Isolation and functional characterization of <Emphasis Type="Italic">Lycopene β-cyclase</Emphasis> (<Emphasis Type="Italic">CYC-B</Emphasis>) promoter from <Emphasis Type="Italic">Solanum habrochaites</Emphasis>

Background  

Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061.     

Cloning and functional expression of thermostable β-glucosidase gene from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis>

A thermostable β-glucosidase (BGLI) was purified from Thermoascus aurantiacus IFO9748, and the gene (bgl1) encoding this enzyme was cloned and expressed in yeast Pichia pastoris. The deduced amino acid sequence encoded by bgl1 showed high similarity with the sequence of glycoside hydrolase family 3. The recombinant enzyme was purified and subjected to enzymatic characterization. Recombinant BGLI retained more than 70% of its initial activity after 1 h of incubation at 60°C and was stable in the pH range 3–8. The optimal temperature for enzyme activity was about 70°C and the optimal pH was about 5. P. pastoris expressing recombinant BGLI became able to utilize cellobiose as a carbon source.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Cloning,expression, and characterization of <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Exiguobacterium</Emphasis> sp. DAU5 and transglycosylation activity

A gram-negative bacterium, designated strain DAU5, was isolated from shrimp shell samples because it demonstrated high β-glucosidase activity. Through 16S rDNA gene sequence analysis the strain was identified as belonging to the genus Exiguobacterium. The β-glucosidase gene of Exiguobacterium sp. DAU5 was successfully cloned by the shotgun method. Nucleotide sequence determination by sodium dodecyl sulfate-ployacrylamide gel electrophoresis indicated that the gene for the enzyme contained 1,350 bp, was coded by 450 amino acids, and was 52 kDa. The polypeptide exhibits significant homology with other bacterial β-glucosidases and belongs to the Glycoside Hydrolase Family 1. The β-glucosidase was purified by a His-fusion purification system. The optimal pH and temperature of the enzyme were 7.0 and 45°C, respectively. The enzyme activity was strongly inhibited by Ca²⁺, and Li⁺, K⁺, Zn²⁺, Mg²⁺, Na²⁺, Ni²⁺, and EDTA partially inhibited the enzyme activity. The BglA showed the highest activity with p-NPG and MUG. However, strain DAU5 β-glucosidase, which is for degradation of oligosaccharides, is expected to be useful for the fermentation of cellulose degradation and the transglycosylation of saccharides.     

Cloning and characterization of a functional flavanone-3ß-hydroxylase gene from <Emphasis Type="Italic">Medicago truncatula</Emphasis>

As a key enzyme in the biosynthesis of flavonols, anthocyanidins and proanthocyanidins, flavanone-3ß-hydroxylase (F3H) plays very important roles in plant stress response. A putative flavanone-3ß-hydroxylase gene from Medicago truncatula (MtF3H), a model legume species, was identified from a bio-data analysis platform. It was speculated to be induced by salt stress based on the outcomes of the analysis platform. The complementary DNA (cDNA) consists of 1499 bp with an open reading frame (ORF) of 1098 bp, which encodes a putative protein of 365 amino acids with a molecular weight of about 41.36 kDa and an isoelectric point of 5.60. To measure the catalytic activity of the protein, the MtF3H gene was ligated to pYES2 vector and heterologously expressed in yeast. The recombinant protein converted naringen into dihydrokaempferol and displayed different enzymatic efficiencies with other flavanones, confirming that MtF3H coding a functional flavanone-3ß-hydroxylase. The expression pattern of the MtF3H gene was analyzed by comparative quantitative RT-PCR and a higher level of expression was observed in the roots than was observed in stems and leaves. Furthermore, the expression was induced by salt stress in the roots, and to a greater extent in the stems, but the response of the gene activity to salt stress in the stems was slower in the first 12 h following treatment when compared to the roots.     

Cloning and characterization of the <Emphasis Type="Italic">Tne</Emphasis>DI restriction–modification system of <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis>

A putative Type II restriction–modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF′ and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Expression and characterization of a cold-active and xylose-stimulated β-glucosidase from <Emphasis Type="Italic">Marinomonas</Emphasis> MWYL1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding a cold-active and xylose-stimulated β-glucosidase of Marinomonas MWYL1 was synthesized and expressed in Escherichia coli. The recombinant enzyme (reBglM1) was purified and characterized. The molecular mass of the purified reBglM1 determined by SDS-PAGE agree with the calculated values (50.6 Da). Optima of temperature and pH for enzyme activity were 40°C and 7.0, respectively. The enzyme exhibited about 20% activity at 5°C and was stable over the range of pH 5.5–10.0. The presence of xylose significantly enhanced enzyme activity even at higher concentrations up to 600 mM, with maximal stimulatory effect (about 1.45-fold) around 300 mM. The enzyme is active with both glucosides and galactosides and showed high catalytic efficiency (k _cat = 500.5 s⁻¹) for oNPGlc. These characterizations enable the enzyme to be a promising candidate for industrial applications.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Mapping and characterization of wheat stem rust resistance genes <Emphasis Type="Italic">SrTm5</Emphasis> and <Emphasis Type="Italic">Sr60</Emphasis> from <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Key message

The new stem rust resistance gene Sr60 was fine-mapped to the distal region of chromosome arm 5A^mS, and the TTKSK-effective gene SrTm5 could be a new allele of Sr22.

Abstract

The emergence and spread of new virulent races of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici; Pgt), including the Ug99 race group, is a serious threat to global wheat production. In this study, we mapped and characterized two stem rust resistance genes from diploid wheat Triticum monococcum accession PI 306540. We mapped SrTm5, a previously postulated gene effective to Ug99, on chromosome arm 7A^mL, completely linked to Sr22. SrTm5 displayed a different race specificity compared to Sr22 indicating that they are distinct. Sequencing of the Sr22 homolog in PI 306540 revealed a novel haplotype. Characterization of the segregating populations with Pgt race QFCSC revealed an additional resistance gene on chromosome arm 5A^mS that was assigned the official name Sr60. This gene was also effective against races QTHJC and SCCSC but not against TTKSK (a Ug99 group race). Using two large mapping populations (4046 gametes), we mapped Sr60 within a 0.44 cM interval flanked by sequenced-based markers GH724575 and CJ942731. These two markers delimit a 54.6-kb region in Brachypodium distachyon chromosome 4 and a 430-kb region in the Chinese Spring reference genome. Both regions include a leucine-rich repeat protein kinase (LRRK123.1) that represents a potential candidate gene. Three CC–NBS–LRR genes were found in the colinear Brachypodium region but not in the wheat genome. We are currently developing a Bacterial Artificial Chromosome library of PI 306540 to determine which of these candidate genes are present in the T. monococcum genome and to complete the cloning of Sr60.

     

Cloning and characterization of a β-1,3-glucan-binding protein from shrimp <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>

The β-1,3-d-glucan binding protein (βGBP), one kind of the pattern recognition proteins (PRPs), was cloned and characterized from the Chinese Shrimp Fenneropenaeus chinensis, and named as FcβGBP-HDL. The results indicated that the full length cDNA of 6713 bp had an open reading frame encoded a polypeptide of 2139 amino acids with two glucanase-like motifs and one RGD motif, while without signal peptide. The calculated molecular mass of mature protein was 240.7 kDa and theoretical pI was 5.95. Sequence comparison of the deduced amino acid sequence of FcβGBP-HDL showed varied identity of 88, 54 and 53% with those of Litopenaeus vannamei βGBP-HDL, Pacifastacus leniusculus βGBP, and Pontastacus leptodactylus βGBP, respectively. qRT-PCR analysis indicated that FcβGBP-HDL was expressed in intestines, hepatopancreas, muscle, gill and hemocytes, and its profile was modified post WSSV challenge. The differential expressions of FcβGBP-HDL in different tissues post WSSV challenge suggested that FcβGBP-HDL might play an important role in shrimp immune and perform differently in different tissues. These data would be helpful to better understand the WSSV-resistance mechanism of farming shrimp.     

Cloning,purification, and characterization of β-galactosidase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> DSM 13

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K _m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na⁺ and K⁺ in the concentration range of 1–100 mM as well as the divalent metal cations Mg²⁺, Mn²⁺, and Ca²⁺ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.     

Cloning and functional characterization of a cation–chloride cotransporter gene <Emphasis Type="Italic">OsCCC1</Emphasis>

Potassium (K⁺) and chloride (Cl⁻) are two essential elements for plant growth and development. While it is known that plants possess specific membrane transporters for transporting K⁺ and Cl⁻, it remains unclear if they actively use K⁺-coupled Cl⁻ cotransporters (KCC), as used in animals, to transport K⁺ and Cl⁻. We have cloned an Oryza sativa cDNA encoding for a member of the cation–Cl⁻ cotransporter (CCC) family. Phylogenetic analysis revealed that plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K⁺–Cl⁻ cotransporters than to other cation–Cl⁻ cotransporters. Real-time PCR revealed that the O. sativa cDNA, which was named OsCCC1, can be induced by KCl in the shoot and root and that the expression level was higher in the leaf and root tips than in any other part of the rice plant. The OsCCC1 protein was located not only in onion plasma membrane but also in O. sativa plasma membrane. The OsCCC1 gene-silenced plants grow more slowly than wild-type (WT) plants, especially under the KCl treatment regime. After 1 month of KCl treatment, the leaf tips of the gene-silenced lines were necrosed. In addition, seed germination, root length, and fresh and dry weight were distinctly lower in the gene-silenced lines than in WT plants, especially after KCl treatment. Analysis of Na⁺, K⁺, and Cl⁻ contents of the gene-silenced lines and WT plants grown under the NaCl and KCl treatment regimes revealed that the former accumulated relatively less K⁺ and Cl⁻ than the latter but that they did not differ in terms of Na⁺ contents, suggesting OsCCC1 may be involved in K⁺ and Cl⁻ transport. Results from different tests indicated that the OsCCC1 plays a significant role in K⁺ and Cl⁻ homeostasis and rice plant development.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Cloning,extracellular expression and characterization of a predominant β-CGTase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. G1 in <Emphasis Type="Italic">E. coli</Emphasis>

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of β-CD.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.