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Applied and Environmental Microbiology, August 2007, p. 4849-4856, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00824-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Ryoma Miyake,1 Jun Kawamoto,1 Yun-Lin Wei,1 Masanari Kitagawa,2 Ikunoshin Kato,2 Tatsuo Kurihara,1* and Nobuyoshi Esaki1*

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan,1 Takara Bio Inc., Seta 3-4-1, Otsu, Shiga 520-9143, Japan2

Received 11 April 2007/ Accepted 18 May 2007

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 ß-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.

* Corresponding author. Mailing address: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Phone for T. Kurihara: 81-774-38-4710. Phone for N. Esaki: 81-774-38-3240. Fax: 81-774-38-3248. E-mail for T. Kurihara: kurihara{at}scl.kyoto-u.ac.jp. E-mail for N. Esaki: esakin{at}scl.kyoto-u.ac.jp

{triangledown} Published ahead of print on 25 May 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, August 2007, p. 4849-4856, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00824-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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