Cloning, sequencing and expression of L-lactate dehydrogenase gene from clostridium thermocellum and isolation, characterization and transformation of various cellulolytic, thermophilic, ethanol-producing bacteria

Özkan, Melek
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 reading frame of 948 base pairs. The amino acid sequence of the C. thermocellum LDH showed high homology to those of Clostridium perfringens, Bacillus stearothermophilus, Thermus caldophilus, and Lactobacillus casei, with respective identities of 57.1 %, 48.9 %, 41.5 %, and 47.1 %. The low G+C ratio (40 %) of the C. thermocellum genome affects codon usage, 111thus codons ending with A or T at the third position are mostly utilized in the C. thermocellum Idh. A putative ribosomal binding site and promoter region located in the 5' flanking region show high similarity to those of other clostridial genes. The gene was successfully expressed in E. coli FMJ39 {Idh mutant) under the lac promoter. C. thermocellum LDH was shown to have a highly reversible activity and to be an allosteric enzyme which is activated by fructose 1,6 diphosphate. As the second part of this research, various thermophilic, cellulolytic, ethanol producers isolated from different environmental samples were characterized in the context of biomass conversion processes; transformability and high ethanol selectivity were the main issues. Transformation efficiencies of the isolates were investigated since the genetic transformation of the type strains of C. thermocellum has not been achieved to date. Two previously described strains and thirteen newly isolated strains were also compared with respect to rates of cellulose degradation, antibiotic sensitivity, morphology and fermentation endproducts. Phylogenetic relationships of the isolates with C. thermocellum were detected using primers designed for 16SrRNA. Homology in the sequences of hydrogenase and phosphotransacetylase/acetate kinase genes were also compared by PCR. Two strains out of thirteen (7-10-1, 7-12-1) did not give any amplicon with the primers used for most of these genes, indicating that they are probably phylogenetically distant from C. thermocellum. Homologs of the cellulose binding domains (CBDs) of cellulosomes of the cellulolytic isolates were amplified by PCR. Sequence analysis of the amplicons for 9 strains showed that the differences in cellulose degradation capacity of the natural isolates was not directly linked to the amino acid sequence of CBDs. Attempts were also made to transform the isolates by PEG electroporation but none of them was transformable. A technique called "mixed culture transformation" was developed with the aim of isolating competent strains of thermophilic, ethanol producing, cellulolytic bacteria which are apparently extremely rare in nature. Although we could not isolate a cellulolytic strain transformable by our techniques, we obtained thermophilic, anaerobic, competent bacteria. It was shown that plasmid pHV33 rearranged in thermophilic anaerobic transformant. Restriction mapping of IVthe rearranged plasmid indicated that four restriction sites were unchanged while many restriction sites had been lost or formed.


Thermostability and regulation of Clostridium thermocellum L-lactate dehydrogenase expressed in Escherichia coli
Assa, P; Ozkan, M; Özcengiz, Gülay (2005-01-01)
In this study, L-lactate dehydrogenase (L-LDH) of Clostridium thermocellum previously cloned aid expressed in Escherichia coli FMJ39 was partially purified and characterised. Optimum temperature and pH of the enzyme were found as 50 degrees C and 7.5, respectively. Different concentrations of Mn2+ did not affect the enzyme activity. Addition of 20-30 mM Mg2+, or the other hand, increased the LDH activity by about 10%. Relatively high concentrations of NaCl (2 M), fructose-1,6-diphosphate (FDP, 5 mM), ATP (1...
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In Streptomyces clavuligerus, the route to the biosynthesis of a-aminoadipic acid (a-AAA) represents an important primary metabolic pathway providing carbon flux to the synthetases of antibiotic formation. This carbon flow comes through the lysine-specific branch of the aspartate pathway and is rate limiting in the formation of cephamycin C, a second generation cephalosporin produced by this organism. In this study, the lysA gene which encodes for an important key enzyme of aspartate pathway; meso-diaminopi...
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Characterization and purification of a bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris
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In this study, a new bacteriocin isolated from a Leuconostoc mesenteroides subsp. cremoris strain was purified to homogeneity by pH mediated cell adsorption-desorption, solid phase extraction with Amberlite XAD-16, cation-exchange chromatography and hydrophobic interaction chromatography. The purification resulted in an electrophoretically pure protein that was smaller than 6 kDa as judged by SDS-PAGE. The bacteriocin was found to be very hydrophobic and cationic and could be adsorbed by synthetic calcium s...
Citation Formats
M. Özkan, “Cloning, sequencing and expression of L-lactate dehydrogenase gene from clostridium thermocellum and isolation, characterization and transformation of various cellulolytic, thermophilic, ethanol-producing bacteria,” Ph.D. - Doctoral Program, Middle East Technical University, 2002.