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.     

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.     

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.     

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.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

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.     

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.     

Characterization of nitrogen substrate limitation on <Emphasis Type="Italic">Escherichia coli</Emphasis>’s growth by parameter identification tools

Carbon-to-nitrogen ratio (CNR) has shown to be a relevant factor in microorganisms growth and metabolites production. It is usual that this factor compromises the productivity yield of different microorganisms. However, CNR has been rarely modeled and therefore the nature of its specific influence on metabolites production has not been understood clearly. This paper describes a parametric characterization of the CNR effect on the Escherichia coli metabolism. A set of parameters was proposed to introduce a mathematical model that considers the biomass, substrate and several byproducts dynamical behavior under batch regimen and CNR influence. Identification algorithm used to calculate the parameters considers a novel least mean square strategy that formalizes the CNR influence in E. coli metabolism. This scheme produced a step-by-step method that was suitable for obtaining the set of parameters that describes the model. This method was evaluated under two scenarios: (a) using the data from a set of numerical simulations where the model was tested under the presence of artificial noises and (b) the information obtained from a set of experiments under different CNR. In both cases, a leave-one-experiment-out cross-validation study was considered to evaluate the model prediction capabilities. Feasibility of the parametric identification method was proven in both considered scenarios.     

Biochemical characterization of <Emphasis Type="Italic">Candida albicans</Emphasis> α-glucosidase I heterologously expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, α-glucosidase I removes the outermost α1,2-glucose unit from the N-linked core Glc₃Man₉GlcNAc₂. We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3′-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed α-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.     

Cyclodextrin enhanced the soluble expression of <Emphasis Type="Italic">Bacillus clarkii</Emphasis> γ-CGTase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Cyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular α-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others. Economic use of these CGTases, particularly γ-CGTase, requires their efficient production. In this study, the effects of chemical chaperones, temperature and inducers on cell growth and the production of soluble γ-CGTase by Escherichia coli were investigated.

Results

The yield of soluble γ-CGTase in shake-flask culture approximately doubled when β-cyclodextrin was added to the culture medium as a chemical chaperone.When a modified two-stage feeding strategy incorporating 7.5 mM β-cyclodextrin was used in a 3-L fermenter, a dry cell weight of 70.3 g·L^??1 was achieved. Using this cultivation approach, the total yield of γ-CGTase activity (50.29 U·mL^??1) was 1.71-fold greater than that observed in the absence of β-cyclodextrin (29.33 U·mL^??1).

Conclusions

Since β-cyclodextrin is inexpensive and nontoxic to microbes, these results suggest its universal application during recombinant protein production. The higher expression of soluble γ-CGTase in a semi-synthetic medium showed the potential of the proposed process for the economical production of many enzymes on an industrial scale.

     

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.     

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.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

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.     

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.     

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.     

<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.     

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.