Anaerobic co-metabolic oxidation of 4-alkylphenols with medium-length or long alkyl chains by <Emphasis Type="Italic">Thauera</Emphasis> sp., strain R5

A 4-alkylphenol-degrading facultative anaerobic bacterium, strain R5, was isolated from paddy soil after enrichment with 4-n-propylphenol, 4-n-butylphenol and 4-hydroxybenzoate (4-HBA) under nitrate-reducing conditions. Strain R5 is a Gram-negative rod bacillus grown on phenolic compounds with short alkyl chains (≤C2), organic acids and ethanol. The sequence of the 16S ribosomal RNA gene revealed that the strain is affiliated with Thauera sp. In the presence of 4-HBA as a carbon source, the strain transformed 4-n-alkylphenols with a medium or long-length alkyl chain (C3–C8) to the corresponding oxidised products as follows: 1-(4-hydroxyphenyl)-1-alkenes, -(4-hydroxyphenyl)-1-alkanones and/or 1-(4-hydroxyphenyl)-1-alcohols. The strain also transformed 4-i-propylphenol and 4-sec-butylphenol to (4-hydroxyphenyl)-i-propene and (4-hydroxyphenyl)-sec-butene but not 4-alkylphenols with tertiary alkyl chains (4-t-butylphenol or 4-t-octylphenol). The biotransformation did not proceed without another carbon source and was coupled with nitrate reduction. Biotransformation activity was high in the presence of p-cresol, 4-ethylphenol, 4′-hydroxyacetophenone and 4-HBA as carbon sources and low in the presence of organic acids and ethanol. We suggest that strain R5 co-metabolically transforms alkylphenols to the corresponding metabolites with oxidised alpha carbon in the alkyl chain during coupling with nitrate reduction.     

Cresols utilization by <Emphasis Type="Italic">Trametes versicolor</Emphasis> and substrate interactions in the mixture with phenol

The ability of the white rot fungus Trametes versicolor strain 1 to degrade and utilize methylated phenols (cresols) was established for the first time in a medium not containing any other carbon components. The data obtained demonstrated the better potential of the strain to assimilate p-cresol instead of o- or m- cresol. The 0.5 g/l p-cresol provided was degraded in full after 96 h. The effect of a dual substrate mixture (0.3 g/l phenol + 0.2 g/l p-cresol) on the growth behavior and degradation capacity of the investigated strain was examined. The cell-free supernatants were analyzed by HPLC. It was established that the presence of p-cresol had not prevented complete phenol degradation but had a significant delaying effect on the phenol degradation dynamics. Phenol hydroxylase, catechol 1.2-dioxygenase and cis,cis-muconate cyclase activities were obtained in conditions of single and mixed substrates cultivation. The influence of different phenolic substrates on phenol hydroxylase activity in Trametes versicolor 1 was established. The mathematical models describing the dynamics of single substrates’ utilization as well as the mutual influence of phenol and p-cresol in the mixture were developed on the bases of Haldane kinetics. The estimated interaction coefficients (I _ph/cr = 4.72, I _cr/ph = 7.46) demonstrated the significant inhibition of p-cresol on phenol biodegradation and comparatively low level of influence of phenol presence on the p-cresol degradation. Molecular 18S RNA gene taxonomy of the investigated strain was performed.     

Degradation of 2-sec-butylphenol: 3-sec-butylcatechol,2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid,and 2-methylbutyric acid as intermediates

Pseudomonas sp. strain HBP1 Prp, a mutant of strain HBP1 that was originally isolated on 2-hydroxybiphenyl, was able to grow on 2-sec-butylphenol as the sole carbon and energy source. During growth on 2-sec-butylphenol, 2-methylbutyric acid transiently accumulated in the culture medium. Its concentration reached a maximum after 20 hours and was below detection limit at the end of the growth experiment. The first three enzymes of the degradation pathway — a NADH-dependent monooxygenase, a metapyrocatechase, and ameta-fission product hydrolase — were partially purified. The product of the the monooxygenase reaction was identified as 3-sec-butylcatechol by mass spectrometry. This compound was a substrate for the metapyrocatechase and was converted to 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid which was identified by gas chromatography-mass spectrometry of its trimethylsilyl-derivative. The cofactor independentmeta-cleavage product hydrolase used 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid as a substrate. All three enzymes showed highest activities for 2-hydroxybiphenyl and its metabolites, respectively, indicating that 2-sec-butylphenol is metabolized via the same pathway as 2-hydroxybiphenyl.     

Isolation and Characterization of 4-tert-Butylphenol-Utilizing Sphingobium fuliginis Strains from Phragmites australis Rhizosphere Sediment

We isolated three Sphingobium fuliginis strains from Phragmites australis rhizosphere sediment that were capable of utilizing 4-tert-butylphenol as a sole carbon and energy source. These strains are the first 4-tert-butylphenol-utilizing bacteria. The strain designated TIK-1 completely degraded 1.0 mM 4-tert-butylphenol in basal salts medium within 12 h, with concomitant cell growth. We identified 4-tert-butylcatechol and 3,3-dimethyl-2-butanone as internal metabolites by gas chromatography-mass spectrometry. When 3-fluorocatechol was used as an inactivator of meta-cleavage enzymes, strain TIK-1 could not degrade 4-tert-butylcatechol and 3,3-dimethyl-2-butanone was not detected. We concluded that metabolism of 4-tert-butylphenol by strain TIK-1 is initiated by hydroxylation to 4-tert-butylcatechol, followed by a meta-cleavage pathway. Growth experiments with 20 other alkylphenols showed that 4-isopropylphenol, 4-sec-butylphenol, and 4-tert-pentylphenol, which have alkyl side chains of three to five carbon atoms with α-quaternary or α-tertiary carbons, supported cell growth but that 4-n-alkylphenols, 4-tert-octylphenol, technical nonylphenol, 2-alkylphenols, and 3-alkylphenols did not. The rate of growth on 4-tert-butylphenol was much higher than that of growth on the other alkylphenols. Degradation experiments with various alkylphenols showed that strain TIK-1 cells grown on 4-tert-butylphenol could degrade 4-alkylphenols with variously sized and branched side chains (ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, tert-octyl, n-nonyl, and branched nonyl) via a meta-cleavage pathway but not 2- or 3-alkylphenols. Along with the degradation of these alkylphenols, we detected methyl alkyl ketones that retained the structure of the original alkyl side chains. Strain TIK-1 may be useful in the bioremediation of environments polluted by 4-tert-butylphenol and various other 4-alkylphenols.4-tert-Butylphenol is an alkylphenol with a tertiary branched side chain of four carbon atoms at the para position of phenol. It is an industrially important chemical and is abundantly and widely used for the production of phenolic, polycarbonate, and epoxy resins. Production of 4-tert-butylphenol in the European Union in 2001 was 25,251 tons (t) (9). In Japan, according to the National Institute of Technology and Evaluation (http://www.safe.nite.go.jp/english/sougou/view/ComprehensiveInfoDisplay_en.faces), production of 4-tert-butylphenol amounted to 27,761 t in 2007. 4-tert-Butylphenol is widely distributed in aquatic environments, including river waters (20), seawaters (17), river sediments (17), marine sediments (23), and effluent samples from sewage treatment plants and wastewater treatment plants (22). Furthermore, 4-tert-butylphenol interacts with estrogen receptors (29, 30, 34, 35, 39) and exhibits other toxic effects on aquatic organisms and humans (4, 15, 16, 25, 26, 42, 43). Therefore, it is necessary to study the biodegradation of 4-tert-butylphenol to understand its fate in the aquatic environment, to establish technologies to treat the waters polluted by it, and to remove it from contaminated environments.Studies of the biodegradation of alkylphenols have focused mainly on branched 4-nonylphenol. Several strains of sphingomonad bacteria, including Sphingomonas sp. strain TTNP3 (38), Sphingobium xenophagum Bayram (11), and Sphingomonas cloacae S-3^T (10), have recently been isolated from activated sludge. These strains can degrade branched 4-nonylphenol and utilize it as a sole carbon source. In addition, several Pseudomonas strains that can degrade medium-chain 4-n-alkylphenols (e.g., 4-n-butylphenol) and utilize them as sole carbon sources have been isolated from activated sludge or contaminated soil; they include Pseudomonas veronii INA06 (1), Pseudomonas sp. strain KL28 (21), and Pseudomonas putida MT4 (36). Biodegradation of branched 4-nonylphenol and 4-n-butylphenol has been well studied, but little is known about the biodegradation of 4-tert-butylphenol, although this compound has a structure similar to those of branched 4-nonylphenol and 4-n-butylphenol. There is only one report on the biotransformation of 4-tert-butylphenol—by resting cells of S. xenophagum strain Bayram grown on technical nonylphenol—but this strain cannot grow on 4-tert-butylphenol (11, 14). To our knowledge, there are no reports of bacteria that utilize 4-tert-butylphenol as the sole carbon source, and the biochemical pathway of 4-tert-butylphenol utilization has not been described.Here we characterize three Sphingobium fuliginis strains—TIK-1, TIK-2, and TIK-3—isolated from rhizosphere sediment of the reed Phragmites australis. These strains could use 4-tert-butylphenol as a sole carbon source. On the basis of additional tests of strain TIK-1, we propose that it degrades 4-tert-butylphenol through 4-tert-butylcatechol along a meta-cleavage pathway. We also show that strain TIK-1 cells grown on 4-tert-butylphenol can degrade a wide range of 4-alkylphenols via a meta-cleavage pathway.     

Biodegradation of phenol and <Emphasis Type="Italic">m</Emphasis>-cresol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 under anaerobic condition

Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.     

The influence of predator regime on reproductive traits in <Emphasis Type="Italic">Gammarus</Emphasis> <Emphasis Type="Italic">pulex</Emphasis> populations

Selection on traits conferring reduced predation may be opposed by selection on other traits associated with reproduction. Here, we examined the hypothesis that traits associated with reproduction in Gammarus pulex are driven by predation. We studied G. pulex originating from ponds with two different kinds of predator regimes: (1) ponds with fish—often large, non-gap-limited predators and (2) ponds without fish where invertebrates are the dominant predators—often small, gap-limited predators with a much more restricted prey size range. We examined the body size of males and females in G. pulex amplexus pairs originating from fish and fishless ponds. We also examined, in the laboratory, their mating success, the number of offspring per female and offspring mortality under different rearing conditions, with or without fish cue. Mating success, defined as the percentage of amplexus pairs that produced live offspring, was higher for G. pulex from fishless ponds independent of rearing condition. Individuals from fish ponds were larger and they produced a higher number of offspring which tended to be related to female body size. Offspring mortality was higher in populations from fish ponds compared to populations from fishless ponds. Despite the higher offspring mortality, females from fish ponds had a higher number of offspring alive after 13 weeks, which is the approximate time it takes for G. pulex to reach maturity. Our data imply that no trade-off between reducing body size to reduce mortality caused by fish and maximising reproductive success exist in G. pulex from fish ponds. The strategy with many offspring may be the correct strategy in fishponds where predation pressure generally is higher than in fishless ponds.     

Morphological and molecular characterization of two ITS groups of <Emphasis Type="Italic">Erysiphe</Emphasis> (Erysiphales) occurring on <Emphasis Type="Italic">Syringa</Emphasis> and <Emphasis Type="Italic">Ligustrum</Emphasis> (Oleaceae)

ITS sequences determined for 53 Erysiphe specimens on Syringa and Ligustrum collected in Europe, East Asia, and North and South America were divided into two ITS groups, S and K types. Phylogenetic analysis showed that these two ITS types do not share a common ancestor and form separate clades. The K type on Ligustrum was identified as Erysiphe ligustri based on the three-dimensional branching pattern of appendages. Morphological observations showed that there are some morphological differences—pigmentation of appendages and number of ascospores per ascus—between the S and K types on Syringa. Based on these morphological observations, the S and K types on Syringa were identified as E. syringae and E. syringae-japonicae, respectively. The recent abundant production of chasmothecia by lilac powdery mildew in Europe was caused by E. syringae-japonicae introduced from East Asia. DNA sequence analyses of the rDNA ITS region and the 28S rDNA, tub2, CYP51, and Chs1 genes did not support an interspecific hybrid origin for E. syringae-japonicae. Haplotype analysis suggested that E. syringae originated in North America and independently migrated to East Asia and Europe/South America.     

Bio-hydrogen production from cellulose by sequential co-culture of cellulosic hydrogen bacteria of <Emphasis Type="Italic">Enterococcus gallinarum</Emphasis> G1 and <Emphasis Type="Italic">Ethanoigenens harbinense</Emphasis> B49

Microbial conversion of lignocellulose to hydrogen is a fascinating way to provide a renewable energy source. A mesophilic bacterium strain G1 that had high cellulose degradation and hydrogen production activity (2.38 mmol H₂ g⁻¹ cellulose) was isolated from rumen fluid and identified as the Enterococcus gallinarum. Hydrogen production from cellulose by using sequential co-cultures of a cellulosic-hydrolysis bacterium G1 and Ethanoigenens harbinense B49 was investigated. With an initial Avicel concentration of 5 g l^−l, the sequential co-culture with G1 and strain Ethanoigenens harbinense B49 produced H₂ yield approximately 2.97 mmol H₂ g⁻¹ cellulose for the co-culture system.     

Response to different oxidants of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">ure2Δ</Emphasis> mutant

Growth of Saccharomyces cerevisiae ure2Δ mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H₂O₂ was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Δ mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the γ-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level.     

Synthesis of orthogonally protected (<Emphasis Type="Italic">3R</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)- and (<Emphasis Type="Italic">3S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

Synthesis and use of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Kinetic limitations during the simultaneous removal of <Emphasis Type="Italic">p</Emphasis>-cresol and sulfide in a denitrifying process

The aim of this study was to evaluate the capacity of a denitrifying consortium to achieve the simultaneous removal of nitrate, sulfide and p-cresol and elucidate the rate-limiting steps in the mixotrophic process. Nitrite reduction appeared as the most evident rate-limiting step in the denitrifying respiratory process. The nitrite reduction rate achieved was up to 57 times lower than the nitrate reduction rate during the simultaneous removal of sulfide and p-cresol. Negligible accumulation of N₂O occurred in the denitrifying cultures corroborating that nitrite reduction was the main rate-limiting step of the respiratory process. A synergistic effect of nitrate and sulfide is proposed to explain the accumulation of nitrite. The study also points at the oxidation of S⁰ as another rate-limiting step in the denitrifying process. Different respiratory rates were achieved with the distinct electron donors provided (p-cresol and sulfide). The oxidation rate of p-cresol (q_CRES) was generally higher (up to 2.6-fold in terms of reducing equivalents) than the sulfide oxidation rate (q_S2−), except for the experiments performed at 100 mg S²⁻ L⁻¹ in which q_S2− was slightly (~1.4-fold in terms of reducing equivalents) higher than q_CRES. The present study provides kinetic information, which should be considered when designing and operating denitrifying reactors to treat industrial wastewaters containing large amounts of sulfurous, nitrogenous and phenolic contaminants such as those generated from petrochemical refineries.     

Comparative analysis of respiratory activity in the wild type strain of <Emphasis Type="Italic">Neurospora crassa</Emphasis> and its photoreceptor complex mutants

Cell respiratory activity of protoplasts obtained from the wild type of Neurospora crassa and photoreceptor complex WCC—white collar 1 (wc-1) and white collar 2 (wc-2)—mutants of Neurospora crassa strains was investigated. Respiration inhibition by KCN in the presence of 25 mM succinate was similar in all strains and did not exceed 83–85% against control. The significant induction of KCN-resistant respiratory pathway occurred under 1% glucose oxidation in wc-1 and wc-2 mutants if compared with the wild type strains. The inhibitors of the main (cytochrome) pathway of electron transfer in mitochondria—1 mM KCN and antimycin A (4 μg/ml)—blocked the respiration rate of the protoplasts from N. crassa wild type by 75%, while the cell respiration of wc-1 and wc-2 strains was suppressed by approximately 50%. The specific inhibitor of alternative oxidase—10 mM salicylhydroxamic acid (SHAM)—in combination with the blockers of mitochondrial electron transfer chain caused the total suppression of respiratory activity of protoplasts in all studied strains. It is supposed that an increase of KCN-resistance in WCC mutants under glucose oxidation is connected with alternative oxidase activation as the result of failure in reception and signal transduction of active oxygen species.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

HMG-CoA reductase limits artemisinin biosynthesis and accumulation in <Emphasis Type="Italic">Artemisia annua</Emphasis> L. plants

In vivo modulation of HMG-CoA reductase (HMGR) activity and its impact on artemisinin biosynthesis as well as accumulation were studied through exogenous supply of labeled HMG-CoA (substrate), labeled MVA (the product), and mevinolin (the competitive inhibitor) using twigs of Artemisia annua L. plants collected at the pre-flowering stage. By increasing the concentration (2–16 μM) of HMG-CoA (3-¹⁴C), incorporation of labeled carbon into artemisinin was enhanced from 7.5 to 17.3 nmol (up to 130%). The incorporation of label (¹⁴C) into MVA and artemisinin was inhibited up to 87.5 and 82.9%, respectively, in the presence of 200 μM mevinolin in incubation medium containing 12 μM HMG-CoA (3-¹⁴C). Interestingly, by increasing the concentration of MVA (2-¹⁴C) from 2 to 18 μM, incorporation of label (¹⁴C) into artemisinin was enhanced from 10.5 to 35 nmol (up to 233%). When HMG-CoA (3-¹⁴C) concentration was increased from 12 to 28 μM in the presence of 150 μM mevinolin, the inhibitions in the incorporation of label (¹⁴C) into MVA and artemisinin were, however, reversed and the labels were found to approach their values in twigs fed with 12 μM HMG-CoA (3-¹⁴C) without mevinolin. In another experiment, 14.2% inhibition in artemisinin accumulation was observed in twigs in the presence of 175 μM fosmidomycin, the competitive inhibitor of 1-deoxy-d-xylulose 5-phosphate reductase (DXR). HMG-CoA reductase activity and artemisinin accumulation were also increased by 18.6 to 24.5% and 30.7 to 38.4%, respectively, after 12 h of treatment, when growth hormones IAA (100 ppm), GA₃ (100 ppm) and IAA + GA₃ (50 + 50 ppm) were sprayed on A. annua plants at the pre-flowering stage. The results obtained in this study, hence, demonstrate that the mevalonate pathway is the major contributor of carbon supply to artemisinin biosynthesis and HMGR limits artemisinin synthesis and its accumulation in A. annua plants.     

Substrate specificity characterization of a thermostable keratinase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> KS-1

A feather-degrading strain of Pseudomonas aeruginosa KS-1 was used in the present study. Its crude cell-free fermentation broth completely degraded chicken feather within 12 h, in the absence of disulphide reductase activity. Keratinase from its extracellular broth was purified and characterized, assuming that it would be a potential β-keratin-degrading enzyme with prospective applications in degradation of β-plaques of prions. The keratinase was purified by using Q-Sepharose anion exchange chromatography and its molecular weight, as determined by SDS–PAGE analysis, was 45 kDa. It was an alkaline, serine protease with pH and temperature optima of 9 and 60°C, respectively. The enzyme was highly thermostable with a t _1/2 > 2 h at 80°C and had a very high K to C (keratinolytic to caseinolytic) ratio of 2.5. Besides feather keratin, it also hydrolyzed a variety of other complex substrates including fibrin, gelatin and meat protein. Its activity on synthetic substrates revealed that it efficiently cleaves them in the order phenylalanine > lysine > alanine > leucine p-nitroanilides. It also cleaved insulin B chain between Val¹²-Glu¹³, Ala¹⁴-Leu¹⁵, Gly²⁰-Glu²¹ and Arg²²-Gly²³ residues.     

Biotechnological production of itaconic acid and its biosynthesis in <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields—dental, ophthalmic, and drug delivery—to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.     

Liquid overlaying improves somatic embryogenesis in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Somatic embryos were induced from in vitro germinating seed-hypocotyls of Catharanthus roseus. The process of embryogenesis has been categorized into a few distinct stages (induction, proliferation, maturation and germination) in which liquid overlaying at varying levels 0 ml (T₀), 0.25 ml (T₁), 0.5 ml (T₂)_, 0.75 ml (T₃) and 1.0 ml (T₄) was applied on solid medium. It was found that liquid overlaying improved proliferation; maturation, germination of embryos in C. roseus. In proliferation stage, particularly in T₂, torpedo embryo number increased significantly (i.e. 129.6%) as compared to control. Liquid overlaying (T₂, T₃ and T₄) also improved embryo maturation and showed early germination even in maturation medium. It also accelerated normal embryo germination frequency particularly in treatment with T₂ and shortened ‘embryo—plantlet’ recovery time. Biochemical analyses revealed more proline, protein and amino acid with increasing level of liquid overlaying as it improved embryo induction, development and faster germination.     

Water relations advantages for invasive <Emphasis Type="Italic">Rubus</Emphasis> <Emphasis Type="Italic">armeniacus</Emphasis> over two native ruderal congeners

Despite species in the Rubus fruticosus complex (wild blackberry) being among the most invasive plants globally in regions with large annual fluctuations in water availability, little is known about their water relations. We compared water relations of a prominent member of the complex, R. armeniacus (Himalayan blackberry), with species native to the Pacific Northwest of North America (PNW), R. spectabilis (salmonberry) and R. parviflorus (thimbleberry). In eight stands of each species located near Portland, Oregon, USA, we measured mid-day hydraulic resistance (R _plant), and daily time series of stomatal conductance (g _s), leaf water potential (Ψ_lf), and environmental conditions at four time periods spanning the 2007 growing season. Although all species maintained Ψ_lf above −0.5 MPa in spring, R. armeniacus maintained less negative Ψ_lf (≥−1.0 MPa) than the natives in summer, a factor attributable to advantages in both its root and shoot systems. R _plant of R. armeniacus was ≤0.1 MPa mmol⁻¹ m² s for the duration of the study, and approximately 25–50% of R _plant for the native species in summer. R. armeniacus had higher g _s compared to the native species throughout the spring and summer, with approximately twice their rates in summer. Our R _plant and g _s results show that R. armeniacus has access to more water during PNW summers than congeneric natives, allowing it to maintain high water-use, and potentially helping it achieve higher growth and reproductive rates. Water relations may therefore be a critical component of the competitive and invasive success of R. armeniacus and other R. fruticosus species worldwide.