Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

다국어 초록 (Multilingual Abstract)

<B>ABSTRACT</B><P>Shuttle vectors carrying the origins of replication that function in <I>Escherichia coli</I> and two capnophilic rumen bacteria, <I>Mannheimia succiniciproducens</I> and <I>Actinobacillus succinogenes</I>, 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 × 10<SUP>6</SUP> and 7.1 × 10<SUP>6</SUP> transformants/μg DNA were obtained with <I>M. succiniciproducens</I> and <I>A. succinogenes</I>, respectively. A 1.7-kb minimal replicon was identified that consists of the <I>rep</I> gene, four iterons, A+T-rich regions, and a <I>dnaA</I> 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 <I>fumC</I> 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.</P>