Mitotic recombination between dispersed but related rRNA genes of Schizosaccharomyces pombe generates a reciprocal translocation

Summary Recombination between dispersed yet related serine tRNA genes of Schizosaccharomyces pombe does occur during mitosis but it is approximately three orders of magnitude less frequent than in meiosis. Two mitotic events have been studied in detail. In the first, a sequence of at least 18 nucleotides has been transferred from the donor sup3 gene on the right arm of chromosome I to the related acceptor gene sup12 on the left arm of the same chromosome, thereby leading to the simultaneous change of 8 bp in the acceptor gene. This event must be explained in terms of recombination rather than mutation. It is assumed that it represents mitotic gene conversion, although it was not possible to demonstrate that the donor gene had emerged unchanged from the event. The second case reflects an interaction between sup9 on chromosome III and sup3 on chromosome I. Genetic and physical analysis allows this event to be described as mitotic gene conversion associated with crossingover. The result of this event is a reciprocal translocation. No further chromosomal aberrations were found among an additional 700 potential intergenic convertants tested. Thus intergenic conversion is much less frequently associated with crossingover than allelic conversion. However, the rare intergenic conversion events associated with crossingover provide a molecular mechanism for chromosomal rearrangements.     

链霉菌Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-聚赖氨酸的研究

目的:考察不同细胞培养方式对Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-聚赖氨酸过程的影响。方法:利用两阶段细胞培养和发酵过程流加方式,建立了两阶段细胞培养转化前体L-赖氨酸合成ε-聚赖氨酸以及转化前体L-赖氨酸耦合甘油发酵生产ε-聚赖氨酸的策略。结果:(1)两阶段细胞培养转化前体L-赖氨酸合成ε-聚赖氨酸策略实现ε-PL积累15 g/L, 转化L-赖氨酸3 g/L;(2)转化前体L-赖氨酸耦合甘油发酵生产ε-聚赖氨酸策略使得ε-PL产量达到33.76 g/L,单位菌体的合成能力提高37.8%,转化L-赖氨酸4 g/L。这表明,上述两种方式下前体L-赖氨酸都能够被Streptomyces sp. M-Z18转化合成ε-聚赖氨酸,但转化效率还有待进一步提高。意义:揭示了Streptomyces sp. M-Z18合成ε-聚赖氨酸的限速步骤在于初级代谢产物L-赖氨酸的合成,这为后续利用代谢工程手段改造菌株提供了方向。     

Gene cloning,expression, and characterization of a novel trehalose synthase from <Emphasis Type="Italic">Arthrobacter aurescens</Emphasis>

Trehalose synthase (TreS) is an intramolecular transglycosylase. It specially catalyzes the conversion of maltose and trehalose. In this study, a novel treS gene, which had a length of 1,797 bp and encoded 598 amino acids, was cloned from Arthrobacter aurescens CGMCC 1.1892 and expressed in Escherichia coli. Thin layer chromatography results indicated that it could catalyze the conversion between maltose and trehalose in one step. However, the ion chromatography results showed that, as a byproduct, about 13% glucose was also produced. The purified recombinant enzyme had a molecular weight of 68 kDa and showed its optimal activity at 35 °C and pH 6.5. This enzyme was not thermostable, and its activity was increased by 1 mM Mg²⁺, Mn²⁺, and Ca²⁺ while strongly inhibited by 5 mM Cu²⁺ and SDS.     

The evolution of plasmodial stage-specific rRNA genes is dominated by gene conversion

Summary Plasmodium species exhibit the unprecedented situation of distinct, stage-specific rRNA sequences. We present an analysis of two pairs of sequences of the small rRNA subunit (Plasmodium falciparum andPlasmodium berghei) and show that these genes do not evolve independently and that in fact their evolution is dominated by gene conversion. This analysis also shows that no extensive stage-specific sequences are conserved in the two species, thus rendering unlikely that the existence of stage-specific rRNA genes results from a requirement for distinct rRNA types.     

Conversion of<Emphasis Type="Italic">G. hansenii</Emphasis> PJK into non-cellulose-producing mutants according to the culture condition

The conversion of a cellulose-producing cell (Cel ⁺) fromGluconacetobacter hansenii PJK (KCTC 10505 BP) to a non-cellulose-producing cell (Cel ⁻) was investigated by measuring the colony forming unit (CFU). This was achieved in a shaking flask with three slanted baffles, which exerted a strong shear stress. The addition of organic acid, such as glutamic acid and acetic acid, induced the conversion of microbial cells from a wild type toCel ⁻ mutants in a flask culture. The supplementation of 1% ethanol to the medium containing an organic acid depressed the conversion of the microbial cells toCel ⁻ mutants in a conventional flask without slanted baffles. The addition of ethanol to the medium containing an organic acid; however, accelerated the conversion of microbial cells in the flask with slanted baffles. TheCel ⁺ cells from the agitated culture were not easily converted intoCel ⁻, mutants on the additions of organic acid and ethanol to a flask without slanted baffles, but some portion of theCel ⁺ cells were converted toCel ⁻ mutants in a flask with slanted baffles. The conversion ratio ofCel ⁺ cells toCel ⁻ mutants was strongly related to the production of bacterial cellulose independently from the cell growth.     

Further evidence of a role for abscisic acid in conversion of somatic embryos ofDaucus carota

Summary Somatic embryogenesis has been shown to be an imperfect recapitulation of stages involved to form embryos from vegetative tissues. Although abscisic acid has been implicated in normalizing development, studies that specifically investigate conversion (vegetative leaf initiation) in somatic embryos are lacking. This report documents a follow-up of a study that implicated abscisic acid as a vital factor in allowing embryos ofDaucus carota to progress to the plantlet stage. Abscisic acid was determined to enhance conversion at doses ranging from 1 to 50 &#x00B5;M. Younger embryo stages were more responsive to abscisic acid application with regards to plantlet recovery. Pulses of abscisic acid were shown to elicit more rapid response with younger embryo stages, indicating more plastic development. Fluridone, an abscisic acid synthesis inhibitor, was shown to dramatically reduce conversion, even at low doses (&lt;5&#x00B5;M). When abscisic acid was applied concurrently with fluridone, partial restoration of conversion was observed. Histologically, fluridone was seen to cause pronounced vacuolation in the shoot apical notch which resulted in the loss of meristematic cells, negating conversion capacity. Quantitation of total cytoplasmic area showed that abscisic acid reduced vacuolar intrusion into the apical notch, while fluridone caused a significant increase in vacuolation of cells in this region. This report documents further evidence of a role for abscisic acid in plantlet establishment from somatic embryos ofDaucus carota.     

Stereoselective epoxidation of <Emphasis Type="Italic">cis</Emphasis>-propenylphosphonic acid to fosfomycin by a newly isolated bacterium <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">simplex</Emphasis> strain S101

In industry, fosfomycin is mainly prepared via chemical epoxidation of cis-propenylphosphonic acid (cPPA). The conversion yield of fosfomycin is less than 50% in the whole process and a large quantity of waste is produced. Biotransformation by microorganisms is an alternative method of preparation. This kind of conversion is more delicate, environmentally friendly, and the conversion yield of fosfomycin would be higher. In this work, an aerobic bacterium capable of transforming cPPA to fosfomycin was isolated. The organism, designated as strain S101, was identified as Bacillus simplex by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. Fosfomycin was assayed by two means, bioassay and gas chromatography (GC). Glycerol was a good carbon source for growth and cPPA conversion of strain S101. When cPPA was used as the sole carbon source, neither growth nor conversion to fosfomycin occurred. The optimum cPPA concentration in the conversion medium was 2,000 μg ml⁻¹. After 6 days of incubation, the concentration of fosfomycin reached its maximum level (1,838.2 μg ml⁻¹), with a conversion ratio of 81.3%. Air was indispensable for the growth but not for the conversion to fosfomycin. Furthermore, vanadium ions were found to be essential for the conversion. High concentrations of cPPA had fewer inhibitory effects on the growth of strain S101.     

Metabolic engineering of Thermobifida fusca for direct aerobic bioconversion of untreated lignocellulosic biomass to 1-propanol

Biofuel production from renewable resources can potentially address lots of social, economic and environmental issues but an efficient production method has yet to be established. Combinations of different starting materials, organisms and target fuels have been explored with the conversion of cellulose to higher alcohols (1-propanol, 1-butanol) being one potential target. In this study we demonstrate the direct conversion of untreated plant biomass to 1-propanol in aerobic growth conditions using an engineered strain of the actinobacterium, Thermobifida fusca. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase was added to T. fusca leading to 1-propanol production during growth on glucose, cellobiose, cellulose, switchgrass and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. These results represent the first demonstration of direct conversion of untreated lignocellulosic biomass to 1-propanol in an aerobic organism and illustrate the potential utility of T. fusca as an aerobic, cellulolytic bioprocess organism.     

Conversion of an RFLP marker for the barley stem rust resistance geneRpg1 to a specific PCR-amplifiable polymorphism

TheRpg1 gene in barley has provided satisfactory levels of stem rust resistance for the last 50 years. The appearance of a new race of stem rust that is virulent toRpg1 has resulted in efforts to incorporate new stem rust resistance genes into barley. Marker-assisted selection may provide the only means of combining this useful gene with resistance genes for which no virulent races have been identified. Several RFLP markers have been identified as linked to theRpg1 locus. One of these, ABG704 was converted into a post-amplification restriction polymorphism. To generate a specific PCR-amplifiable polymorphism the sequence of the ABG704 locus from four barley cultivars was determined. Primers were developed that can detect a single-base difference between resistant and susceptible cultivars. The successful conversion of an RFLP marker to an allele-specific PCR-based marker not only demonstrates that this type of conversion is possible for cereals, but also results in an immediately useful marker for application to plant breeding programmes.     

Expression of a cephalosporin C esterase gene in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> for the direct production of deacetylcephalosporin C

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.     

Molecular cloning and differential expression of an γ-aminobutyrate transaminase gene, OsGABA-T, in rice (Oryza sativa) leaves infected with blast fungus

γ-Aminobutyrate transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde. Using differential display PCR and cDNA library screening, a full-length GABA-T cDNA (OsGABA-T) was isolated from rice (Oryza sativa) leaves infected with an incompatible race of Magnaporthe grisea. The deduced amino acid sequence comprises 483 amino acid residues and shares 85–69% identity with GABA-T sequences from other plants. OsGABA-T expression is induced by blast fungus infection, mechanical wounding and ultraviolet radiation in rice leaves and is not detected in normal rice organs. This gene is also induced by defense signal molecules such as salicylic acid and abscisic acid, but not by jasmonic acid. Our data suggest that OsGABA-T (GABA shunt) may play a role in restricting the levels of cell death during the host–pathogen interaction.     

Anti-Prelog Reduction of Prochiral Carbonyl Compounds by Oenococcus oeni in a Biphasic System

An aqueous-organic biphasic system was established and used with whole cells of Oenococcus oeni to reduce 2-octanone to (R)-2-octanol. The conversion reached 99% when the Tris/borate buffer was increased from 50 mM to 300 mM in the aqueous phase. In addition, the conversion increased as the log P value of the organic solvent changed from 0.5 to 6.6. Under optimized conditions, the conversion of (R)-2-octanol reached 99% from 0.5 M 2-octanone with an optical purity of 99% e.e. The biphasic system allows the anti-Prelog reduction of aliphatic and aromatic ketones to furnish (R)-configurated alcohols in high optical purity as well.     

Propagation of mulberry variety – S54 by synseeds of axillary bud

Synseeds were produced from aseptic axillary buds of mulberry variety – S₅₄ (Morus indica L.) to investigate their in vitro and ex vitro conversion potential. The synseeds when cultured on Linsmaier and Skoog's basal medium supplemented with 6-benzyl amino purine (8.88 μM) and 2,3,5-tri iodo benzoic acid (2 μM), produced shoots after 21 days exhibiting 48.2±0.60 in vitro conversion response. The synseeds ex␣vitro conversion response was found to be 45.5±0.76 on soilrite mix containing half strength LS nutrients after 21 days of sowing. Further, synseeds stored at 4 °C for 1–4 months resulted in maximum conversion response under both in vitro and ex vitro conditions, followed by development into complete plantlets without any significant loss of conversion potential. Plants regenerated from the synseeds of axillary bud were hardened, acclimatized and established in soil with varying survival frequency.     

Cloning of a Novel Omega-6 Desaturase from Flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and Its Functional Analysis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.     

The growth of external AM fungal mycelium in sand dunes and in experimental systems

We estimated the biomass and growth of arbuscular mycorrhizal (AM) mycelium in sand dunes using signature fatty acids. Mesh bags and tubes, containing initially mycelium-free sand, were buried in the field near the roots of the dune grass Ammophila arenaria L. AM fungal mycelia were detected at a distance of about 8.5 cm from the roots after 68 days of growth by use of neutral lipid fatty acid (NLFA) 16:1ω5. The average rate of mycelium extension during September and October was estimated as 1.2 mm day⁻¹. The lipid and fatty acid compositions of AM fungal mycelia of isolates and from sand dunes were analysed and showed all to be of a similar composition. Phospholipid fatty acids (PLFAs) can be used as indicators of microbial biomass. The mycelium of G. intraradices growing in glass beads contained 8.3 nmol PLFAs per mg dry biomass, and about 15% of the PLFAs in G. intraradices, G. claroideum and AM fungal mycelium extracted from sand dunes, consisted of the signature PLFA 16:1ω5. We thus suggest a conversion factor of 1.2 nmol PLFA 16:1ω5 per mg dry biomass. Calculations using this conversion factor indicated up to 34 μg dry AM fungal biomass per g sand in the sand dunes, which was less than one tenth of that found in an experimental system with Glomus spp. growing with cucumber as plant associate in agricultural soil. The PLFA results from different systems indicated that the biomass of the AM fungi constitutes a considerable part of the total soil microbial biomass. Calculations based on ATP of AM fungi in an experimental growth system indicated that the biomass of the AM fungi constituted approximately 30% of the total microbial biomass. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lipase-catalysed synthesis of olvanil in organic solvents

The release rate of vanillylamine from its hydrochloride salt was the limiting step in the lipase-catalysed synthesis of olvanil, a capsaicin analogue amide, in organic solvents. When the tertiary amine base concentration (N,N-diisopropylethylamine) was increased from 20 mM to 360 mM, the initial rate of amide synthesis increased proportionally. At a 12 molar excess of N,N-diisopropylethylamine and 30 min of preincubation, both the initial rate and total conversion were the same as those with free vanillylamine (80% conversion in 20 h). This result was independent of the organic solvent used. It is also shown that N,N-diisopropylethylamine does not enhance lipase activity.     

Enhanced aerobic respiration improves <Emphasis Type="Italic">in vitro</Emphasis> coconut embryo germination and culture

Summary Germination of coconut (Cocos nucifera L.) zygotic embryos was tested in liquid or solid medium. Percent germination was greater when embryos were cultured in solid medium, particularly when embryos were placed with their micropyle end facing upwards in relation to the vial orientation, independently of orientation in relation to gravity. This occurred because solid medium allowed embryos to be positioned with their micropyle end exposed to the ambient atmosphere of the vial. Germination of embryos facing upwards was suppressed when the ambient atmosphere was replaced with N₂ or when aerobic respiration inhibitors were added to the medium, whereas it was not suppressed when the atmosphere was replaced with a N₂/O₂ (50∶50, v/v) mixture, thus indicating that aerobic respiration is required for embryos to germinate. Germination in facing upwards mode also resulted in improved conversion and plantlet development. These improvements in recovery are important since they will help to avoid unintentional selection due to low in vitro germination and corresponding conversion of a population subset. These results set the basis for the improvement of current protocols for in vitro coconut embryo culture, allowing safe movement of germoplasm both in terms of phytosanitation and conservation of genetic diversity in a population of interest.     

Effect of Solvents and Kinetic Parameters on Synthesis of Ethyl Propionate Catalysed by Poly (AAc-co-HPMA-cl-MBAm)-Matrix-Immobilized Lipase of Pseudomonas aeruginosa BTS-2.

Summary A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa BTS-2 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel network. The hydrogel showed approximately 95% binding efficiency for lipase (specific activity 1.96 U mg⁻¹). The immobilized enzyme achieved 65.1% conversion of ethanol and propionic acid (100 mM each) into ethyl propionate in n-nonane at 65 °C in 9 h. When alkane of C-chain length lower than n-nonane was used as the organic solvent, the conversion of ethanol and propionic acid into ethyl propionate decreased with a decrease in the log P value of alkanes. The immobilized lipase retained approximately 30% of its original catalytic activity after five cycles of reuse for esterification of ethanol and propionic acid into ethyl propionate at temperature 65 °C in 3 h. Addition of a molecular sieve (3 ?) to the reaction mixture enhanced the formation of ethyl propionate to 89.3%. Moreover, ethanol and propionic acid when taken a molar ratio of 3:1 further promoted the conversion rate to 94%. However, an increase in the molar ratio of propionic acid with respect to ethanol resulted in a decline of ethyl propionate synthesis.