Characterization of a New Stearoyl-acyl Carrier Protein Desaturase Gene from Jatropha curcas

A new full-length cDNA of stearoyl-acyl carrier protein desaturase was obtained by RT-PCR and RACE techniques from developing seeds of Jatropha curcas. Sequence alignment showed that its deduced amino acid sequence had high similarity with other stearoyl-acyl carrier protein desaturases. The gene was functionally expressed in E. coli and the desaturating activity of recombinant protein was easily detected when assayed in vitro with added spinach ferredoxin. Southern blot analysis indicated that the gene was a member of a small gene family. Northern blot analysis revealed it was highly expressed in developing fruits of J. curcas. Revisions requested 16 December 2005; Revisions received 6 February 2006     

The Occurrence of Antibiotic Resistance Genes in Taq Polymerases and a Decontamination Method Applied to the Detection of Genetically Modified Crops

Different antibiotic resistance (AR) genes, such as Bla, Tet and NPTII, contaminate commercially available Taq polymerases. The specificity of the AR gene PCR can be increased when using a restriction enzyme-based decontamination of polymerase. The elimination of Taq polymerase contamination allows the use of PCR tests to screen seeds (corn) and processed food for the presence of genetically modified organisms (GMO) based on the detection of AR genes. Without a decontamination procedure for AR genes, PCR screening tests should be interpreted with caution. Revisions requested 1 November 2005; Revisions received 28 November 2005     

Stable expression and visualization of Mat-8 (FXYD-3) tagged with a fluorescent protein in Chinese Hamster Ovary (CHO)-K1 cells

A type I transmembrane protein, Mat-8 (FXYD-3), was tagged with fluorescent protein, Discosoma red fluorescent protein, at the carboxyl terminal cytoplasmic tail, and stably expressed in Chinese Hamster Ovary (CHO)-K1 cells. The fluorescence signal was distributed in intracellular membranes, being not only detected around the nuclear envelope but also partly overlapping with an endoplasmic reticulum marker. Subcellular fractionation by density gradient centrifugation supported this partial overlapping. The spherical structures observed were not colocalized with markers for lysosomes, endosomes, and Golgi bodies, suggesting that Mat-8 is distributed in a distinct endoplasmic reticulum region and the nuclear envelope after synthesis on membrane-bound ribosomes.Revisions requested 15 April 2005; Revisions received 11 May 2005     

High-level Expression of a Synthetic Gene Encoding a Sweet Protein,Monellin, in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE. Revisions requested 13 April 2005 and 26 May 2005; Revisions received 19 May 2005 and 30 August 2005     

Characterization of pbpA and pbp2 Encoding Penicillin-binding Proteins Located on the Downstream of Clavulanic Acid Gene Cluster in Streptomyces clavuligerus

Two genes, pbpA (orf18) and pbp2 (orf19) located on the downstream of clavulanic acid (CA) gene cluster of Streptomyces clavuligerus were cloned into pET-28a(+), and confirmed to encode a family of high molecular-weight penicillin-binding proteins (PBPs). Both genes were amplified from genomic DNA by PCR and expressed in E. coli BL21 (DE3). Hydropathy plots of the proteins revealed a single stretch of hydrophobic amino acids indicating them to be transmembrane proteins. Pbp2 had lower affinity to penicillin G compared to PbpA, and was essential to the cell growth in contrast to PbpA. Revisions requested 3 November 2005; Revisions received 13 December 2005     

Cloning and Sequence Analysis of the Gene Encoding (6-4)photolyase from Dunaliella salina

DNA photolyase can repair UV-induced DNA damage in a light-dependent manner. A cDNA of (6-4)photolyase from Dunaliella salina (GenBank accession number: AY845324) was cloned, sequenced and its amino acid sequence was deduced. The derived amino acid sequence showed high homology with other (6-4)photolyases and a predicted 3D model was constructed by homology modeling. Revisions requested 20 May 2005 and 18 August 2005; Revisions received 2 August 2005 and 28 November 2005     

One-step Concentration and Partial Purification of Aspergillus kawachii Non-acidic Polygalacturonases by Adsorption to Glass Fiber Microfilters

The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved. Received 27 July 2005; Revisions requested 24 August 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Characterization of an Anti-Thioredoxin Monoclonal Antibody

An anti-E. coli thioredoxin monoclonal antibody, IMM-3C6, which showed high specificity to thioredoxin as assessed by indirect ELISA, was generated using hybridoma technology. The affinity constant of IMM-3C6 to thioredoxin was 0.40×10⁹ m⁻¹ and its sensitivity to thioredoxin fusion protein in dot blotting was 50 ng. In sandwich ELISA, it detected thioredoxin fusion protein between 16 and 150 ng/ml. By using IMM-3C6 as the ligand, thioredoxin fusion protein was successfully purified by affinity chromatography. IMM-3C6 was confirmed to be a useful tool for immunoassay and purification of thioredoxin fusion proteins. These authors contributed equally to the work. Received 21 September 2005; Revisions requested 7 October 2005; Revisions received 10 November 2005; Accepted 11 November 2005     

Cloning and DNA-binding properties of ethylene response factor,LeERF1 and LeERF2, in tomato

Two new genes, LeERF1 andLeERF2, were isolated from a tomato (Lycopersicon esculentum cv. Lichun) cDNA library. Phylogenetic analysis indicated that they encoded Ethylene Responsive Element Binding Proteins (EREBPs), characterized by a conserved ERF (ethylene response factor) domain of specific binding plant cis-acting elements GCC box. Both LeERF1 and LeERF2 proteins were obtained via prokaryotic expression and purification. Electrophoretic mobility shift assay showed that LeERF1 and LeERF2 protein could bind to the promoter of the NP24 gene coding for pathogenesis-related protein osmotin precursor but not the mutant promoter where its GCC box was deleted. Polyclonal antibodies of LeERF1 and LeERF2 blocked their binding in vitro.Revisions requested 4 January 2005; Revisions received 28 January 2005     

Using Redox Potential to Detect Microbial Activities During Clavulanic Acid Biosynthesis in <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>

Production of clavulanic acid (CA) by Streptomyces clavuligerus in a shake-flask culture increased from 92 to 180 mg l⁻¹ with an increased O₂ transfer efficiency (0.039 → 0.058 s⁻¹), which maintained the redox potential values above −250 mV. Compared with traditional measures, such as dissolved O₂ concentration and respiratory activity, the redox potential can easily be determined and correlates closely with CA production. It can therefore be used to monitor microbial activities during biosyntheses of secondary metabolites. Revisions requested 5 April 2005 and 19 July 2005; Revisions received 19 July 2005 and 9 September 2005     

Quantification of S-adenosyl Methionine in Microbial Cell Extracts

A sensitive method for quantification of S-adenosyl methionine (SAM) in microbial cell extracts was developed and applied to Corynebacterium glutamicum. The method is based on SAM being completely hydrolyzed into ¹⁸O-homoserine when extracted in boiling H₂¹⁸O and thus can be clearly distinguished by GC-MS analysis from naturally labeled homoserine present in the cell extract. Additional quantification of the total homoserine pool, representing both SAM and homoserine, via HPLC allows separate determination of both metabolites. Received 23 August 2005; Revisions requested 30 August 2005; Revisions received 26 September 2005; Accepted 31 October 2005     

Expression and Secretion of Bifidobacterium adolescentis Amylase by Bifidobacterium Longum

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli–Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium. Both authors contributed equally Received 17 June 2005; Revisions requested 13 July 2005 and 26 September 2005; Revisions received 12 September 2005 and 8 November 2005; Accepted 11 November 2005     

A cultivation technique for <Emphasis Type="Italic">E. coli</Emphasis> fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O₂ transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.Revisions requested 13 April 2005; Revisions received 6 May 2005     

Production of a Cellulase in Silkworm Larvae using a Recombinant Bombyx mori nucleopolyhedrovirus lacking the Virus-encoded Chitinase and Cathepsin Genes

The expression efficiency of an insect-derived cellulase was assayed in silkworm larvae infected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) mutants lacking the virus-encoded chitinase (chiA) and/or cathepsin (v-cath) genes. Expression was increased by approx. 10% in mutants lacking chiA or v-cath and 17% in a mutant lacking both chiA and v-cath compared with that of the unmodified recombinant BmNPV. The recombinant BmNPV lacking both chiA and v-cath can therefore be used for a large-scale production of foreign proteins in silkworms. Revisions requested 27 September 2005 and 16 December 2005; Revisions received 6 December 2005 and 3 February 2006     

Purification and Properties of a β-1,3-glucanase from Chaetomium sp. that is Involved in Mycoparasitism

A β-1,3-glucanase was detected, using laminarin as substrate, in the culture broth of Chaetomium sp. Major activity was associated with a 70 kDa protein band visualized on a polyacrylamide gel. β-1,3-Glucanase was purified by a one-step, native gel purification procedure. Optimal activity was observed at pH 6.0 and 30 °C (over 30 min). It could degrade cell walls of plant pathogens including Rhizoctonia solani, Gibberella zeae, Fusarium sp., Colletotrichum gloeosporioides and Phoma sp. The N-terminal amino acid residues of the purified β-1,3-glucanase are PYQLQTP, which do not exhibit homology to other fungal β-1,3-glucanases suggesting it may be a novel enzyme. Received 20 July 2005; Revisions requested 2 August 2005 and 27 September 2005; Revisions received 16 September 2005 and 3 November 2005; Accepted 6 November 2005     

Development of Transformation System in Monascus Purpureus using an Autonomous Replication Vector with Aureobasidin A Resistance Gene

To enhance the variety of genetic tools and thus to promote molecular genetic study, aureobasidin A and its resistance gene were adopted as a new marker system together with the incorporation of the Gateway system to facilitate the introduction of long heterologous DNA fragments into Monascus purpureus. The minimum inhibitory concentration of aureobasidin A against Monascus was 0.05 μg/ml and a transformation efficiency of 17 colonies/μg DNA was obtained by the protoplast-PEG method with the vector pAUR316, containing the aureobasidin A resistance gene. Southern analysis of the transformants confirmed that pAUR316 exists as an independent vector, demonstrating that the AMA1 sequence acts as the autonomous replication sequence in M. purpureus. Through the use of the Gateway system, a polyketide synthase gene (7.8 kbp) responsible for citrinin biosynthesis was introduced. As a result, the transformants showed 1.5-fold higher production of citrinin than the wild-type strain. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 2 November 2005; Accepted 3 November 2005     

Polymeric and Compositional Properties of Novel Extracellular Microbial Polyglucosamine Biopolymer from New Strain of Citrobacter sp. BL-4

A novel polyglucosamine polymer, PGB-2, was produced extracellularly from a new strain Citrobacter sp. BL-4 using pH-stat fed batch cultivation. It was composed of 97.3% glucosamine and 2.7% rhamnose; its average molecular weight, solubility in 2% acetic acid and viscosity were 20 kDa, 5 g l⁻¹ and 2.9 cps, respectively. FT-IR and ¹H NMR spectra of PGB-2 revealed a close identity with chitosan from crab shells. Received 20 September 2005; Revisions requested 6 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Expression of Galactose Permease and Pyruvate Carboxylase in Escherichia coli ptsG Mutant Increases the Growth Rate and Succinate Yield under Anaerobic Conditions

In Escherichia coli, disruption of ptsG, which encodes the glucose-specific permease of the phosphotransferase transport system (PTS) protein EIICB^Glc, is crucial for high succinate production. This mutation can, however, cause very slow growth and low glucose consumption rates. The ptsG mutant (TUQ2), from wild type E. coli W1485, and E. coli galP (encoding galactose permease) and glk (encoding glucose kinase) gene expression plasmids were constructed. TUQ2 increased the generation time to approximately 4 h and gave a higher final cell density of 0.5 g/l by expression of galP. However, glk expression had no effect on the mutant. After expression of pyruvate carboxylase (PYC) and galactose permease, the ptsG mutant showed higher succinate yield (1.2 mol/mol glucose) and the specific rate of glucose consumption from 0.33 to 0.6 g/1 h. Received 31 August 2005; Revisions requested 27 September 2005; Revisions received 1 November 2005; Accepted 2 November 2005 An erratum to this article is available at .     

A simplified method for assay of hydrogenase activities of H<Subscript>2</Subscript> evolution and uptake in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis>

Assay of hydrogenase activity pertaining to H₂ production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobater aerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H₂ uptake and evolution activities of the intact cells, respectively. This method enabled H₂ uptake and evolution activities of the intact cells to be easily detected. This is also the first report of the presence of H₂ uptake hydrogenase activity in E. aerogenes.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 12 April 2005 and 17 May 2005