Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

• Published in: Applied and Environmental Microbiology Vol. 73; no. 15; pp. 4849 - 4856
• Main Authors: Miyake, Ryoma, Kawamoto, Jun, Wei, Yun-Lin, Kitagawa, Masanari, Kato, Ikunoshin, Kurihara, Tatsuo, Esaki, Nobuyoshi
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.08.2007
• Subjects: Adaptation, Physiological; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Biotechnology - methods; Cold Temperature; Deltaproteobacteria - enzymology; Deltaproteobacteria - genetics; Desulfotalea psychrophila; DNA Primers; Enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Glucosidases - genetics; Glucosidases - metabolism; Heat-Shock Response; Microbiology; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; Physiology and Biotechnology; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Shewanella; Shewanella - enzymology; Shewanella - genetics; Shewanella - physiology; Temperature; Cold; Vibrionaceae; Bacteria; Gene expression; Shewanella; Protein; Low temperature
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.00824-07

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.