Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and characterized, we have cloned two additional genes, amyB and amyC, coding for amylases of this thermophile, into Escherichia coli and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coli promoters. Almost all activity was detected in the intracellular fraction in the E. coli cells. The molecular mass and NH2-terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coli transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67,000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59,000). The optimal temperatures for AmyB and AmyC activities on soluble starch were 80 degrees C and 70 degrees C, respectively. Both AmyB and AmyC showed a pH optimum of 5.5. AmyB and AmyC showed a different pattern of starch hydrolysis when examined by thin-layer chromatography. Some homology in the amino acid sequences with the functional regions of Taka-amylase A was found in both AmyB and AmyC. The codon usage in the amyA, amyB and amyC genes was highly biased, which reflects the fact that the guanine-plus-cytosine (G + C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the codons was non-random; the G + C content of the first position of codons is significantly high, whereas that of the third position is somewhat low. In addition, codons consisting only of A and T were preferentially used in this thermophile.     

Transglycosylation activity of Dictyoglomus thermophilum amylase A

Amylase A from Dictyoglomus thermophilum is a thermophilic enzyme and has about 40% identity with 4-alpha-glucanotransferase (GTase) from Thermococcus litoralis, and both of these enzymes belong to family 57 glycosyl hydrolase. Since the transglycosylation activity of T. litoralis GTase has been well characterized, the substrate specificity and reaction products of amylase A from D. thermophilum were examined. alpha-1,4 Glucan was produced from maltooligosaccharides, and glucoamylase-resistant molecules (cycloamyloses) were produced from longer chain amylose (average molecular mass 200 kDa). It has been reported that amylase A from D. thermophilum hydrolyzes starch, but in this study it was found that the enzyme was also able to use maltooligosaccharides and long chain amylose as substrate and has transglycosylation activity.     

Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate.

A genomic library of the Dictyoglomus sp. strain Rt46B.1 was constructed in the phage vector lambda ZapII and screened for xylanase activity. A plaque expressing xylanase activity, designated B6-77, was isolated and shown to contain a genomic insert of 5.3 kb. Subcloning revealed that the xylanase activity was restricted to a internal 1,507-bp PstI-HindIII fragment which was subsequently sequenced and shown to contain a single complete open reading frame coding for a single-domain xylanase, XynA, with a putative length of 352 amino acids. Homology comparisons show that XynA is related to the family F group of xylanases. The temperature and pH optima of the recombinant enzyme were determined to be 85 degrees C and pH 6.5, respectively. However, the enzyme was active across a broad pH range, with over 50% activity between pH 5.5 and 9.5. XynA was shown to be a true endo-acting xylanase, being capable of hydrolyzing xylan to xylotriose and xylobiose, but it could not hydrolyze xylobiose to monomeric xylose. XynA was also shown to hydrolyze xylan present in Pinus radiata kraft pulp, indicating that it may be of use as an aid in pulp bleaching. The equivalent xylanase gene was also isolated from the related bacterium Dictyoglomus thermophilum, and DNA sequencing showed these genes to be identical, which, together with the 16S small-subunit rRNA gene sequencing data, indicates that Rt46B.1 and D. thermophilum are very closely related.     

Cloning, nucleotide sequences, and overexpression in Escherichia coli of tandem copies of a tryptophanase gene in an obligately symbiotic thermophile, Symbiobacterium thermophilum.

Symbiobacterium thermophilum, a thermophilic bacterium, is a thermostable tryptophanase producer that can grow only in coculture with a specific Bacillus strain. Two thermostable tryptophanase genes, tna-1 and tna-2, that are located close to each other were cloned into Escherichia coli from S. thermophilum by the DNA-probing method. The nucleotide and deduced amino acid sequences indicate that Tna1 and Tna2 share 92% identical amino acids in a total of 453 amino acids. By means of DNA manipulation with E. coli host-vector systems, Tna1 and Tna2 were produced in very large amounts in enzymatically active forms. Comparison of the NH2-terminal amino acid sequences and the enzymatic properties of the tryptophanases purified from the original S. thermophilum strain and these two tryptophanases from recombinant E. coli cells suggest that in S. thermophilum, only Tna2 is produced and tna-1 is silent. Notwithstanding the great similarity in amino acid sequence between Tna1 and Tna2, the two enzymes differ markedly in activation energy for catalysis and thermostability.     

Cloning, nucleotide sequences, and overexpression in Escherichia coli of tandem copies of a tryptophanase gene in an obligately symbiotic thermophile, Symbiobacterium thermophilum.

Symbiobacterium thermophilum, a thermophilic bacterium, is a thermostable tryptophanase producer that can grow only in coculture with a specific Bacillus strain. Two thermostable tryptophanase genes, tna-1 and tna-2, that are located close to each other were cloned into Escherichia coli from S. thermophilum by the DNA-probing method. The nucleotide and deduced amino acid sequences indicate that Tna1 and Tna2 share 92% identical amino acids in a total of 453 amino acids. By means of DNA manipulation with E. coli host-vector systems, Tna1 and Tna2 were produced in very large amounts in enzymatically active forms. Comparison of the NH2-terminal amino acid sequences and the enzymatic properties of the tryptophanases purified from the original S. thermophilum strain and these two tryptophanases from recombinant E. coli cells suggest that in S. thermophilum, only Tna2 is produced and tna-1 is silent. Notwithstanding the great similarity in amino acid sequence between Tna1 and Tna2, the two enzymes differ markedly in activation energy for catalysis and thermostability.     

Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum

The nucleotide sequence of two novel plasmids isolated from the extreme thermophilic anaerobic bacterium Anaerocellum thermophilum DSM6725 (A. thermophilum), growing optimally at 70 degrees C, has been determined. pBAS2 was found to be a 3653 bp plasmid with a GC content of 43%, and the sequence revealed 10 open reading frames (ORFs). The two largest of these, namely Orf21 and Orf41, showed similarity to a Bacillus plasmid recombinase and a Pseudoalteromonas plasmid replication protein, respectively. A sequence with homology to double stranded replication origins from rolling circle plasmids was found, but no single stranded intermediates, characteristic of rolling circle replication, were found on Southern blots. The larger plasmid, pBAL, was found to be a 8294 bp plasmid with a GC content of 39%. It revealed 17 ORFs, of which three showed similarity at the amino acid (aa) level to known proteins. Orf22 showed the strongest similarity (33% aa) to replication proteins from large multiresistance Staphylococcal and Lactococcal plasmids, all of which are believed to replicate via a theta-like replication mechanism. Orf32 showed similarity to both DNA repair proteins and DNA polymerases with highest similarity to DNA repair protein from Campylobacter jejuni (25% aa). Orf34 showed similarity to sigma factors with highest similarity (28% aa) to the sporulation specific Sigma factor, Sigma 28(K) from Bacillus thuringiensis.     

Cloning and nucleotide sequence of the maltopentaose-forming amylase gene from Pseudomonas sp. KO-8940.

The gene coding for the maltopentaose-(G5)-forming amylase of Pseudomonas sp. KO-8940 was cloned into Escherichia coli and its nucleotides were sequenced. It was expected that a long open reading frame composed of 1,842-bp that encoded 614 amino acid residues for secretory precursor polypeptide including the typical signal sequence with an NH2-terminal was the gene. An extract of Escherichia coli carrying the cloned G5-forming amylase gene had amylolytic activity with which produced only G5 from starch, the same as that of the donor strain enzyme. In the deduced primary structure of this enzyme, the four conserved regions of many alpha-amylases were found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases.     

Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila.

The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA. The amylase gene is transcribed from its own promoter in Escherichia coli, producing a gene product of Mr 49,000. The amylase gene product is secreted to the periplasm of E. coli; however, it is not excreted. Nucleotide sequencing revealed an open reading frame of 1,392 base pairs corresponding to a protein of 464 amino acid residues. A potential signal peptide of 21 amino acid residues is present at the NH2 terminal of the predicted protein. Three regions of homology with other procaryotic and eucaryotic alpha-amylases were detected within the predicted amino acid sequence.     

Cloning and nucleotide sequence of a carbomycin-resistance gene from Streptomyces thermotolerans

Two plasmids (pOJ158 and pOJ159) containing DNA fragments from the carbomycin(Cb)-producing strain Streptomyces thermotolerans were identified in Streptomyces griseofuscus based on their ability to confer resistance to Cb. The Cb-resistance determinants on pOJ158 and pOJ159 were designated carA and carB, respectively. In S. griseofuscus, pOJ159 also confers resistance to spiramycin, rosaramicin, lincomycin, and vernamycin B, but not to tylosin; in Streptomyces lividans, pOJ159 additionally confers resistance to erythromycin and oleandomycin. The carB gene was localized on pOJ159 to a 1.25-kb region whose nucleotide sequence was determined. The sequence has a G + C content of 68% and contains the coding sequence for carB and portions of the 5' and 3' untranslated regions. A comparison of the amino acid sequence of the protein encoded by carB (as deduced from the nucleotide sequence) with the deduced amino acid sequence of the RNA methylase from Streptomyces erythraeus (encoded by ermE) revealed extensive homology, suggesting that carB also encodes an RNA methylase. The region 5' to the coding sequence does not contain a small ORF or regions of complementarity that are commonly associated with translationally regulated macrolide-lincosamide-streptogramin B resistance genes. The 3' untranslated region contains an inverted repeat sequence that potentially can form a stable RNA stem-loop structure with a calculated delta G of -70 kcal.     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

Cloning and nucleotide sequence of a hsp70 gene from Streptomyces griseus

The cultivation of Streptomyces griseus 2247 at the growth-limited temperature (37°C) or in liquid medium containing 5% ethanol (toxic for growth) revealed the presence of heat-induced proteins in the total cellular proteins. Among them, a 70 kDal protein was isolated and its N-terminal amino acid sequence was determined. The 70 kDal protein possessed a possible ATP-binding site in the N-terminus, which was conserved among the HSP70 family. A DNA fragment encoding the HSP70 homologue was isolated from a genomic library of S. griseus 2247 strain using an oligonucleotide probe based on the N-terminal amino acid sequence of the 70 kDal protein. DNA sequence analysis of the cloned gene revealed an open reading frame consisting of 618 amino acid residues. The deduced amino acid sequence is highly homologous to the HSP70 family proteins; it is 59.8 % identical to Clostridium perfringens HSP70, 59.7% to the Bacillus megaterium DnaK protein, 58.4% to the Methanosarcina mazei DnaK protein, 58.1% to Synechocystis HSP70, 52.8% to the DnaK protein of Escherichia coli, and about 50% to some of the mitochondrial heat shock proteins. The cloned gene could encode the HSP70 of S. griseus.     

The phylogenetic position of Dictyoglomus thermophilum based on 16S rRNA sequence analysis

Abstract The phylogenetic position of Dictyoglomus thermophilum has been determined by comparative sequence analysis of in vitro amplified 16S rRNA genes from the type strain as well as from a Dictyoglomus isolate. Results indicate that it forms a deep branch within the phylum of Thermotogales or may even represent its own phylum. It does not contain signature sequences within the 16S rRNA which could relate it to the Thermotogales group.     

Cloning and nucleotide sequence of the gene (amyP) for maltotetraose-forming amylase from Pseudomonas stutzeri MO-19.

The gene (amyP) coding for maltotetraose-forming amylase (exo-maltotetraohydrolase) of Pseudomonas stutzeri MO-19 was cloned. Its nucleotide sequence contained an open reading frame coding for a precursor (547 amino acid residues) of secreted amylase. The precursor had a signal peptide of 21 amino acid residues at its amino terminus. An extract of Escherichia coli carrying the cloned amyP had amylolytic activity with the same mode of action as the extracellular exo-maltotetraohydrolase obtained from P. stutzeri MO-19. A region in the primary structure of this amylase showed homology with those of other amylases of both procaryotic and eucaryotic origins. The minimum 5' noncoding region necessary for the expression of amyP in E. coli was determined, and the sequence of this region was compared with those of Pseudomonas promoters.     

Cloning, expression, and nucleotide sequence of a gene encoding a second thioredoxin from Corynebacterium nephridii

A gene encoding thioredoxin in Corynebacterium nephridii was cloned in Escherichia coli by complementation of a thioredoxin mutant. Transformants that appeared to complement were analyzed for the presence of thioredoxin by the coupled assay using methionine sulfoxide reductase. Of 18 transformants, four contained high levels of thioredoxin activity. Transformants containing plasmids pLCN2 and pLCN4 were unable to support replication of T7 phage, in spite of their thioredoxin activities, and were studied in more detail. The plasmid pLCN2 contains a 1.85-kilobase Sau3AI insert, whereas pLCN4 contains a 10-kilobase TaqI insert. These plasmids complement all phenotypes of a thioredoxin-deficient strain except for replication of T7 phage. The nucleotide sequence of a 620-base pair HinfI fragment encoding thioredoxin derived from either plasmid indicated that the protein derived from this DNA is different from the thioredoxin of C. nephridii previously reported (Meng, M., and Hogenkamp, H.P.C. (1981) J. Biol. Chem. 256, 9174-9182). The amino acid sequence predicted from the nucleotide sequence shows a high degree of homology with other procaryotic thioredoxins. However, the new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site and a third half-cystine residue in the carboxyl-terminal domain of the protein. The molecular weight of this thioredoxin, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is smaller than that estimated from the DNA sequence, suggesting that processing may have occurred.     

Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum

We cloned and expressed a gene encoding a thermostable cellobiose dehydrogenase (CDH) from the thermophilic ascomycete Myriococcum thermophilum. The 2904 bp long open reading frame contained six introns located either close to the 5′- or 3′-end of the ORF. The corresponding cDNA of 2487 bp was cloned into the expression vector pPICZαB to achieve inducible heterologous expression and secretion of the recombinant flavocytochrome in the methylotrophic yeast Pichia pastoris. Transformants were selected on media with normal and 10-fold increased zeocin concentration, and selected clones were tested for inducible extracellular production of the recombinant oxidoreductase. The maximally obtained volumetric activity was 0.25 U/ml in YPM (rich) medium and 2.15 U/ml in production stage (minimal) medium in a fed-batch fermentation. Recombinant CDH was purified in two consecutive chromatographic steps leading to a final specific activity of up to 7.4 U/mg protein at 40 °C. Kinetic properties of the recombinant CDH were characterized and the temperature optimum for the recombinant CDH was determined at 63 °C. Certain properties of the sequence of MtCDH are discussed in context with thermal and proteolytic stability.     

Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae

The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.     

极端耐热木聚糖酶基因在枯草芽孢杆菌中的整合表达

目的:为了在枯草芽孢杆菌中整合表达极端耐热木聚糖酶。方法:将嗜热网球菌(Dictyoglomus thermophilum)Rt46B.1的极端耐热木聚糖酶基因xynB通过穿梭载体pDL整合到B.subtilis168染色体上,使其实现表达。结果:极端耐热木聚糖基因在枯草芽孢杆菌中成功整合并表达。结论:基因工程菌B.subtilis168-xynB能外泌表达极端耐热木聚糖酶,且表达水平为0.732IU/mL,比在大肠杆菌中的高。酶学性质表明,此酶分子量约为24kD,其最适反应温度为85℃,最适反应pH值为6.5,且在弱碱性条件下稳定。