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Efficient Production of an Alginate Lyase in Bacillus subtilis with Combined Strategy: Vector and Host Selection, Promoter and Signal Peptide Screening, and Modification of a Translation Initiation Region.
- Source :
-
Journal of agricultural and food chemistry [J Agric Food Chem] 2024 Sep 04; Vol. 72 (35), pp. 19403-19412. Date of Electronic Publication: 2024 Aug 24. - Publication Year :
- 2024
-
Abstract
- Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis ( B. subtilis ), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, P <subscript>nprE</subscript> exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SP <subscript>ydhT</subscript> demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis . These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis .
- Subjects :
- Vibrio genetics
Vibrio enzymology
Genetic Vectors metabolism
Genetic Vectors genetics
Genetic Vectors chemistry
Peptide Chain Initiation, Translational
Bacillus subtilis genetics
Bacillus subtilis enzymology
Bacillus subtilis metabolism
Promoter Regions, Genetic
Polysaccharide-Lyases genetics
Polysaccharide-Lyases metabolism
Polysaccharide-Lyases chemistry
Bacterial Proteins genetics
Bacterial Proteins metabolism
Bacterial Proteins chemistry
Protein Sorting Signals genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1520-5118
- Volume :
- 72
- Issue :
- 35
- Database :
- MEDLINE
- Journal :
- Journal of agricultural and food chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 39180506
- Full Text :
- https://doi.org/10.1021/acs.jafc.4c03532