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     

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     

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     

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     

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.     

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     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

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     

Synthesis of (<Emphasis Type="Italic">R</Emphasis>)-2-trimethylsilyl-2-hydroxyl-ethylcyanide catalyzed with (<Emphasis Type="Italic">R</Emphasis>)-oxynitrilase from loquat seed meal

The synthesis of optically active (R)-2-trimethylsilyl-2-hydroxyl-ethylcyanide by asymmetric trans-cyanation of acetyltrimethylsilane with acetone cyanohydrin in a biphasic system was achieved using (R)-oxynitrilase from loquat seed meal. Diisopropyl ether was the most suitable organic phase among the organic solvents examined. The optimal concentration of acetyltrimethylsilane, concentration of crude enzyme, volume ratio of the aqueous to the organic phase, temperature and the buffer pH value were 14 mM, 61.4 U ml^-1, 13% (v/v), 30 °C and 4, respectively. The substrate conversion and the product enantiomeric excess were 95% and 98% under the optimized conditions. Acetyltrimethylsilane was a better substrate of the enzyme than its carbon counterpart. Revisions requested 24 August 2004; Revisions received 12 November 2004     

Kinetic Modelling of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> ssp. <Emphasis Type="Italic">rhamnosus</Emphasis> Growth and Lactic Acid Production in Batch Cultures Under Various Medium Conditions

Enrichment of medium with yeast extract and tryptone increased growth and lactic acid production in batch cultures of Lactobacillus casei ssp. rhamnosus. A reliable kinetic model that explicitly expresses the strong relationship between microbial growth, lactic acid production and medium enrichment is provided and validated using experimental data obtained with six different medium compositions. Revisions requested 2 February 2005 and 26 July 2005; Revisions received 25 July 2005 and 9 September 2005     

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     

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     

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     

Purification and characterization of two ascorbate peroxidases of rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.) expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g^–1 dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min^–1 mg^–1 protein, respectively. The K_m values for ascorbate were 4 and 1 mM, respectively, and those for H₂O₂ were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.Revisions requested 27 September 2004; Revisions received 12 November 2004     

Antioxidant and antibacterial activities of lichen <Emphasis Type="Italic">Usnea ghattensis</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Various solvent extracts of the lichen Usnea ghattensis showed good antioxidant activity. A methanol extract prevented lipid peroxidation by 87% followed by 65% in Trolox at 20 μg/ml. It also showed superoxide anion scavenging activity and free radical scavenging activity 56% and 73%, respectively. The known antioxidants butylated hydroxytoluene (BHT), butylated hydroxyanisol (BHA) and quercetin at similar concentrations showed superoxide anion scavenging activity of 68, 59 and 47% and free radical scavenging activity 83, 77 and 69%, respectively. In addition, these extracts were inhibitory against Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis and Staphylococcus aureus with MIC values of 5–10 μg/ml.Received after revisions 10 May 2005     

Propagation of <Emphasis Type="Italic">Haemaria discolor</Emphasis> via <Emphasis Type="Italic">in vitro</Emphasis> seed germination

In vitro propagation protocol for Haemaria discolor (Ker) Lindl. var. dawsoniana by artificial cross-pollination and asymbiotic germination of seeds has been developed. Fruit set (100 %) was obtained when the pollinia and ovules of various aged flowers were used for pollination. In vitro germination of seeds obtained from capsules of various ages was achieved on half-strength Murashige and Skoog’s (MS) medium supplemented with 3 % sucrose and 0.85 % agar. The germinated seedlings were cultured on half-strength MS medium with 0.2 % activated charcoal, 8 % banana homogenate, 0.1 mg dm⁻³ 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 1 mg dm⁻³ α-naphthaleneacetic acid (NAA). Ninety-six percent of plantlets survived after hardening in greenhouse.This research was supported by grant (91AS-3.1.1-CI-C3) from the Council of Agriculture, Executive Yuan of Taiwan and grants (NSC-89-2317-B055-002 and NSC-91-2317-B324-001) from the National Science Council of Taiwan. This paper is Agricultural Research Institute Contribution No. 2158.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

<Emphasis Type="Italic">Catenaria anguillulae</Emphasis> Sorokin: a Natural Biocontrol Agent of <Emphasis Type="Italic">Meloidogyne graminicola</Emphasis> Causing Root Knot Disease of Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Summary Catenaria anguillulae parasitized and killed the eggs and second stage juveniles (J₂) of Meloidogyne graminicola under natural conditions. The percentage of infection in eggs was higher than J₂ of M.␣graminicola, which ranged between 0–50.3% and 0–18.9% in 2004 and 0–46.6% and 0–21.7% in 2005, respectively. The higher parasitism of eggs and J₂ was recorded from those fields in which plants were severely infected with M. graminicola. The degree of parasitism of eggs and J₂ by C. anguillulae varied with severity of root knot disease. The fields with a higher root gall index recorded a higher percentage of infection in eggs and J₂ of M. graminicola. In general, old galls when teased and incubated, recorded higher parasitism of eggs and juveniles than young galls.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Methyl <Emphasis Type="Italic">tert</Emphasis>-butyl Ether and <Emphasis Type="Italic">tert</Emphasis>-butyl Alcohol Degradation by <Emphasis Type="Italic">Fusarium solani</Emphasis>

Fusarium solani degraded methyl tert-butyl ether (MTBE) and other oxygenated compounds from gasoline including tert-butyl alcohol (TBA). The maximum degradation rate of MTBE was 16 mg protein h and 46 mg/g protein h for TBA. The culture transformed 77% of the total carbon to ¹⁴CO₂. The estimated yield for MTBE was 0.18 g dry wt/g MTBE.