Engineering sigma factors and chaperones for enhanced heterologous lipoxygenase production in Escherichia coli.
High-throughput screening
Molecular chaperone
Soluble expression
σ factor
Journal
Biotechnology for biofuels and bioproducts
ISSN: 2731-3654
Titre abrégé: Biotechnol Biofuels Bioprod
Pays: England
ID NLM: 9918300888906676
Informations de publication
Date de publication:
10 Oct 2022
10 Oct 2022
Historique:
received:
21
06
2022
accepted:
30
09
2022
entrez:
10
10
2022
pubmed:
11
10
2022
medline:
11
10
2022
Statut:
epublish
Résumé
Lipoxygenase (EC. 1.13.11.12, LOX) can catalyze the addition of oxygen into polyunsaturated fatty acids to produce hydroperoxides, which are widely used in the food, chemical, and pharmaceutical industries. In recent years, the heterologous production of LOX by Escherichia coli has attracted extensive attention. However, overexpressed recombinant LOX in E. coli aggregates and forms insoluble inclusion bodies owing to protein misfolding. In this study, a split green fluorescent protein-based screening method was developed to screen sigma (σ) factors and molecular chaperones for soluble LOX expression. Three mutant libraries of Skp, GroES, and RpoH was analyzed using the high-throughput screening method developed herein, and a series of mutants with significantly higher yield of soluble heterologous LOX were obtained. The soluble expression level of LOX in the isolated mutants increased by 4.2- to 5.3-fold. Further, the highest LOX activity (up to 6240 ± 269 U·g-DCW In this study, a high-throughput screening method was developed for improved soluble LOX expression. The obtained positive mutants of the regulatory factor were analyzed and employed for the expression of other heterologous proteins, thus providing a potential solution for the inclusion-body protein.
Sections du résumé
BACKGROUND
BACKGROUND
Lipoxygenase (EC. 1.13.11.12, LOX) can catalyze the addition of oxygen into polyunsaturated fatty acids to produce hydroperoxides, which are widely used in the food, chemical, and pharmaceutical industries. In recent years, the heterologous production of LOX by Escherichia coli has attracted extensive attention. However, overexpressed recombinant LOX in E. coli aggregates and forms insoluble inclusion bodies owing to protein misfolding.
RESULTS
RESULTS
In this study, a split green fluorescent protein-based screening method was developed to screen sigma (σ) factors and molecular chaperones for soluble LOX expression. Three mutant libraries of Skp, GroES, and RpoH was analyzed using the high-throughput screening method developed herein, and a series of mutants with significantly higher yield of soluble heterologous LOX were obtained. The soluble expression level of LOX in the isolated mutants increased by 4.2- to 5.3-fold. Further, the highest LOX activity (up to 6240 ± 269 U·g-DCW
CONCLUSION
CONCLUSIONS
In this study, a high-throughput screening method was developed for improved soluble LOX expression. The obtained positive mutants of the regulatory factor were analyzed and employed for the expression of other heterologous proteins, thus providing a potential solution for the inclusion-body protein.
Identifiants
pubmed: 36217152
doi: 10.1186/s13068-022-02206-x
pii: 10.1186/s13068-022-02206-x
pmc: PMC9552429
doi:
Types de publication
Journal Article
Langues
eng
Pagination
105Subventions
Organisme : National Natural Science Foundation of China
ID : 32172153
Organisme : National Key Research and Development Program of China
ID : 2019YFA0904900
Organisme : Natural Science Foundation of Jiangsu Province
ID : BK20202002
Informations de copyright
© 2022. The Author(s).
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