Cloning and Expression of Fructosyl-amine Oxidase from Marine Yeast <Emphasis Type="Italic">Pichia</Emphasis> Species N1-1

The gene encoding the fructosyl-amine oxidase (FAOD) from the marine yeast Pichia sp. N1-1 was cloned and expressed in Escherichia coli. Partial amino acid sequence analysis of the Pichia sp. N1-1 FAOD allowed the design of oligonucleotide primers for the amplification of the gene by inverse polymerase chain reaction. The FAOD gene was found to be devoid of introns and to encode a 48-kDa protein composed of 429 amino acid residues. The FAD-binding consensus sequence GXGXXG and the FAD covalent attachment-site cysteine residue have been identified within the predicted amino acid sequence. Comparisons with the amino acid sequences of other eukaryotic FAODs showed only 30% to 40% identities, establishing that the isolated Pichia N1-1 gene encodes a unique FAOD. Recombinant FAOD expression levels in E. coli reached 0.48 U/mg of soluble protein, which is considerably greater than native expression levels by inducing Pichia sp. N1-1 with fructosyl-valine (f-Val). The kinetic properties of the recombinant enzyme were almost indistinguishable from those of the native enzyme. We previously reported on the construction of a number of effective Pichia sp. N1-1 FAOD-based biosensors for measuring f-Val, a model compound for glycated hemoglobin. The further development of these biosensor systems can now greatly benefit from protein engineering and recombinant expression of the FAOD from Pichia N1-1.Note: The previous online version (January 20, 2005) of this article appeared with the legends of Figures 1 and 2 transposed. This version contains the figures with their appropriate legends.     

Ethanol production from cellulosic materials by genetically engineered <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

To confer the ability to ferment cello-oligosaccharides on the ethanol-producing bacterium, Zymomonas mobilis, the -glucosidase gene from EmRuminococcus albus, tagged at its N-terminal with the 53-amino acid Tat signal peptide from the periplasmic enzyme glucose–fructose oxidoreductase from Z. mobilis, was introduced into the strain. The tag enabled 61% of the -glucosidase activity to be transported through the cytoplasmic membrane of the recombinant strain which then produced 0.49 g ethanol/g cellobiose. Revisions requested 9 November 2004; Revisions received 10 December 2004; Accepted 13 December 2004     

Production of an anti-fungal substance for biological control of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> causing phytophthora blight in red-peppers by <Emphasis Type="Italic">Streptomyces halstedii</Emphasis>

The culture broth of Streptomyces halstedii AJ-7 suppressed the growth of Phytophthora capsici, which causes phytophthora blight in red-peppers, with less than 1% survival of the pathogen after 12 h of treatment. The low molecular fraction ( 10 kDa) of the culture broth retained anti-fungal activity against P. capsici after being held at 100 °C for 6 h.Revisions requested/26 August 2004; Revisions received 16 August 2004/10 December 2004     

Cloning and expression of <Emphasis Type="SmallCaps">d</Emphasis> -lactonohydrolase cDNA from <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A d -lactonohydrolase gene of about 1.1 kb was cloned from Fusarium moniliforme. The ORF sequence predicted a protein of 382 amino acids with a molecular mass of about 40 kDa. An expression plasmid carrying the gene under the control of the triose phosphate isomerase gene promotor was introduced into Saccharomyces cerevisiae, and the d -lactonohydrolase gene was successfully expressed in the recombinant strains.Revisions requested 10 September 2004; Revisions received 15 October 2004The nucleotide sequence data reported in this paper has been assigned accession number AY728018 in the GeneBank database.     

Survival of <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> during heating,drying and storage in the dried state when growth has occurred in the presence of sucrose or monosodium glutamate

Spray-dried cells of Lactobacillus sakei CTC 494 survived ca. 60% longer in the spray dried state when cells were grown in the presence of 20g sucrose l^–1or 12.5g monosodium glutamate l^–1. No significant differences were observed in viability during storage in the freeze dried state with the addition of these compounds to the growth medium, nor in survival during a heat treatment (55°C). Both sucrose and glutamate in the growth medium suppressed intracellular accumulation of total amino acids and changed the overall pattern of the individual amino acids. Glutamate in the growth medium enhanced intracellular glutamate by ca. 38%. Revisions requested 4 November 2004; Revisions received 13 December 2004     

Continuous production of neo-fructooligosaccharides by immobilization of whole cells of <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

To produce neo-fructooligosaccharides (neo-FOSs) in a 500ml continuous packed-bed reactor using whole cell immobilization of Penicillium citrinum KCCM 11663, the optimum reaction conditions were 50 °C, pH 6 with 600 g sucrose l^-1 being fed as substrate at 1.3 ml min^-1 . Under these conditions, the maximum neo-FOSs production was 49 g l^-1. In a packed-bed reactor, continuous production of neo-FOSs was possible for 50 d indicating a potential for industrial production. Revisions requested 6 September 2004/14 October 2004; Revisions received 7 October 2004/29 November 2004     

Enhancement of photorespiration in immobilized <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Immobilization of Chlamydomonas reinhardtii in alginate increases its photorespiration rate. In the immobilized cells, the photorespiratory enzyme, phosphoglycolate phosphatase, was 75% higher than in freely suspended cells. Thus, the immobilized cells produced glycolate at twice the rate than in freely suspended cells when treated with aminooxyacetate (a transaminase inhibitor). With immobilized cells in a batch reactor, 270mol glycolatemg^–1 Chl was produced after 12h.Revisions requested 27 October 2004; Revisions received 13 December 2004     

A new isolate of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis>that degrades 2-chloronitrobenzene

A strain of Pseudomonas stutzeri ZWLR2-1 was isolated from soil contaminated with chloronitrobenzenes and identified by 16S rDNA sequencing. This bacterium released chloride and nitrite into the medium when grown on 0.5mm 2-chloronitrobenzene. PCR amplification and DNA sequencing revealed a DNA fragment encoding a polypeptide homologous to the -subunit of ring-hydroxylating dioxygenasesRevisions requested 01 December 2004; Revisions received 9 December 2004     

A novel psychrotrophic fungus, <Emphasis Type="Italic">Mortierella minutissima</Emphasis>, for <Emphasis Type="BoldSmallCaps">D</Emphasis>-limonene biotransformation

Of 98 strains of moulds, isolated from arctic soils, Mortierella minutissima 01, grew the best on agar plates with limonene vapor. Perillyl alcohol and perillic acid were the main products of limonene biotransformation. Maximal yield of perillyl alcohol (125mgl^–1) occurred in medium containing 0.8% substrate, at 15°C, pH 6 and after 4–5 d. Revisions requested 27 October 2004; Revisions received 27 November 2004     

Dehalogenation of chlorinated aromatic compounds using a hybrid bioinorganic catalyst on cells of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis>

A novel bioinorganic catalyst was obtained via reduction of Pd(II) to Pd⁰ on to the surface of cells of Desulfovibrio desulfuricans at the expense of H₂. Palladised biomass, supplied with formate or H₂ as an electron donor, catalysed the dehalogenation of 2-chlorophenol and polychlorinated biphenyls. In the example of 2,3,4,5-tetrachlorobiphenyl, the bioinorganic catalyst promoted a rate of chloride release of 9.33 ± 0.17 nmol min^–1 mg ^–1and only ~5% of this value was obtained using chemically reduced or commercially available Pd ⁰. In the case of 2,2,4,4,6,6-hexachlorobiphenyl the rate was more than four orders of magnitude faster than the degradation reported using a sulfidogenic culture. Negligible chloride release occurred from any of the chloroaromatic compounds using biomass alone, or from palladised biomass challenged with hexane carrier solvent only. Analysis of the spent solution showed that in addition to catalysis of reductive dehalogenation the new material was able to remove very effectively the organic residua, with neither any PCB nor any breakdown products identifiable by GC/MS.Revisions requested 8 September 2004; Revisions received 21 October 2004;     

<Emphasis Type="Italic">In vitro</Emphasis> DNA-binding properties of a manganese response regulator (ManR) from <Emphasis Type="Italic">Anabaena</Emphasis> sp.

ManR of Anabaena sp. PCC 7120 is a manganese response regulator. Two ManR molecules bind to the specific DNA sequences at the same time, which was demonstrated by our previous results. From size exclusion chromatography, ManR exits as monomer in solution. Therefore, cooperative interactions of ManR–ManR play a role in DNA binding of the ManR, suggesting that ManR molecules bind co-operatively to DNA. When serial deletions of N-terminal of the ManR were also carried out the mutant proteins, ManRC111, ManRC130 and ManRC158, had completely lost the in DNA binding activity. Mutants ManRC 196, ManRC206, ManRC221 and ManRC230, however, could specifically bind to DNA, indicating that the amino acid residues between Val₁₆ and Ile₇₈ of the N-terminal of ManR are necessary for the DNA binding activity of C-terminal domain.Revisions requested 20 Ocotober 2004/15 November 2004; Revisions received 10 November/13 December 2004     

Purification and characterization of ferredoxin-NADP<Superscript>+</Superscript> reductase encoded by <Emphasis Type="Italic">Bacillus subtilis yumC</Emphasis>

From Bacillus subtilis cell extracts, ferredoxin-NADP⁺ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with K_m values of 0.57 M vs >200 M. K_cat values of YumC with NADPH were 22.7 s^–1 and 35.4 s^–1 in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.     

Expression of <Emphasis Type="Italic">Escherichia coli</Emphasis> AppA2 phytase in four yeast systems

To develop an effective fermentation system for producing Escherichia coliphytase AppA2, we expressed the enzyme in three inducible yeast systems: Saccharomyces cerevisiae (pYES2), Schizosaccharomyces pombe (pDS472a), and Pichia pastoris (pPICZ A), and one constitutive system: P. pastoris (pGAPZA). All four systems produced an extracellular functional AppA2 phytase with apparent molecular masses ranging from 51.5 to 56 kDa. During 8-day batch fermentation in shaking flasks, the inducible Pichia system produced the highest activity (272 units ml^–1 medium), whereas the Schizo. pombesystem produced the lowest activity (2.8 units ml^–1). The AppA2 phytase expressed in Schizo. pombehad 60–75% lower K_mfor sodium phytate and 28% higher heat-stability at 65 °C than that expressed in other three systems. However, all four recombinant AppA2 phytases had pH optimum at 3.5 and temperature optimum at 55 °C and similar efficacy in hydrolyzing phytate–phosphate from soybean meal.Revisions requested 18 November 2004; Revisions received 7 January 2005     

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     

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     

High level expression of kringle 5 fragment of plasminogen in <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">pastoris</Emphasis>

Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300mgl^-1. Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5.Revisions requested 9 November 2004; Revisions received 2 December 2004     

Oxidation of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids by Acetobacter and Serratia strains

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110g 2-furoic acid l^-1 from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2 biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86--91% molar yields.Revisions requested 21 July 2004; Revisions received 7 September 2004     

Changes of primary sequence and secondary structure proximal to the 5′ end of the stop codon substantially increases the expression of the variable region of an antibody in <Emphasis Type="Italic">E. coli</Emphasis>

The sequence context at the 5 end of the stop codon may influence the efficiency of termination and translation. To increase the expression of a designed variable region of an antibody (named as VH5) against tumor necrosis factor (TNF), two nucleotides (TC) at 25 and 26 nucleotides (nt) upstream of termination codon were substituted with AG, respectively. The free energy of 70 nt (arbitrarily defined from the 32 nt upstream of termination codon to 38 nt downstream) was changed from –13.5 kcal mol^-1 to –17.3 kcal mol^-1. The expression level was increased from 1 ± 0.3% to 10 ± 1.2% of total cellular protein. Although the precise mechanism of this phenomenon remains to be elucidated, this report provides an alternative means to increase the expression of a foreign gene in E. coli. Revisions requested 18 October 2004; Revisions received 26 November 2004     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Comparative study of Cr(VI) uptake and reduction in industrial effluent by Ochrobactrum intermedium and Brevibacterium sp.

Two chromium-resistant bacterial strains, CrT-1 and CrT-13, tolerant up to 40mg K₂CrO₄ ml^–1 on nutrient agar, 25mgml^–1 in nutrient broth, and up to 10mgml^–1 in acetate-minimal media, were identified as Ochrobactrum intermedium and Brevibacterium sp., respectively, on the basis of 16S rRNA gene sequencing. Uptake of chromate was greater in living cells than in heat-killed on dried cells. CrT-1 reduced 82%, 28% and 16% of Cr(VI) at 100, 500, and 1000gml^–1 after 24h while CrT-13 reduced 41%, 14% and 9%. Other heavy metals at low concentrations did not affect these reductions. At 150 and 300gml^–1 in an industrial effluent sample Cr(VI) was reduced by 87% and 71%, respectively, with CrT-1 and by 68% and 47% with CrT-13.Revisions requested 17 May 2004; Revisions received 2 July 2004