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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Main Authors: Yuqi Zhu, Pengdong Sun, Chunjian Li, Yu Zhang, Yu Wang, Jingyuan Li, Yanfeng Liu, Jian Chen, Yang Deng
Format: Article
Language:English
Published: Tsinghua University Press 2024-07-01
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.
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institution Kabale University
issn 2097-0765
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publishDate 2024-07-01
publisher Tsinghua University Press
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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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