Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum

Özcengiz, Gülay
Lin, WR
Ozcengiz, E
Westenberg, D
Lynd, LR
Demain, AL
Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that region.


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In this study, the structural gene for L-lactate dehydrogenase (LDH; EC. 1.1. 1.27) was cloned and characterized for the first time from Clostridium thermocellum 27405 in Escherichia coli. A 357 bp PCR product of predicted size was obtained with degenerate primers designed for conserved regions of Idh genes of different organisms. This amplicon was used as a probe to screen a Lambda Zap II phage library of C. thermocellum genomic DNA. One positive clone contained an insert of 2.5 kb which included an open r...
Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization
Ozkan, M; Yilmaz, EI; Lynd, LR; Özcengiz, Gülay (Canadian Science Publishing, 2004-10-01)
The structural gene for L-lactate dehydrogenase (LDH) (EC. from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap 11 phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. ...
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Citation Formats
G. Özcengiz, W. Lin, E. Ozcengiz, D. Westenberg, L. Lynd, and A. Demain, “Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum,” JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, pp. 145–149, 1998, Accessed: 00, 2020. [Online]. Available: