Cloning, expression, and characterization of pyrrolidone carboxyl peptidase from the archaeon Thermococcus litoralis

The gene encoding pyrrolidone carboxyl peptidase (Pcp) has been cloned from the hyperthermophilic archaeon Thermococcus litoralis. The recombinant enzyme has been expressed in Escherichia coli, purified, and char-acterized. The T. litoralis Pcp demonstrates strong sequence homology to previously characterized bacterial Pcps. Some investigations have been carried out on enzyme substrate specificity and stability. Received: July 4, 2000 / Accepted: July 21, 2000     

Cloning of a Gene Encoding Acetate Kinase from Methanosarcina mazei 2-P Isolated from a Japanese Paddy Field Soil

The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His₆-tagged acetate kinase purified from E. coli JM109 was optimal at 35°C. Received: 28 January 2002 / Accepted: 27 February 2002     

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity. Received: September 17, 1999 / Accepted: March 21, 2000     

Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale

The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale. Received: 19 September 1996 / Accepted: 21 May 1997     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10

The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20–29% identity with zinc-containing, NAD⁺-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD⁺- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity. Received: 5 March 1999 / Received last revision: 10 May 1999 / Accepted: 16 May 1999     

Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824

Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added. Journal of Industrial Microbiology & Biotechnology (2001) 27, 220–227. Received 12 September 2000/ Accepted in revised form 26 June 2001     

Expression of the creatininase gene from Pseudomonas putidaRS65 in Escherichia coli

The gene encoding creatininase from Pseudomonas putida RS65 was cloned, sequenced and expressed in Escherichia coli. One plasmid containing a 7.0-kb HindIII insert was selected by its ability to express creatininase activity. After deletion of the adjacent restriction fragments, a 1.1-kb SphI fragment, which contained the full length of the creatininase gene, was subcloned into a pUC18 vector and the nucleotide sequence of the creatininase gene was determined. The gene consists of 771 base pairs and encodes a protein of 257 amino acids. The constitutive creatininase productivity of E. coli DH5α (pCRN741) cultured in broth was about 8.5-fold higher than that of P. putida RS65 cultured in a creatinine-containing medium. The creatininase gene was expressed efficiently in E. coli from its own promoter. Journal of Industrial Microbiology & Biotechnology (2000) 24, 2–6. Received 02 April 1999/ Accepted in revised form 31 July 1999     

Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca²⁺ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect. Received: 25 February 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli

To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity. Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998     

An answer to a complex problem: cloning the mouse t-complex responder

The t-complex is maintained in wild mouse populations by its high transmission (up to 99%) from heterozygous males and provides an example of ``meiotic drive'. Its molecular basis has remained obscure despite long and intensive study. In a major advance, the t-complex responder gene, thought to be the key gene on which several distorters act, has now been cloned. Received: 14 April 2000 / Accepted: 4 May 2000     

A cellulase gene from a new alkalophilic Bacillus sp. (strain N186-1). Its cloning,nucleotide sequence and expression in Escherichia coli

 Several alkalophilic Bacillus spp. strains were selected for their capacity to produce alkaline cellulases. Culture supernatants of these strains showed optimal cellulase activities between pH 8 and 9 and they were stable from pH 6 to pH 12. A cellulase gene (celB1) from the alkalophilic Bacillus sp. strain N186-1 was cloned in Escherichia coli using polymerase chain reaction techniques. The cloned gene was present in a 2.539-bp HindIII fragment and its nucleotide sequence was determined. The coding sequence showed an open-reading frame encoding 389 amino acids. The amino acid sequence, deduced from the nucleotide sequence, permitted us to include it in family 5 (or A) of the glycosyl hydrolases. The complete open-reading frame of celB1 was cloned in the plasmid pET-11d and expressed in E. coli BL21 (DE3), in which a protein of 39 kDa was obtained in the cytoplasm; however, no endoglucanase activity was detected. A second construction in pET-12a allowed the production of a 39-kDa protein located in the periplasmic space of E. coli that had endoglucanase activity. The protein produced has optimal activity at pH 7 and 50°C and it retains more than 70% of its activity after incubation for 1 h at pH 12. Received: 27 December 1995/Received revision: 14 March 1996/Accepted: 25 March 1996     

Malate dehydrogenase from the green gliding bacterium Chloroflexus aurantiacus is phylogenetically related to lactic dehydrogenases

The gene encoding malate dehydrogenase (MDH) from Chloroflexus aurantiacus was cloned, sequenced, and analyzed. The mdh gene corresponded to a polypeptide of 309 amino acids with a molecular mass of 32,717 Da. The primary structure and the coenzyme-binding domain showed a high degree of similarity to lactate dehydrogenase (LDH), whereas the conserved amino acids that participate in substrate binding were those typical of MDHs. Using PCR techniques, the mdh gene was cloned in the expression vector pET11a, and large amounts of active C. aurantiacus MDH were produced in Escherichia coli after induction with isopropyl β-d-thiogalactoside. The expressed enzyme thus obtained was purified and retained full activity at 55° C. High levels of expression of mdh were also observed when the gene and its flanking sequences were cloned into pUC18/19, indicating that the putative σ⁷⁰ promoter sequences found upstream of the C. aurantiacus mdh functioned in E. coli. When these sequences were deleted, the expression in E. coli was reduced dramatically. Received: 24 October 1995 / Accepted: 23 February 1996     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

Multi-copy repression of Serratia marcescens nuclease expression by dinI

The dinI homolog of S. marcescens was cloned from a plasmid library by virtue of its ability to inhibit nuclease expression from the S. marcescens nucA gene integrated in the genome of E. coli. The S. marcescens DinI protein is 68% identical to DinI of E. coli. It has a similar effect on other SOS regulated genes and likely exerts it effect on nuclease expression, which is most pronounced as the cells entered stationary phase, through inhibition of basal SOS expression. Received: 12 April 2001 / Accepted: 14 May 2001     

Cloning and Characterization of a Campylobacter jejuni Iron-Uptake Operon

We report that C. jejuni modifies its outer membrane protein (OMP) repertoire when cultivated under iron-limiting conditions such as during incubation with epithelial cells. To identify genes encoding de novo expressed OMPs, a C. jejuni cosmid library was screened with antisera raised against proteins expressed in the presence of epithelial cells. A single clone was identified encoding an 80-kDa antigen. Sequence analysis of subclones identified an operon of three open reading frames (ORFs) encoding proteins that are homologous to the E. coli ferrichrome uptake system encoded by the fhu locus. Under low-iron conditions, C. jejuni expressed the 80-kDa OMP, indicating that its expression is regulated by the presence of iron. Southern blot analysis indicated that six of eleven isolates of C. jejuni harbor a fhuA homolog which, like all other DNA in this region sequenced thus far, is strikingly GC-rich (65%) compared with the C. jejuni genome (35% G+C). Received: 19 June 2000 / Accepted: 30 August 2000     

Molecular characterization of xynX, a gene encoding a multidomain xylanase with a thermostabilizing domain from Clostridium thermocellum

A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined. The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa). Sequence analysis of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain. A deletion mutant of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability. In terms of half-life at 70 °C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme, XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain. The optimum temperature of XynX-C was about 5–10 °C lower than that of XynX-TC. Received: 12 January 2000 / Received revision: 24 April 2000 / Accepted: 1 May 2000     

Enhanced Tolerance Against Salt-Stress and Freezing-Stress of Escherichia coli Cells Expressing Algal bbc1 Gene

The expression of the eukaryotic bbc1 (breast basic conserved) gene (the bbc1 gene of the marine green alga Chlamydomonas sp. W-80 strain) enhanced the tolerance against salt-stress and freezing-stress in E. coli cells. The expression of the BBC1 protein in the E. coli cells carrying the algal bbc1 gene and that in the Chlamydomonas W-80 cells were examined by Western blotting analysis. The result suggests that the eukaryotic BBC1 protein expressed in the E. coli cells has a protective function against the cellular dehydration. Received: 24 April 2000 / Accepted: 21 August 2000     

The structure of the α-galactosidase gene loci in Thermus brockianus ITI360 and Thermus thermophilus TH125

The Thermus thermophilus TH125 α-galactosidase gene, agaT, and flanking sequences were cloned in Escherichia coli and sequenced as well as flanking sequences of the previously cloned agaT from Thermus brockianus ITI360. Different structures of putative α-galactosidase operons in the two Thermus strains were revealed. Downstream of and overlapping with the α-galactosidase genes of both strains, a gene was identified that is similar to the galactose-1-phosphate uridylyltransferase gene (galT) of E. coli and Streptomyces lividans. Upstream of the agaT of T. brockianus ITI360, four open reading frames were observed. The deduced translation products displayed similarity to components of bacterial binding protein-dependent transport systems and a β-galactosidase. No galactoside utilization genes were identified upstream of agaT in T. thermophilus TH125. The inactivation of the α-galactosidase genes of both strains by insertional mutagenesis led to an inability to use melibiose or galactose as a single carbohydrate source. An attempt was made to isolate a gene encoding the enzyme responsible for para-nitrophenyl-(pNP-) β-galactoside hydrolyzing activity in T. thermophilus TH125. A gene designated bglT was cloned and expressed in E. coli. The inactivation of the bglT gene led to 55% reduction of the pNP-β-galactoside hydrolyzing activity in the mutant strain in comparison to the wild type. Received: April 28, 1999 / Accepted: September 9, 1999     

Cloning of the Pichia anomala SEC61Gene and Its Expression in a Saccharomyces cerevisiae sec61 Mutant

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene. Received: 24 May 2002 / Accepted: 10 July 2002     

Characterization of the Porphyromonas gingivalis FtsZ Containing a Novel GTPase Activity

The FtsZ protein is a GTPase that is essential for cell division. We have cloned, sequenced, and expressed the FtsZ (PgFtsZ) gene from the Porphyromonas gingivalis, an oral, anaerobic, rod-shaped bacterium implicated in progressive periodontal disease. The PgFtsZ gene consisted of 1374 bp and coded for an acidic protein with a calculated molecular mass of 50,253 Da. The deduced amino acid sequence exhibited a significant homology with E. coli FtsZ (54% identical residues). Like other prokaryotic FtsZs, PgFtsZ possessed the clear motifs for GTP binding (GGGTGTG) and hydrolysis (NLDFADV). When PgFtsZ was overexpressed in E. coli, cell division was inhibited. Recombinant PgFtsZ was purified to homogeneity and characterized. The purified PgFtsZ exhibited GTPase activity even in the absence of Mg²⁺, and completely retained its activity with EDTA. Furthermore, Na⁺ and K⁺ ions inhibited its GTPase activity in a dose-dependent manner. These results suggest that PgFtsZ contains an atypical GTPase activity that has not been previously described. Received: 25 May 2001 / Accepted: 8 August 2001