Metabolic characteristics and lipid composition of yeastlike cells and mycelium of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of γ-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Activity of NAD-dependent isocitrate dehydrogenase, isocitrate lyase, and malate dehydrogenase in Mucor circinelloides var. lusitanicus INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of gamma-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of gamma-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> malate dehydrogenase,a novel solubility enhancer for heterologous proteins synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.     

Overexpression of the <Emphasis Type="Italic">plg</Emphasis>1 gene encoding pectin lyase in <Emphasis Type="Italic">Penicillium griseoroseum</Emphasis>

The pectin lyase (PL) is an industrially important enzyme since it is used for maceration and clarification in the process of fruit juice production in food industries. In order to increase the yields of pectin lyase we cloned the plg1 (pectin lyase 1) from Penicillium griseoroseum gene under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) and the terminator region of the tryptophan synthetase (trpC) gene from Aspergillus nidulans (plasmid pAN52-Plg1) and transformed this construct into the P. griseoroseum strain PG63. One of the pAN52-Plg1 multi-copy transformants (strain 105) grown in culture medium containing glucose or sugar cane juice showed PL activities of 4,804 or 5,202 U ml⁻¹ respectively, which represented 57- and 132-fold increases. In addition, the apparent specific activity of PL produced by this strain was much higher than the one observed for a commercial pectinase preparation. Evaluation of the extracellular proteins in the culture supernatant of strain 105 by SDS-PAGE showed the presence of a clear and strong band of approximately 40 kDa that probably corresponds to PL. The enzyme yields reported here demonstrate that the system we developed is able to express pectin lyase at levels comparable to, or exceeding, previously reported data.     

Characterization of malate dehydrogenase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrobaculum islandicum</Emphasis>

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K _m values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80°C and pH 8.0) were 15 and 86 μM, respectively, with specific activity as high as 470 U mg⁻¹. Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD⁺ were used as substrates and was not detected with NADP⁺. A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.     

Overexpression of Acetyl-CoA Carboxylase Gene of <Emphasis Type="Italic">Mucor rouxii</Emphasis> Enhanced Fatty Acid Content in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Overexpression of alginate lyase of <Emphasis Type="Italic">Pseudoalteromonas elyakovii</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>, purification,and characterization of the recombinant alginate lyase

The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively. The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl₂, NaCl, KCl, CaCl₂, BaCl₂ and MnCl₂, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO₄, AgNO₃, and CoCl₂. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries.     

Kinetic model of functioning and regulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> isocitrate dehydrogenase

To describe published experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH), a Rapid Equilibrium Random Bi Ter mechanism involving the formation of two dead-end enzyme complexes is proposed and a kinetic model of enzyme functioning is constructed. The enzyme is shown to be regulated through reversible phosphorylation by IDH kinase/phosphatase; the latter, in its turn, is controlled by IDH substrates and also by a number of central metabolites—pyruvate, 3-phosphoglycerate, and AMP—reflecting the energy demand of the cell. Using the model, it is shown that an increase in the concentration of the above effectors raises the fraction of active IDH and thus enhances the Krebs cycle flux. The ratio between the free and the phosphorylated forms of IDH is more sensitive to AMP, NADP, and isocitrate than to pyruvate and 3-phosphoglycerate. The model also predicts changes in the ratio between phosphorylated and active forms of IDH in the Krebs cycle that occur with a change in the energy and biosynthetic loads on E. coli cells.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Identification of the <Emphasis Type="Italic">bkdAB</Emphasis> gene cluster,a plausible source of the starter-unit for virginiamycin M production in <Emphasis Type="Italic">Streptomyces virginiae</Emphasis>

The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain α-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for virginiamycin production. Gene disruption of bkdA completely abolished the production of virginiamycin M (a polyketide-peptide antibiotic), while the production of virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the virginiamycin M production, indicating that the bkdAB cluster is essential for virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the α and β subunits, S. virginiae bkdA seemed to encode the fused form of the α and β subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli. Supply of an additional bkdA gene under the strong and constitutive promoter ermE* in the wild-type strain of S. virginiae resulted in enhanced production of virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of virginiamycin M.