Versatile <Emphasis Type="Italic">Rhodococcus equi</Emphasis>–<Emphasis Type="Italic">Escherichia coli</Emphasis> shuttle vectors

Rhodococcus equi is an intracellular pathogen of macrophages, causing disease in young foals, humans, and sporadically other animals. Although R. equi is easy to grow and manipulate, the analysis of virulence is hampered by a lack of molecular tools. This paper describes the development of a number of versatile plasmids for use in R. equi. Plasmids pREV2 and pREV5 use origins of replication derived from the Mycobacterium fortuitum plasmids pAL5000 and pMF1. These plasmids and their derivatives are compatible in R. equi, allowing their use for analysis of gene function in trans. The stability of these plasmids in R. equi in the absence of selection for the plasmid borne antibiotic resistance markers, and their integrity following passage through Escherichia coli and R. equi was determined.     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

Synthesis of theanine from glutamic acid <Emphasis Type="Italic">γ</Emphasis>-methyl ester and ethylamine catalyzed by <Emphasis Type="Italic">Escherichia coli</Emphasis> having <Emphasis Type="Italic">γ</Emphasis>-glutamyltranspeptidase activity

Glutamic acid γ-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing γ-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45°C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h.     

New recombinant producer of human ω-amidase based on <Emphasis Type="Italic">Escherichia coli</Emphasis>

A new artificial gene encoding human ω-amidase (Nit2) adapted for highly efficient expression in E. coli has been established. A pQE-Nit2 plasmid construct controlled by the T5 promoter has been engineered for its expression. The nit2 gene within the pQE-Nit2 construct has optimized codon usage and an artificial 6His-tag sequence inserted directly after the ATG initiation codon. This tag provides the possibility of single-step purification of a product via metal chelate chromatography. The codon-usage optimization involves the inclusion of several codons of extremely rare occurrence in natural E. coli ORFs within a 30 a.a-long N-terminal region. Other codons included in the N-terminus have moderate occurrence in E. coli. The subsequent sequence of the artificial gene has been composed of the most frequently occurring codons in E. coli. The recombinant producer based on the pQE-Nit2 construct allowed purification of the enzyme with an activity of 6.2 ± 0.2 μmol/min/mg protein, which corresponds to or slightly exceeds the specific activity of rat liver Nit2. The omega-amidase preparation is necessary for the screening of potential inhibitors that can be used as candidate drugs to cure hyperammonemia disorders in liver pathologies and oncological diseases.     

Characterization flavanone 3β-hydroxylase expressed from <Emphasis Type="Italic">Populus euphratica</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in dihydroflavonol production

Flavanone 3β-hydroxylase plays very important role in the biosynthesis of flavonoids. A putative flavanone 3β-hydroxylase gene (Pef3h) from Populus euphratica was cloned and over-expressed in Escherichia coli. Induction performed with 0.1 mM IPTG at 20°C led to localization of PeF3H in the soluble fraction. Recombinant enzyme was purified by Ni-NTA affinity. The optimal activity of PeF3H was revealed at pH 7.6 and 35°C. The purified enzyme was stable over pH range of 7.6–8.8 and had a half-life of 1 h at 50°C. The activity of PeF3H was significantly enhanced in the presence of Fe²⁺ and Fe³⁺. The K _M and V _max for the enzyme using naringenin as substrate were 0.23 mM and 0.069 μmoles mg–1min-1, respectively. The K _m and V _max for eriodictyol were 0.18 mM and 0.013 μmoles mg^–1min^–1, respectively. The optimal conditions for naringenin bioconversion in dihydrokaempferol were obtained: OD₆₀₀ of 3.5 for cell concentration, 0.1 mM IPTG, 5 mM α-ketoglutaric acid and 20°C. Under the optimal conditions, naringenin (0.2 g/L) was transformed into 0.18 g/L dihydrokaempferol within 24 h by the recombinant E. coli with a corresponding molar conversion of 88%. Thus, this study provides a promising flavanone 3β-hydroxylase that may be used in biosynthetic applications.     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

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 purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Reactive oxygen and nitrogen species’ effect on <Emphasis Type="Italic">lux</Emphasis>-biosensors based on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H₂O₂ > OCl^– > NO^? > RОO^? > ONOO^–> O₂^?- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO^? > H₂O₂ > ONOO^– > RОO^? > OCl^– > O₂^?- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS′::lux possessed a wider range of sensitivity, including H₂O₂ and OCl^–, along with O₂^?- and NO^?. Among the used reactive oxygen and nitrogen species, H₂O₂ showed the highest induction activity concerning to the plasmids pKatG′::lux, pSoxS′::lux and pRecA′::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison with the E. coli lux-biosensors.     

Molecular polymorphism of α-galactosidase <Emphasis Type="Italic">MEL</Emphasis> genes of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts

Molecular genetic analysis of melibiose-fermenting Saccharomyces strains isolated from fermentative processes and natural sources in different world regions was conducted to deduce the evolutionary diversity of Saccharomyces yeasts and find new α-galactosidase MEL genes. The species S. bayanus, S. mikatae, and S. paradoxus were shown to have a single copy of MEL and not accumulate polymeric genes, unlike some S. cerevisiae populations. The polymeric genes MELp1 and MELp2 were identified in S. paradoxus for the first time. Genes identical by 98.7% are located on the chromosomes X and VI, respectively. Phylogenetic analysis indicates that MEL genes of the Saccharomyces yeasts are species-specific.     

Influence of N-Terminal Truncations on the Functional Expression of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> γ-Glutamyltranspeptidase in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacterial strain BF36^T, isolated from the biofilm of a tufa deposit in a hard water rivulet, was characterized by a polyphasic taxonomic approach. Cells of these organisms were Gram-negative, motile, nonpigmented, rod-shaped, non-endospore-forming, and facultatively anaerobic. Cells, organized in loose consortia, were coated by a massive slime layer. Phylogenetic analyses using 16S rRNA gene sequences showed that strain BF36^T was a member of the family Enterobacteriaceae, class Gammaproteobacteria, displaying a moderate degree of relationship (96.5% sequence similarity) to Sodalis glossinidius and “Sodalis pallipedes,” intracellular symbionts of the tsetse fly Glossinis morsitans morsitans. Dendrograms of relationship generated by different algorithms consistently grouped isolate BF36^T with Sodalis glossinidius, Pragia fontium, Budvicia aquatica, Serratia rubideae, and Brenneria spp (94.7–95.8% similarity) which also share many common metabolic properties. Differences between strain BF36^T and Sodalis glossinidius DSM 13495^T are seen in motility and in the pattern of substrates utilized. Membership to the family was also confirmed by a fatty acid profile consisting of major amounts of C_16:0 and C_16:1ω7, by the presence of isoprenoids of the ubiquinone Q8 and menaquinone MK8 types and a DNA G + C content of 54.2 mol%. The decision to classify strain BF36^T into a new genus Biostraticola gen. nov. is based on its distant phylogenetic position as compared to any other representative of the family and the significant phenotypic differences to its nearest phylogenetic neighbor, Sodalis glossinidius. BF36^T represents the type species, for which the name Biostraticola tofi sp. nov. is proposed. The type strain is BF36^T (DSM 19580^T; CIP109699^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BF36^T is AM774412.     

Bioinformatics Characterization of Potential New Beta-Glucuronidase from <Emphasis Type="Italic">Streptococcus</Emphasis> <Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.     

Whole-cell bioreduction of aromatic α-keto esters using <Emphasis Type="Italic">Candida tenuis</Emphasis> xylose reductase and <Emphasis Type="Italic">Candida boidinii</Emphasis> formate dehydrogenase co-expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP⁺ or NAD⁺ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR.     

Characterization of β-1,3-galactosyl-<Emphasis Type="Italic">N</Emphasis>-acetylhexosamine phosphorylase from <Emphasis Type="Italic">Propionibacterium acnes</Emphasis>

Homologs of the β-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) gene (gnpA) were cloned from the genomic DNA of Propionibacterium acnes JCM6425 and P. acnes JCM6473, showing 99.9% and 97.9% nucleotide sequence identity, respectively, with the ppa0083 gene from the genome-sequenced P. acnes KPA171202. No gnpA gene was detected in the genomic DNA of type strain P. acnes ATCC25746. The recombinant enzyme from P. acnes JCM6425 (GnpA) showed approximately 70 times higher specific activity of phosphorolysis on galacto-N-biose (Galβ1→3GalNAc, GNB) than that on lacto-N-biose I (Galβ1→3GlcNAc). K _m value for GnpA on GNB was high, but GnpA did not exhibit activity on any derivatives of GNB examined. These results indicate that GnpA is GalHexNAcP which should be classified as galacto-N-biose phosphorylase. The large k _cat value of GnpA on GalNAc suggests that GnpA would be a useful catalyst for the synthesis of GNB.     

Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (<Emphasis Type="Italic">Smhppd</Emphasis>) from <Emphasis Type="Italic">Salvia</Emphasis> <Emphasis Type="Italic">miltiorrhiza</Emphasis> hairy root cultures

A novel 4-hydroxyphenylpyruvate dioxygenase gene (designated as Smhppd) was cloned from hairy roots of Salvia miltiorrhiza Bung. The full-length cDNA of Smhppd was 1,736 bp long with an ORF (open reading frame) that putatively encoded a polypeptide of 481 amino acids, with a predicted molecular mass of 52.54 kDa. The deduced amino acid sequence of the Smhppd gene shared high homology with other known HPPDs. Analysis of Smhppd genomic DNA revealed that it contained two exons and one intron. The analysis of Smhppd promoter region was also presented. Southern-blot analysis revealed that the Smhppd was a low-copy gene in S. miltiorrhiza. Real-time quantitative PCR analysis indicated that Smhppd was constitutively expressed in roots, stems and leaves of S. miltiorrhiza, with the high expression in roots. In addition, Smhppd expreession level under different stress condition was also analyzed during the hairy root culture period, including signaling components for plant defence responses, such as methyl jasmonate and salicylic acid, as well as an abiotic elicitor, Ag⁺ and a biotic elicitor, yeast extract. This study will enable us to further understand the role Smhppd plays in the synthesis of active pharmaceutical compounds in S. miltiorrhiza at molecular level.