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Applied and Environmental Microbiology, September 2007, p. 5411-5420, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.01382-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria{triangledown} ,{dagger}

Yu-Sin Jang,1,4 Young Ryul Jung,3 Sang Yup Lee,1,2* Ji Mahn Kim,1 Jeong Wook Lee,1 Doo-Byoung Oh,3 Hyun Ah Kang,3 Ohsuk Kwon,3 Seh Hee Jang,1 Hyohak Song,1 Sang Jun Lee,1 and Kyu Young Kang4

Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program) and BioProcess Engineering Research Center,1 Department of Biosystems and Bioinformatics Research Center, Korea Advanced Institute of Science and Technology (KAIST), 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea,2 Omics and Integration Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Oun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea,3 Division of Applied Life Science, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, 900 Gajwa-dong, Jinju 600-701, Republic of Korea4

Received 21 June 2007/ Accepted 26 June 2007

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 106 and 7.1 x 106 transformants/µg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

* Corresponding author. Mailing address: Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea. Phone: 82-42-869-3930. Fax: 82-42-869-8800. E-mail: leesy{at}kaist.ac.kr

{triangledown} Published ahead of print on 6 July 2007.

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

Applied and Environmental Microbiology, September 2007, p. 5411-5420, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.01382-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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