Nitrilase Expression Research Articles

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL-1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.

Highly Efficient Biosynthesis of Nicotinic Acid by Immobilized Whole Cells of E. coli Expressing Nitrilase in Semi-Continuous Packed-Bed Bioreactor

A recombinant E. coli, expressing nitrilase from Acidovorax facilis 72W with dual-site expression plasmid pRSFduet (E. coli pRSF-AfNit2), was constructed. It showed higher soluble expression of nitrilase than that in the pET21a plasmid. The recombinant nitrilase can efficiently catalyze the hydrolysis of 3-cyanopyridine to nicotinic acid. The whole cells of E. coli pRSF-AfNit2 were immobilized by using sodium alginate/glutaraldehyde/polyethylene imine as the best immobilized reagents. The immobilized cells showed 95% activity recovery and excellent mechanical strength, with improved thermal stability and pH stability. They also retained 82% of initial activity after nearly two months of storage at 4 °C. A semi-continuous packed-bed bioreactor (sPBR) filled with the immobilized cells was studied for efficient production of nicotinic acid. After optimization, the highest space–time yield of 1576 g/(L·d) was obtained on 0.8 M substrate concentration at 2 mL/min of flow rate. The sPBR was repeatedly operated for 41 batches, keeping 100% conversion in the presence of 30 mM CaCl2. Finally, 95 g of nicotinic acid were obtained at 90% yield after separation and purification. The developed technology has potential application value.

Multi-omics analyses reveal new insights into nutritional quality changes of alfalfa leaves during the flowering period.

High-quality alfalfa is an indispensable resource for animal husbandry and sustainable development. Its nutritional quality changes dramatically during its life cycle and, at present, no molecular mechanisms for nutrient metabolic variation in alfalfa leaves at different growth stages have been clearly reported. We have used correlation and network analyses of the alfalfa leaf metabolome, proteome, and transcriptome to explore chlorophyll, flavonoid, and amino acid content at two development stages: budding stage (BS) and full-bloom stage (FBS). A high correlation between the expression of biosynthetic genes and their metabolites revealed significant reductions in metabolite content as the plant matured from BS to FBS. l-Glutamate, the first molecule of chlorophyll biosynthesis, decreased, and the expression of HemA, which controls the transformation of glutamyl-tRNA to glutamate 1-semialdehyde, was down-regulated, leading to a reduction in leaf chlorophyll content. Flavonoids also decreased, driven at least in part by increased expression of the gene encoding CYP75B1: flavonoid 3'-monooxygenase, which catalyzes the hydroxylation of dihydroflavonols and flavonols, resulting in degradation of flavonoids. Expression of NITRILASE 2 (NIT2) and Methyltransferase B (metB), which regulate amino acid metabolism and influence the expression of genes of the glycolysis-TCA pathway, were down-regulated, causing amino acid content in alfalfa leaves to decrease at FBS. This study provides new insights into the complex regulatory network governing the content and decrease of chlorophyll, amino acids, flavonoids, and other nutrients in alfalfa leaves during maturation. These results further provide a theoretical basis for the generation of alfalfa varieties exhibiting higher nutritional quality, high-yield cultivation, and a timely harvest.

Optimization of the medium composition and product extraction for R-mandelic acid using recombinant Escherichia coli expressing Alcaligenes sp. nitrilase

The aim of this study is to investigate the effects of the components of the medium on nitrilase expression in recombinant Escherichia coli ( E. coli), which is applied in the hydrolysis of racemic mandelonitrile (MN) to R-mandelic acid (MA). In addition, the separation of R-MA from the reaction mixture is studied. The glycerol medium is screened, and compositions are optimized in single-factor experiments. The nitrilase activity is 1.96-fold higher than before using the optimal medium containing peptone 15 g/L, yeast extract 12 g/L, NaCl 10 g/L, glycerol 15 g/L, (NH4)2SO4 5 g/L, KH2PO4 10 mM, K2HPO4·3H2O 10 mM, and MgSO4·7H2O 15 mM. During the acid-base extraction of R-MA, the optimal alkaline pH, the alkaline extractant ratio, the optimal acidic pH, the acidic extractant ratio and the amount of activated carbon are 10.0, 1:0.5 (once), 2.50, 1:2 (twice) and 1%, respectively. This study provides a basis for the industrial production of R-MA.

Salt-Specific Gene Expression Reveals Elevated Auxin Levels in Arabidopsis thaliana Plants Grown Under Saline Conditions.

Soil salinization is increasing globally, driving a reduction in crop yields that threatens food security. Salinity stress reduces plant growth by exerting two stresses on plants: rapid shoot ion-independent effects which are largely osmotic and delayed ionic effects that are specific to salinity stress. In this study we set out to delineate the osmotic from the ionic effects of salinity stress. Arabidopsis thaliana plants were germinated and grown for two weeks in media supplemented with 50, 75, 100, or 125 mM NaCl (that imposes both an ionic and osmotic stress) or iso-osmolar concentrations (100, 150, 200, or 250 mM) of sorbitol, that imposes only an osmotic stress. A subsequent transcriptional analysis was performed to identify sets of genes that are differentially expressed in plants grown in (1) NaCl or (2) sorbitol compared to controls. A comparison of the gene sets identified genes that are differentially expressed under both challenge conditions (osmotic genes) and genes that are only differentially expressed in plants grown on NaCl (ionic genes, hereafter referred to as salt-specific genes). A pathway analysis of the osmotic and salt-specific gene lists revealed that distinct biological processes are modulated during growth under the two conditions. The list of salt-specific genes was enriched in the gene ontology (GO) term “response to auxin.” Quantification of the predominant auxin, indole-3-acetic acid (IAA) and IAA biosynthetic intermediates revealed that IAA levels are elevated in a salt-specific manner through increased IAA biosynthesis. Furthermore, the expression of NITRILASE 2 (NIT2), which hydrolyses indole-3-acetonitile (IAN) into IAA, increased in a salt-specific manner. Overexpression of NIT2 resulted in increased IAA levels, improved Na:K ratios and enhanced survival and growth of Arabidopsis under saline conditions. Overall, our data suggest that auxin is involved in maintaining growth during the ionic stress imposed by saline conditions.

Extracellular expression of natural cytosolic nitrilase from Rhodococcus zopfii through constructing a transmembrane tunnel structure in Escherichia coli cells

Nitrilases are a class of industrially important hydrolases that convert nitriles to carboxylic acids. As a natural cytosolic enzyme, production of nitrilase suffer from inclusion body and cumbersome purification procedure. Extracellular expression of recombinant proteins offers advantages including improved protein folding and simplified downstream bioprocesses, which is more suitable for industrial production. Herein, extracellular expression of nitrilase (RzNIT) from Rhodococcus zopfii in Escherichia coli cells was investigated using active and passive transport strategies. The active transport of RzNIT mediated by N-terminal fused signal peptides and secretory proteins had negative effects on both solubility and activity. For the passive transport, PhiX174 protein E was co-expressed to form a 40−70 nm transmembrane tunnel structure in cell membranes. RzNIT was spontaneously released from cytoplasm into extracellular medium through the constructed hole. Under the optimum conditions, the extracellular RzNIT activity reached 7.6 U/mL (86 % of its total activity) toward 3-cyanopyridine. The calculated concentration of extracellular RzNIT reached 0.13 mg/mL. The obtained results provide a novel and efficient strategy for extracellular production of recombinant nitrilases.

Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber.

For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32–47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

Optimization of the Expression of Nitrilase from Alcaligenes denitrificans in Rhodococcus rhodochrous to Improve the Efficiency of Biocatalytic Synthesis of Ammonium Acrylate

A Rhodococcus rhodochrous strain, M33-2nit, has been constructed with two copies of the nitrilase gene from A. denitrificans В-9582 under the control of nitrile hydratase promoter from R. rhodochrous M8. The optimized cultivation of this strain made it possible to obtain the enzyme in a concentration of up to 17 g of dry cells/L with a specific activity of up to 7 U/mg cdw with a two-substrate culturing scheme in a fed-batch reactor with the sequential addition of glucose and acetate. The capacities of A. denitriificans В-9582 and R. rhodochrous M33-2nit cells to synthesize ammonium acrylate from acrylonitrile under conditions imitating industrial synthesis are compared. It is shown that R rhodochrous M33-2nit cells can synthesize ammonium acrylate under higher rates of acrylonitrile feeding than A. denitriificans В-9582 cells. The potential to obtain a highly concentrated solution of ammonium acrylate (450 g/L) with R. rhodochrous M33-2nit cells as a biocatalyst was demonstrated. The conversion of acrylonitrile to ammonium acrylate reached 99.5%.

Bacterial nitrilases and their regulation.

Commercially, nitrilases are valuable biocatalysts capable of converting a diverse range of nitriles to carboxylic acids for the greener synthesis of chemicals and pharmaceuticals. Nitrilases are widespread in nature and are both important components of metabolic pathways and a response to environmental factors such as natural or manmade nitriles. Nitrilases are often grouped together on a genome in specific gene clusters that reflect these metabolic functions. Although nitrilase induction systems are still poorly understood, it is known that a powerful Rhodococcal transcription regulator system permits accumulation of intracellular nitrilase of up to 30-40% of total soluble protein in wild type Rhodococcous rhodochrous and host Streptomyces strains. Nitrilase expression inducer molecules encompass a broad range of aliphatic, aromatic and heteroaromatic nitriles, as well as some secondary and tertiary amides that are resistant to nitrilase degradation.

Erratum to: Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis.

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.

Arabidopsis NITRILASE 1 Contributes to the Regulation of Root Growth and Development through Modulation of Auxin Biosynthesis in Seedlings.

Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant Arabidopsis thaliana a second group of Brassicaceae-specific nitrilases (NIT1-3) has been found. This so-called NIT1-subfamily has been associated with the conversion of indole-3-acetonitrile (IAN) into the major plant growth hormone, indole-3-acetic acid (IAA). However, apart of reported functions in defense responses to pathogens and in responses to sulfur depletion, conclusive insight into the general physiological function of the NIT-subfamily nitrilases remains elusive. In this report, we test both the contribution of the indole-3-acetaldoxime (IAOx) pathway to general auxin biosynthesis and the influence of altered nitrilase expression on plant development. Apart of a comprehensive transcriptomics approach to explore the role of the IAOx route in auxin formation, we took a genetic approach to disclose the function of NITRILASE 1 (NIT1) of A. thaliana. We show that NIT1 over-expression (NIT1ox) results in seedlings with shorter primary roots, and an increased number of lateral roots. In addition, NIT1ox plants exhibit drastic changes of both free IAA and IAN levels, which are suggested to be the reason for the observed phenotype. On the other hand, NIT2RNAi knockdown lines, capable of suppressing the expression of all members of the NIT1-subfamily, were generated and characterized to substantiate the above-mentioned findings. Our results demonstrate for the first time that Arabidopsis NIT1 has profound effects on root morphogenesis in early seedling development.

Cloning and functional characterization of nitrilase from Fusarium proliferatum AUF-2 for detoxification of nitriles

A fungal nitrilase gene from Fusarium proliferatum AUF-2 was cloned through reverse transcription-PCR. The open reading frame consisted of 903 bp and potentially encoded a protein of 301 amino acid residues with a theoretical molecular mass of 33.0 kDa. The encoding gene was expressed in Escherichia coli strain BL21 and the recombinant protein with His6-tag was purified to electrophoretic homogeneity. The purified enzyme exhibited optimal activity in the range of 35-40 °C and pH 8.0. EDTA, Mg(2+), Zn(2+), Ca(2+), Fe(2+), Fe(3+) and Mn(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+) and Ni(2+) had inhibitory effect on nitrilase activity. Ag(+) ions showed a strong inhibitory effect on the recombinant nitrilase activity. This nitrilase was specific towards aliphatic, heterocyclic and aromatic nitriles. The kinetic parameters V(max) and K(m) for benzonitrile substrate were determined to be 14.6 μmol/min/mg protein and 1.55 mM, respectively. Homology modelling and molecular docking studies provided an insight into the substrate specificity and the proposed catalytic triad for recombinant nitrilase consisted of Glu-54, Lys-133 and Cys-175. This is the first report on the cloning and heterologous expression of nitrilase from Fusarium proliferatum.

Erratum to: Purification and characterization of heterologously expressed nitrilases from filamentous fungi

Two works cited in the previous article (Kaplan et al. 2006, 2011) were corrected (Kaplan et al. 2013a) or retracted (Kaplan et al. 2013b). The reason was the falsity of data (N-terminal aa sequence and theMALDI-TOF analysis) by KBwhose ethics of scientific workwas examined in 2012 by the Ethics Commission of the Charles University in Prague and the Institute of Microbiology of the Academy of Sciences of the Czech Republic. The Commission found scientific misconduct of KB in several cases. Therefore, we wish to correct the following parts of the previous article: Introduction: “The nitrilase studied by us in Aspergillus niger K10 was the first fungal nitrilase to be purified from both its native... and heterologous producer...” Actually, the enzymes purified from the native and heterologous producer were different. The enzyme natively expressed in this strain was purified and characterised (Kaplan et al. 2006) and its partial aa sequence was recently published (Kaplan et al. 2013a). The recombinant enzyme was recently examined for its substrate specificity in whole cells (Kaplan et al. 2013c), and was found to be a cyanide hydratase with a significant nitrilase activity. Materials and methods, Nitrilase expression: Construction of plasmid pOK101 referred toKaplan et al. 2011 (retracted article). Therefore, we wish to describe this method here: To construct plasmid pOK101, NdeI-HindIII fragment of the PCR product amplified from cDNA of Aspergillus niger K10 (forward primer 5′-GCC ATATGG CAC CMG TCT TRA AGA AGTACA A3′, M=A or C; R=A or G, and reverse primer, 5′-GCA AGC TTT TAC TAG TTC TCC GAATCC ACG GT-3′) was ligated into vector pET-30a(+) (Novagen). Results, Variability of fungal nitrilases: The statement that in the nitrilase fromA . niger K10 “both its amino acid sequence and its biochemical properties have been already known” referred to Kaplan et al. 2011 (retracted article). For substrate specificity of this enzyme please refer to Kaplan et al. 2013c (see also above). Results, Nitrilase in Aspergillus niger K10: The hypothesis that the difference of substrate specificities in the natively expressed and the recombinant enzymes were caused by misfolding of the latter referred to Kaplan et al. 2011 (retracted article). Actually, the two enzymes were different in terms of their primary structure (see above). The online version of the original article can be found at http://dx.doi.org/ 10.1007/s00253-011-3525-7.

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis

(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (phi). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.

Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis.

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.

Diverse range of gene activity during Arabidopsis thaliana leaf senescence includes pathogen-independent induction of defense-related genes.

To determine the range of gene activities associated with leaf senescence, we have identified genes that show preferential transcript accumulation during this developmental stage. The mRNA levels of a diverse array of gene products increases during leaf senescence, including a protease, a ribosomal protein, two cinnamyl alcohol dehydrogenases, a nitrilase and glyoxalase II. Two of the genes identified are known to be pathogen-induced. The senescence specificity of each gene was determined by characterization of transcript accumulation during leaf development and in different tissues. The increased expression of nitrilase in senescent leaves is paralleled by an increase in free indole-3-acetic acid (IAA) levels. Additionally, we have demonstrated that the induction of defense-related genes during leaf senescence is pathogen-independent and that salicylic acid accumulation is not essential for this induction. Our data indicate that the induction of certain genes involved in plant defense responses is a component of the leaf senescence program.