Molecular characterization and utilization of the CAK1 filamentous viruslike particle derived from Clostridium beijerinckii |
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Authors: | Li Y Blaschek H P Tan D |
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Institution: | (1) Biotechnology and Bioengineering Group, Department of Food Science and Human Nutrition, University of Illinois, 1207 West Gregory Drive, Urbana, IL 61801, USA, US |
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Abstract: | An examination of the replication origin and stability determinant associated with the CAK1 filamentous viruslike particle
recovered from Clostridium beijerinckii NCIMB 6444 was carried out. Seven deletion derivatives, pCKE, pCEP1, pDT5, pCKP, pDTH102, pYL102E and pYL102, were constructed
and transformed into C. beijerinckii NCIMB 8052. The successful transformation of pCKE, pDT5, pCKP, pDTH102, pYL102E and pYL102 into C. beijerinckii 8052, together with the corresponding recovery of single-stranded DNA from Escherichia coli indicated that the double- and single-stranded replication origins are located on a 0.4-kb CAK1 DNA fragment. Sequence analysis
of the putative 0.4-kb replication origin region of CAK1 reveals a nick site containing 22 base pairs that has homology with
plasmids pC194 and pUB110 and suggests the presence of a 2.0-kb DNA region involved in stability. The putative Rep protein
of CAK1 contains three conserved motifs and three essential residues of the catalytic site in agreement with Rep proteins
associated with the pC194 family. The utility of the developed CAK1-derived phagemid designated pYL102E was evaluated by using
it to examine heterologous expression of: (1) the manA gene derived from Thermoanaerobacterium polysaccharolyticum in E. coli and C. beijerinckii NCIMB 8052 and (2) the sol operon derived from Clostridium acetobutylicum DSM 792 in C. beijerinckii SA-2. Journal of Industrial Microbiology & Biotechnology (2002) 28, 118–126 DOI: 10.1038/sj/jim/7000225
Received 12 September 2000/ Accepted in revised form 23 October 2001 |
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Keywords: | : Clostridium beijerinckii shuttle vectors CAK1 viruslike particle DNA |
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