | doaj-art-837619b7d0cd46f89faf03e753c02c3c |
| institution | DOAJ |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-837619b7d0cd46f89faf03e753c02c3c2025-08-20T03:21:31ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-05-011610.3389/fmicb.2025.16052291605229Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese productionYing HanLiu-Qun ZhangDe-Ming RaoLei LeiJiang-Ke YangChymosin (EC3.4.23.4), primarily sourced from calf abomasum, serves as a 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 high-density 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.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1605229/fullcalf chymosinin silico engineeringsignal peptidepromotergene dosage |
| spellingShingle | Ying Han Liu-Qun Zhang De-Ming Rao Lei Lei Jiang-Ke Yang Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production Frontiers in Microbiology calf chymosin in silico engineering signal peptide promoter gene dosage |
| title | Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production |
| title_full | Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production |
| title_fullStr | Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production |
| title_full_unstemmed | Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production |
| title_short | Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production |
| title_sort | multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin btchy in kluyveromyces lactis for cheese production |
| topic | calf chymosin in silico engineering signal peptide promoter gene dosage |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1605229/full |
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