Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening
Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA...
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Tsinghua University Press
2024-07-01
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| Series: | Food Science and Human Wellness |
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| Online Access: | https://www.sciopen.com/article/10.26599/FSHW.2022.9250192 |
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| author | Yuqi Zhu Pengdong Sun Chunjian Li Yu Zhang Yu Wang Jingyuan Li Yanfeng Liu Jian Chen Yang Deng |
| author_facet | Yuqi Zhu Pengdong Sun Chunjian Li Yu Zhang Yu Wang Jingyuan Li Yanfeng Liu Jian Chen Yang Deng |
| author_sort | Yuqi Zhu |
| collection | DOAJ |
| description | Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (PaprE, P43, and Pglv) were compared and applied. The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk. |
| format | Article |
| id | doaj-art-d865c495d0634005afe567830f0cf556 |
| institution | Kabale University |
| issn | 2097-0765 2213-4530 |
| language | English |
| publishDate | 2024-07-01 |
| publisher | Tsinghua University Press |
| record_format | Article |
| series | Food Science and Human Wellness |
| spelling | doaj-art-d865c495d0634005afe567830f0cf5562025-01-10T06:56:48ZengTsinghua University PressFood Science and Human Wellness2097-07652213-45302024-07-011342310231610.26599/FSHW.2022.9250192Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screeningYuqi Zhu0Pengdong Sun1Chunjian Li2Yu Zhang3Yu Wang4Jingyuan Li5Yanfeng Liu6Jian Chen7Yang Deng8College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, ChinaCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, ChinaCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, ChinaCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, ChinaCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, ChinaCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, ChinaQingdao Special Food Research Institute, Qingdao 266109, ChinaQingdao Special Food Research Institute, Qingdao 266109, ChinaCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, ChinaAlpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (PaprE, P43, and Pglv) were compared and applied. The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.https://www.sciopen.com/article/10.26599/FSHW.2022.9250192bacillus subtilisalpha-lactalbuminbioengineering milksignal peptidepromoter screening |
| spellingShingle | Yuqi Zhu Pengdong Sun Chunjian Li Yu Zhang Yu Wang Jingyuan Li Yanfeng Liu Jian Chen Yang Deng Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening Food Science and Human Wellness bacillus subtilis alpha-lactalbumin bioengineering milk signal peptide promoter screening |
| title | Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening |
| title_full | Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening |
| title_fullStr | Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening |
| title_full_unstemmed | Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening |
| title_short | Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening |
| title_sort | enhanced extracellular production of alpha lactalbumin from bacillus subtilis through signal peptide and promoter screening |
| topic | bacillus subtilis alpha-lactalbumin bioengineering milk signal peptide promoter screening |
| url | https://www.sciopen.com/article/10.26599/FSHW.2022.9250192 |
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