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     

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     

Coenzyme Q<Subscript>10</Subscript> production by <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in stirred tank and in airlift bioreactor

A higher Coenzyme Q₁₀ (CoQ₁₀) concentration of 25.04 mg/l was found in airlift bioreactor than the value of 18.11 mg/l obtained in stirred tank under the aerobic-dark cultivation of Rhodobacter sphaeroides. Aeration rate didn’t show obvious impact to CoQ₁₀ production in airlift bioreactor. The fed-batch operation in airlift bioreactor could increase the biomass concentration and led to the maximum CoQ₁₀ concentration of 33.91 mg/l measured, but a lower CoQ₁₀ cell content (3.5 mg CoQ₁₀/DCW) was observed in the fed-batch operation as compared to the batch operation. To enhance the CoQ₁₀ content, an aeration-change strategy was proposed in the fed-batch operation of airlift bioreactor. This strategy led to the maximum CoQ₁₀ concentration of 45.65 mg/l, a 35% increase as compared to the simple fed-batch operation. The results of this study suggested that a fed-batch operation in airlift bioreactor accompanying aeration-change could be suitable for CoQ₁₀ production.     

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l⁻¹ and a productivity of 0.11 g l⁻¹ h⁻¹. Revisions requested 1 September 2005 and 11 November 2005; Revisions received 7 November 2005 and 4 January 2006     

Influence of temperature on the production of an archaeal thermoactive alcohol dehydrogenase from Pyrococcus furiosus with recombinant Escherichia coli

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD₆₀₀ of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD₆₀₀ of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD₆₀₀ of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular.     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH, a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin, is a promising therapeutic candidate for thromboembolic diseases. To develop SFH into a new thrombolytic agent, scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies. Here, we describe a pilot-scale cultivation and purification process for the production of SFH. A high-cell-density fed-batch cultivation for the production of SFH in E. coli was developed in a 40-L bioreactor, which produced about 1.1 g/L of recombinant protein. SFH was purified to homogeneity from the E. coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography, with over 99% purity and 54% recovery. Moreover, the residual endotoxin content was less than 0.5 EU/mL. The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay, respectively.     

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     

Methyl Oleate Modulates <Emphasis Type="Italic">LIP2</Emphasis> Expression in the Lipolytic Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Methyl oleate was used as a primary carbon source and as an alternative inducer for the production of an extracellular lipase, Lip2, in Y. lipolytica strain LgX64.81 grown in a 20-l bioreactor. The lipase-encoding gene, LIP2, was investigated during culture on methyl oleate using a pLIP2–LacZ reporter fusion and we provide evidence for the involvement of methyl oleate in its regulation. Revisions requested 7 July 2005; Revisions received 30 August 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     

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     

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     

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     

Production of biomass and recombinant human-like collagen in <Emphasis Type="Italic">Escherichia coli</Emphasis> processes with different CO<Subscript>2</Subscript> pulses

The evolution of CO₂ in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O₂-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO₂ concentration of 12.7% in the effluent gas was detected. The CO₂ pulse injection experiments showed that: (1) a 20% CO₂ pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases slightly stimulated growth; and (2) CO₂ inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20% CO₂ pulse. The higher the CO₂ concentration and/or the longer the duration of the CO₂ pulse, the stronger the stimulatory or inhibitory effects. An erratum to this article can be found at     

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     

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     

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     

Production of isopropanol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.     

Rheology and Shear Stress of Centaurea calcitrapa Cell Suspension Cultures Grown in Bioreactor

Centaurea calcitrapa suspension cultures were grown either in Erlenmeyer flasks or in a mechanically stirred bioreactor. Its rheological behaviour, when fitted to the Oswald–de Waele model (power law), showed pseudoplastic characteristics in both cases. The flow behaviour index (n) decreased over the course of a growth cycle and the consistency index (K) increased, reached a value of 1.81 N sⁿ m⁻² run on 2 l bioreactor. Bioreactor cultivation of C. calcitrapa cells at different agitation rates (30, 60, 100 and 250 rpm), highlighted the influence of shear forces on cell viability loss (90–34%) and phenol accumulation (74–140 μg l⁻¹), due to increased stirring speeds. Analysis of these results suggests that this cell line is shear-sensitive. An empirical exponential correlation was defined between apparent viscosity and biomass concentration, under the studied conditions, giving the possibility to estimate the prevailing broth regime and to optimize bioreactor design. Revisions requested 10 October 2005; Revisions received 19 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     

Fully integrated L-phenylalanine separation and concentration using reactive-extraction with liquid-liquid centrifuges in a fed-batch process with<Emphasis Type="Italic"> E. coli</Emphasis>

A novel in situ product recovery (ISPR) approach for the (fully) integrated separation of L-phenylalanine (L-phe) from a 20 l fed-batch process with the recombinant L-tyrosine auxotrophic strain E. coli F-4/pF81 is presented. The strain was rationally constructed for the production of the aromatic amino acid. Glucose and tyrosine control is used. A reactive extraction system consisting of kerosene, the cation-selective carrier D₂EHPA and sulphuric acid, all circulating in liquid-liquid centrifuges, is applied for the on-line L-phe separation from cell- and protein-free permeate. Permeate is drained off from the bioreactor bypass. Using the novel ISPR approach, a significantly extended product formation period at 0.25 mmol/(g*h) together with a reduced by-product formation and a 28% relative glucose/L-phe yield increase is observed. Thus, the ISPR approach is superior to the reference non-ISPR process and even offers extraction rates approximately three times higher than the published membrane-based process.