Cloning and Expression of Clostridium acetobutylicum Endoglucanase, Cellobiase and Amino Acid Biosynthesis Genes in Escherichia coli

Zappe, Harold; Jones, David T.; Woods, David R.

doi:10.1099/00221287-132-5-1367

Cited by 34 publications

(29 citation statements)

References 27 publications

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“…The structure of pEcoR251 (a gift from M. Zabeau, Plant Genetic Systems, Gent, Belgium) is similar to other plasmids utilizing inactivation of the EcoRI endonuclease gene as a selective marker (e.g. Cheng & Modrich, 1983) and has been described previously (Zappe et al, 1986). B.jibrisolvens H17c was grown in M10 medium (Caldwell & Bryant, 1966).…”

Section: Methodsmentioning

confidence: 99%

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a -glucosidase

Lin

Rumbak

Zappe

et al. 1990

Journal of General Microbiology

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The cloning, expression and nucleotide sequence of a 3-74 kb DNA segment on pLS215 containing a P-glucosidase gene (bglA) from Butpivibrio Jibrisolvens H17c was investigated. The B. fibrisolvens bglA open reading frame (ORF) of 2490 bp encoded a P-glucosidase of 830 amino acid residues with a calculated M, of 91800. In Escherichiu coli C6OO(pLS215) cells the P-glucosidase was localized in the cytoplasm and these cells produced an additional protein with an apparent Mr of approximately 94000. The bglA gene was expressed from its own regulatory region in E. coli and a single mRNA initiation point was identified upstream of the bglA ORF and adjacent to a promoter consensus sequence. The primary structure of the P-glucosidase showed > 40 yo similarity with a domain of 237 amino acids present in the P-glucosidases of Kluyveromyces fragilis and Clostridium thermocellum. The B. fibrisolvens P-glucosidase. hydrolysed cellobiose to a limited extent, cellotriose to cellobiose and glucose, and cellotetraose and cellopentaose to predominantly glucose.

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Section: Methodsmentioning

confidence: 99%

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a -glucosidase

Lin

Rumbak

Zappe

et al. 1990

Journal of General Microbiology

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“…Since the 1.4 kb PCR product in pC2-engB contains only the nucleotide sequence of the engB gene together with its putative RBS, the transcription initiation must be from the lac promoter on the pUC vector, although transcription can probably be initiated from a Clostridium promoter sequence by the E . coli RNA polymerase (Zappe et al, 1986).…”

Section: F Foong and Othersmentioning

confidence: 99%

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Foong

Hamamoto²,

Shoseyov³

et al. 1991

Journal of General Microbiology

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An endoglucanase gene, engB, from ClostridYum cellulovorans, previously cloned into pUCl9, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichiu coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosomebinding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulolytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.

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“…Genes involved in amino acid biosynthesis (64), glutamine synthetase (57), alcohol dehydrogenase (62), xylanase (65), endoglucanase (64), and cellobiase (64) have all been cloned from a gene library for C. acetobutylicum P262 by direct selection for expression in E. coli. These results would suggest that E. coli-like promoters and ribosome binding sites are widespread in C. acetobutylicum, as has been reported for a number of genes from other gram-positive bacteria (21,38,40).…”

Section: Resultsmentioning

confidence: 99%

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli

et al. 1991

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A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the i-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-3-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichwa coil. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the ,B-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium 3-galactosidase gene in E. coli was not subject to glucose repression. By using transposon TnS mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for P-galactosidase expression in E.coi. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and KkbsieUa pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus H, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but TnS insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of 13-galactosidase in E. coli.Clostridium acetobutylicum is a gram-positive obligate anaerobe that is able to utilize a variety of carbohydrates as substrates for production of acetone, butanol, and ethanol, commercially important solvents, by way of the ABE fermentation (32). It has been shown that several strains of C. acetobutylicum are able to utilize whey or deproteinized whey, containing lactose, as a substrate for solvent production (16, 36). There are two major pathways for the uptake and utilization of lactose in bacteria, one involving uptake of lactose by ,-galactoside permease followed by hydrolysis of the lactose to glucose and galactose by P-galactosidase and the other involving uptake of the lactose by a lactose phosphoenol pyruvate-dependent phosphotransferase system followed by hydrolysis of the lactose to glucose and galactose-6-phosphate by phospho-,B-galactosidase (12, 43). The latter pathway is common in gram-positive bacteria such as streptococci (8) and staphylococci (12,43). Recent work has shown that C. acetobutylicum strains grown on lactose have both 0-galactosidase and phospho-,-galactosidase activities, each showing a different pattern of induction during the ABE fermentation (63). Levels of phospho-,B-galactosidase were highest during the early acidogenic phase of the ABE fermentation, whereas the ,B-galactosidase was induced later in the ABE fermentation, with maximum levels coinciding w...

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Cloning and Expression of Clostridium acetobutylicum Endoglucanase, Cellobiase and Amino Acid Biosynthesis Genes in Escherichia coli

Cited by 34 publications

References 27 publications

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a -glucosidase

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a -glucosidase

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli

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