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Title 

Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria

Authors 

Y S JangY R JungS Y LeeJ M KimJ W LeeDoo-Byoung OhHyun Ah KangOh Suk KwonS H JangH SongSang Jun LeeK Y Kang

Publisher 

American Society for Microbiology

Issue Date 

2007

Citation 

Applied and Environmental Microbiology, vol. 73, no. 17, pp. 5411-5420

Keywords 

organic acidssuccinic acidcoliform bacteriumorganic acidbacterial genebacterium culturemannheimia succiniciproducensrumen microorganismshuttle vectorgenetic vectors

Abstract 

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 × 106 and 7.1 × 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.

ISSN 

0099-2240

Link 

http://dx.doi.org/10.1128/AEM.01382-07

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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