1. Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli
- Author
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Seok-Hyun Hong, Sun-Ki Kim, Tae-Gu Kang, Yung-Hun Yang, and Gi-Beom Jeon
- Subjects
Signal peptide ,DNA, Bacterial ,Glycoside Hydrolases ,Caldicellulosiruptor ,Bioengineering ,Carboxypeptidases ,Peptidoglycan ,Protein Sorting Signals ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,chemistry.chemical_compound ,Industrial Microbiology ,Bacterial Proteins ,Cellulase ,Protein Domains ,medicine ,Extracellular ,Escherichia coli ,Glycoside hydrolase ,Secretion ,Cellulose ,Caldicellulosiruptor bescii ,Signal recognition particle ,Secretory Pathway ,biology ,urogenital system ,biology.organism_classification ,Recombinant Proteins ,Protein Transport ,Biochemistry ,chemistry ,Fermentation ,Mutation ,Signal Recognition Particle ,Biotechnology - Abstract
Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.
- Published
- 2020