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     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

Production of active pigment epithelium-derived factor in<Emphasis Type="Italic">E. coli</Emphasis>

Human pigment epithelium-derived factor (PEDF), a neurotrophic factor, is the most potent natural inhibitor of angiogenesis. To produce the active PEDF, the gene coding for the human PEDF protein was expressed in E. coli. The rPEDF protein was expressed at 457 mg l^–1 as a soluble protein. The yield of purified GST fusion protein was 14 mg ll^–1. Purified rPEDF inhibited tube formation in endothelial cells.Revisions requested 30 November 2004; Revisions received 25 January 2005     

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     

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     

Expression and secretion of a single-chain sweet protein monellin in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by <Emphasis Type="Italic">sacB</Emphasis> promoter and signal peptide

The sweet protein monellin gene was expressed in Bacillus subtilis under the control of the Bacillus subtilis sacB promoter and signal peptide sequence. A 294-bp DNA fragment, coding for sweet protein monellin, was ligated into the Escherichia coli/B. subtilis shuttle vector pHPC, producing pHPMS, which was subsequently transformed into B. subtilis QB1098, DB104, and DB403. The peptide efficiently directed the secretion of monellin from the recombinant B. subtilis cells. A maximum yield of monellin of 0.29 g protein l⁻¹ was obtained from the supernatant of B. subtilis DB403 harboring pHPMS. SDS-PAGE confirmed the purity of the recombinant product.     

Desalting minimal amounts of DNA for electroporation in <Emphasis Type="Italic">E. coli</Emphasis>: a comparison of different physical methods

Recovery and purification of ligated plasmid DNA is a critical and limiting step in modern molecular cloning techniques prior to electroporation in E. coli. Four different DNA purification and desalting methods were compared in regard to simple handling, reproducibility, efficiency and cost-effectiveness using picogram and nanogram amounts of DNA. Microcolumn purification of DNA was up to two orders of magnitude more efficient compared to gel filtration, ethanol precipitation or drop dialysis.Revisions requested 14 April 2005; Revisions received 10 May 2005     

Production of Bioactive Human β-defensin-3 in Escherichia coli by Soluble Fusion Expression

A codon optimized mature human β-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l⁻¹, i.e. 0.21 g mature HBD3 l⁻¹. Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli. Revisions requested 13 December 2005; Revisions received 24 January 2006     

Isolation and characterization of a fructosyl-amine oxidase from an <Emphasis Type="Italic">Arthrobacter </Emphasis>sp.

An Arthrobacter sp. was isolated that, when induced by fructosyl-valine, expressed a fructosyl-amine oxidase (FAOD) that was specific for -glycated amino acids. The N-terminal amino acid sequence of the purified oxidase was determined and used to design oligonucleotides to amplify the gene by inverse PCR. Expression of the gene in Escherichia coli produced 0.23 units FAOD per mg protein, over 30-fold greater than native expression levels, with properties almost indistinguishable from the native enzyme. The presence of FAOD was confirmed in other Arthrobacter ssp.Revisions requested 8 September 2004; Revisions received 4 November 2004     

Polyclonal antibody against <Emphasis Type="Italic">Manduca sexta</Emphasis> chitinase and detection of chitinase expressed in transgenic cotton

The chitinase gene of Manduca sexta was cloned into the expression vector, pET-28a, and expressed in Escherichia coli BL21 (DE3) host cells. The protein product was expressed in inclusion bodies. After denaturation and renaturation procedures using a Ni²⁺-NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of the renatured protein was confirmed by Western blotting. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed chitinase and was used to quantify its presence in transgenic cotton being developed to resist attack by various insects.Revisions requested 24 September 2004; Revisions received 18 November 2004     

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 .     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

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     

Biotransformation of racemic diisophorone by <Emphasis Type="Italic">Cephalosporium aphidicola</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Racemic diisophorone (500 mg) was converted by Cephalosporium aphidicola and Neurospora crassa over 10 days at 25 °C to 8β-hydroxydiisophorone in yields of 10% (52 mg) and 20% (103 mg), respectively. The structure was established by IR, specific rotation, mass spectral, 1D and 2D-NMR studies.Revisions requested 2 March 2005 and 21 April 2005; Revisions received 8 April 2005 and 10 May 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     

Stable RNA Interference in Spodoptera frugiperda Cells by a DNA Vector-based Method

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 22 November 2005; Accepted 25 November 2005     

Production of H<Subscript>2</Subscript> from sucrose by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains carrying the pUR400 plasmid,which encodes invertase activity

Escherichia coli HD701, a hydrogenase-upregulated strain, has the potential for industrial-scale H₂ production but is unable to metabolise sucrose, which is a major constituent of many waste materials that could be used as feedstocks for H₂ production processes. A 70 kb plasmid (pUR400), which carries the genes necessary for sucrose transport into the cell and its metabolism, was conjugated into E. coli strains HD701 and FTD701 [a derivative of HD701 which has a deletion of the tatC gene of the twin arginine transport (Tat) protein system] from an E. coli K12 strain. Comparative studies on H₂ evolution by FTD701 and HD701, with and without the pUR400 plasmid, were made using sucrose as substrate. The parental strains did not evolve H₂, although HD701/pUR400 and FTD701/pUR400 evolved 1.27 ± 0.09 and 1.38 ± 0.05 ml H₂ mg dry wt^–1 l culture^–1, respectively over 10 h. This work provides the choice for using a recombinant E. coli strain, which produces H₂ from sucrose, as an alternative to coupling-in an upstream invertase, and hence this provides a simpler method for the bioproduction of H₂ from sucrose.Revisions requested 24 August 204; Revisions received 21 October 2004     

Expression and secretion of a single-chain sweet protein,monellin, in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by an α-factor signal peptide

The sweet protein monellin gene was expressed in Saccharomyces cerevisiae under the control of the GAL1 promoter and α-factor signal peptide sequence of S. cerevisiae. The gene, which was obtained through mutation of the synthesized single-chain monellin gene, was cloned into an E. coli-yeast shuttle vector pYES2.0 which carries the galactose-inducible promoter GAL1. Then the α-factor signal peptide of S. cerevisiae was linked also, resulting in the secreting expression vector pYESMTA. The recombinant plasmid was subsequently transformed into strain S. cerevisiae INVsc1. The peptide efficiently directed the secretion of monellin from the recombinant yeast cell. A maximum yield of active monellin was 0.41 g l⁻¹ of the supernatant from INVsc1 harboring pYESMTA.     

Cloning, Expression, and Characterization of a DNA Ligase from a Hyperthermophilic Archaeon Thermococcus Sp.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed an ORF of 1689 bases encoding 562 amino acids that showed a high similarity to DNA ligases from other hyperthermophilic archaea. The ligase, which was designated TNA1_lig (Thermococcus sp. NA1 ligase), was cloned and expressed in Escherichia coli. The recombinant TNA1_lig was purified by metal affinity chromatography. The optimum ligase activity of the recombinant TNA1_lig occurred at 80 °C and pH 7.5. The enzyme was activated by MgCl₂ and ZnCl₂ but was inhibited by MnCl₂ and NiCl₂. Additionally, the enzyme was activated by either ATP or NAD⁺. Revisions requested 27 October 2005; Revisions received 14 December 2005     

High-level Expression of an α-l-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the Production of Xylobiose from Xylan

To efficiently produce xylobiose from xylan, high-level expression of an α-l-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an α-l-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine–Dalgarno sequence. A maximum activity of 12 U mg⁻¹ was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed α-l-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and α-l-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 °C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of α-l-arabinofuranosidase gave only half this yield. Revisions requested 27 October 2005; Revisions received 5 September 2005