Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria.
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 10(6) and 7.1 x 10(6) transformants/mug 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.
Jang
Yu-Sin
YS
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program) and BioProcess Engineering Research Center, Republic of Korea.
Jung
Young Ryul
YR
Lee
Sang Yup
SY
Kim
Ji Mahn
JM
Lee
Jeong Wook
JW
Oh
Doo-Byoung
DB
Kang
Hyun Ah
HA
Kwon
Ohsuk
O
Jang
Seh Hee
SH
Song
Hyohak
H
Lee
Sang Jun
SJ
Kang
Kyu Young
KY
D000188:Actinobacillus / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D031121:Mannheimia / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D012417:Rumen / Q000382:microbiology*; D019802:Succinic Acid / Q000378:metabolism*; D014169:Transformation, Bacterial
D019802:Succinic Acid