Monday, May 5, 2008
7-15

Development of shuttle vectors for succinic acid producing rumen bacteria

Yu-Sin Jang, Jeong Wook Lee, and Sang Yup Lee. Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, Department of BioSystems and Bioinformatics Research Center, Institute for the Biocentury, Korea Advanced Institute of Science and Technology (KAIST), 335 Gwahangno, Yuseong-gu, Daejeon, South Korea

Shuttle vectors carrying the origins of replication that function in Escherichia coli and two succinic acid producing capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. They were found to be stably maintained with 10 copies number in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 120 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0×106 and 7.1×106 transformants/microgram DNA were obtained in M. succiniciproducens and A. succinogenes, respectively. The 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 conformed 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. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (2005-01294). Further supports by the LG Chem Chair Professorship, IBM SUR program, Microsoft, and by the KOSEF through the Center for Ultramicrochemical Process Systems are appreciated.].

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