Immobilization of aldoxime dehydratases on metal affinity resins and use of the immobilized catalysts for the synthesis of nitriles important in fragrance industry.

Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.

Scanning aldoxime dehydratase sequence space and characterization of a new aldoxime dehydratase from Fusarium vanettenii.

The aim of this work was to map the sequence space of aldoxime dehydratases (Oxds) as enzymes with great potential for nitrile synthesis. Microbes contain an abundance of putative Oxds but fewer than ten Oxds were characterized in total and only two in fungi. In this work, we prepared and characterized a new Oxd (protein gb|EEU37245.1 named OxdFv) from Fusarium vanettenii 77-13-4. OxdFv is distant from the characterized Oxds with a maximum of 36% identity. Moreover, the canonical Oxd catalytic triad RSH is replaced by R141-E187-E303 in OxdFv. R141A and E187A mutants did not show significant activities, but mutant E303A showed a comparable activity as the wild-type enzyme. According to native mass spectrometry, OxdFv contained almost 1 mol of heme per 1 mol of protein, and was composed of approximately 88% monomer (41.8 kDa) and 12% dimer. A major advantage of this enzyme is its considerable activity under aerobic conditions (25.0 ± 4.3 U/mg for E,Z-phenylacetaldoxime at pH 9.0 and 55 °C). Addition of sodium dithionite (reducing agent) and Fe2+ was required for this activity. OxdFv favored (aryl)aliphatic aldoximes over aromatic aldoximes. Substrate docking in the homology model of OxdFv showed a similar substrate specificity. We conclude that OxdFv is the first characterized Oxd of the REE type.

Sigma regulatory network in Rhodococcus erythropolis CCM2595.

The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed.

Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum.

The sigma H (σΗ) and sigma E (σE) subunits of Corynebacterium glutamicum RNA polymerase belong to Group 4 of sigma factors, also called extracytoplasmic function (ECF) sigma factors. Genes of the C. glutamicum σΗ regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the σE regulon, which is involved in cell surface stress response, have not been explored until now. Using the C. glutamicum RES167 strain and its derivative C. glutamicum ΔcseE with a deletion in the anti-σΕ gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum ΔcseE compared to C. glutamicum RES167. To confirm the functional link between σΕ and the corresponding promoters, we tested selected promoters using the in vivo two-plasmid system with gfpuv as a reporter gene and by in vitro transcription. Analyses with RNAP+σΗ and RNAP+σΕ, which were previously shown to recognize similar promoters, proved that the σΗ and σE regulons significantly overlap. The σE-controlled genes were found to be involved for example in protein quality control (dnaK, dnaJ2, clpB, and clpC), the regulation of Clp proteases (clgR), and membrane integrity maintenance. The single-promoter analyses with σΗ and σΕ revealed that there are two groups of promoters: those which are exclusively σΗ-specific, and the other group of promoters, which are σΗ/σE-dependent. No exclusively σE-dependent promoter was detected. We defined the consensus sequences of exclusively σΗ-regulated promotors to be -35 GGAAt and - 10 GTT and σΗ/σE-regulated promoters to be -35 GGAAC and - 10 cGTT. Fifteen genes were found to belong to the σΗ/σΕ regulon. Homology modeling showed that there is a specific interaction between Met170 in σΗ and the nucleotides -31 and - 30 within the non-coding strand (AT or CT) of the σΗ-dependent promoters. In σE, Arg185 was found to interact with the nucleotides GA at the same positions in the σE-dependent promoters.

Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus.

Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from Trametes versicolor and NitAb from Agaricus bisporus were purified and characterized as the representatives of this type of fungal NLase. Both enzymes were slightly more similar to NIT4 type than to NIT1/NIT2/NIT3 type of plant NLases in terms of their amino acid sequences. Expression of the synthetic genes in Escherichia coli Origami B (DE3) was induced with 0.02 mM isopropyl ß-D-1-thiogalactopyranoside at 20 °C. Purification of NitTv1 and NitAb by cobalt affinity chromatography gave ca. 6.6 mg and 9.6 mg of protein per 100 mL of culture medium, respectively. Their activities were determined with 25 mM of nitriles in 50 mM Tris/HCl buffer, pH 8.0, at 30 °C. NitTv1 and NitAb transformed ß-cyano-L-alanine (ß-CA) with the highest specific activities (ca. 132 and 40 U mg-1, respectively) similar to plant NLase NIT4. ß-CA was transformed into Asn and Asp as in NIT4 but at lower Asn:Asp ratios. The fungal NLases also exhibited significant activities for (aryl)aliphatic nitriles such as 3-phenylpropionitrile, cinnamonitrile and fumaronitrile (substrates of NLase NIT1). NitTv1 was more stable than NitAb (at pH 5-9 vs. pH 5-7). These NLases may participate in plant-fungus interactions by detoxifying plant nitriles and/or producing plant hormones. Their homology models elucidated the molecular interactions with various nitriles in their active sites.

Genetic and Functional Diversity of Nitrilases in Agaricomycotina.

Nitrilases participate in the nitrile metabolism in microbes and plants. They are widely used to produce carboxylic acids from nitriles. Nitrilases were described in bacteria, Ascomycota and plants. However, they remain unexplored in Basidiomycota. Yet more than 200 putative nitrilases are found in this division via GenBank. The majority of them occur in the subdivision Agaricomycotina. In this work, we analyzed their sequences and classified them into phylogenetic clades. Members of clade 1 (61 proteins) and 2 (25 proteins) are similar to plant nitrilases and nitrilases from Ascomycota, respectively, with sequence identities of around 50%. The searches also identified five putative cyanide hydratases (CynHs). Representatives of clade 1 and 2 (NitTv1 from Trametes versicolor and NitAg from Armillaria gallica, respectively) and a putative CynH (NitSh from Stereum hirsutum) were overproduced in Escherichia coli. The substrates of NitTv1 were fumaronitrile, 3-phenylpropionitrile, ß-cyano-l-alanine and 4-cyanopyridine, and those of NitSh were hydrogen cyanide (HCN), 2-cyanopyridine, fumaronitrile and benzonitrile. NitAg only exhibited activities for HCN and fumaronitrile. The substrate specificities of these nitrilases were largely in accordance with substrate docking in their homology models. The phylogenetic distribution of each type of nitrilase was determined for the first time.

Overproduction and characterization of the first enzyme of a new aldoxime dehydratase family in Bradyrhizobium sp.

Almost 100 genes within the genus Bradyrhizobium are known to potentially encode aldoxime dehydratases (Oxds), but none of the corresponding proteins have been characterized yet. Aldoximes are natural substances involved in plant defense and auxin synthesis, and Oxds are components of enzymatic cascades enabling bacteria to transform, utilize and detoxify them. The aim of this work was to characterize a representative of the highly conserved Oxds in Bradyrhizobium spp. which include both plant symbionts and members of the soil communities. The selected oxd gene from Bradyrhizobium sp. LTSPM299 was expressed in Escherichia coli, and the corresponding gene product (OxdBr1; GenBank: WP_044589203) was obtained as an N-His6-tagged protein (monomer, 40.7 kDa) with 30-47% identity to Oxds characterized previously. OxdBr1 was most stable at pH ca. 7.0-8.0 and at up to 30 °C. As substrates, the enzyme acted on (aryl)aliphatic aldoximes such as E/Z-phenylacetaldoxime, E/Z-2-phenylpropionaldoxime, E/Z-3-phenylpropionaldoxime, E/Z-indole-3-acetaldoxime, E/Z-propionaldoxime, E/Z-butyraldoxime, E/Z-valeraldoxime and E/Z-isovaleraldoxime. Some of the reaction products of OxdBr1 are substrates of nitrilases occurring in the same genus. Regions upstream of the oxd gene contained genes encoding a putative aliphatic nitrilase and its transcriptional activator, indicating the participation of OxdBr1 in the metabolic route from aldoximes to carboxylic acids.

Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032.

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σC, σD, σE, σH, and σM are mostly involved in stress responses. The role of σD consists in the control of cell wall integrity. The σD regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential σD-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σD-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAACA/G and -10 GAT. Surprisingly, nearly all of these σD-dependent promoters were also active to a much lower extent with σH in vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the σH-dependent promoters is different (-35 GGAAT/C and -10 GTT). In addition to the activity of σH at the σD-controlled promoters, we discovered separated or overlapping σA- or σB-regulated or σH-regulated promoters within the upstream region of 8 genes of the σD-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σD-dependent genes. Computer modeling revealed that σH binds to the promoter DNA in a similar manner as σD to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σD and Lys 53 in σH, bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the σD-controlled genes also belong to the σH-regulon and are also transcribed from the overlapping or closely located housekeeping (σA-regulated) and/or general stress (σB-regulated) promoters.

Biodegradation of phenol and its derivatives by engineered bacteria: current knowledge and perspectives.

Biodegradation of phenolic compounds is a promising alternative to physical and chemical methods used to remove these toxic pollutants from the environment. The ability of various microorganisms to metabolize phenol and its derivatives (alkylphenols, nitrophenols and halogenated derivatives) has therefore been intensively studied. Knowledge of the enzymes catalyzing the individual reactions, the genes encoding these enzymes and the regulatory mechanisms involved in the expression of the respective genes in bacteria serves as a basis for the development of more efficient degraders of phenols via genetic engineering methods. Engineered bacteria which efficiently degrade phenolic compounds were constructed in laboratories using various approaches such as cloning the catabolic genes in multicopy plasmids, the introduction of heterologous genes or broadening the substrate range of key enzymes by mutagenesis. Efforts to apply the engineered strains in in situ bioremediation are problematic, since engineered strains often do not compete successfully with indigenous microorganisms. New efficient degraders of phenolic compounds may be obtained by complex approaches at the organism level, such as genome shuffling or adaptive evolution. The application of these engineered bacteria for bioremediation will require even more complex analysis of both the biological characteristics of the degraders and the physico-chemical conditions at the polluted sites.

Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial producer of various amino acids and other metabolites. The C. glutamicum genome encodes seven sigma subunits (factors) of RNA polymerase: the primary sigma factor SigA (σA), the primary-like σB and five alternative sigma factors (σC, σD, σE, σH and σM). We have developed in vitro and in vivo methods to assign particular sigma factors to individual promoters of different classes. In vitro transcription assays and measurements of promoter activity using the overexpression of a single sigma factor gene and the transcriptional fusion of the promoter to the gfpuv reporter gene enabled us to reliably define the sigma factor dependency of promoters. To document the strengths of these methods, we tested examples of respective promoters for each C. glutamicum sigma factor. Promoters of the rshA (anti-sigma for σH) and trxB1 (thioredoxin) genes were found to be σH-dependent, whereas the promoter of the sigB gene (sigma factor σB) was σE- and σH-dependent. It was confirmed that the promoter of the cg2556 gene (iron-regulated membrane protein) is σC-dependent as suggested recently by other authors. The promoter of cmt1 (trehalose corynemycolyl transferase) was found to be clearly σD-dependent. No σM-dependent promoter was identified. The typical housekeeping promoter P2sigA (sigma factor σA) was proven to be σA-dependent but also recognized by σB. Similarly, the promoter of fba (fructose-1,6-bisphosphate aldolase) was confirmed to be σB-dependent but also functional with σA. The study provided demonstrations of the broad applicability of the developed methods and produced original data on the analyzed promoters.

Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications.

The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and ß-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors.

Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles.

The aim of this study was to discover new nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic nitrilase, arylacetonitrilase, aliphatic nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetonitrilases and an aromatic nitrilase, respectively.

Catabolism of Phenol and Its Derivatives in Bacteria: Genes, Their Regulation, and Use in the Biodegradation of Toxic Pollutants.

Phenol and its derivatives (alkylphenols, halogenated phenols, nitrophenols) are natural or man-made aromatic compounds that are ubiquitous in nature and in human-polluted environments. Many of these substances are toxic and/or suspected of mutagenic, carcinogenic, and teratogenic effects. Bioremediation of the polluted soil and water using various bacteria has proved to be a promising option for the removal of these compounds. In this review, we describe a number of peripheral pathways of aerobic and anaerobic catabolism of various natural and xenobiotic phenolic compounds, which funnel these substances into a smaller number of central catabolic pathways. Finally, the metabolites are used as carbon and energy sources in the citric acid cycle. We provide here the characteristics of the enzymes that convert the phenolic compounds and their catabolites, show their genes, and describe regulatory features. The genes, which encode these enzymes, are organized on chromosomes and plasmids of the natural bacterial degraders in various patterns. The accumulated data on similarities and the differences of the genes, their varied organization, and particularly, an astonishingly broad range of intricate regulatory mechanism may be read as an exciting adventurous book on divergent evolutionary processes and horizontal gene transfer events inscribed in the bacterial genomes. In the end, the use of this wealth of bacterial biodegradation potential and the manipulation of its genetic basis for purposes of bioremediation is exemplified. It is envisioned that the integrated high-throughput techniques and genome-level approaches will enable us to manipulate systems rather than separated genes, which will give birth to systems biotechnology.

Induction and carbon catabolite repression of phenol degradation genes in Rhodococcus erythropolis and Rhodococcus jostii.

Rhodococcus erythropolis CCM2595 is able to efficiently utilize phenol and other aromatic compounds. We cloned and sequenced its complete gene cluster - catA, catB, catC, catR, pheR, pheA2, pheA1 - involved in the ortho-cleavage pathway of phenol. The activity of the key enzyme of the phenol degradation pathway, two-component phenol hydroxylase, was found to be induced by phenol. When both phenol and succinate were present in the medium, phenol hydroxylase activity decreased substantially. To analyze the regulation of phenol degradation at the transcriptional level, the transcriptional fusions of the divergently oriented promoters PpheA2 and PpheR with the gfpuv reporter gene were constructed. The promoters driving expression of the genes of the pheR-pheA2pheA1 cluster were localized by determining the respective transcriptional start points. Measurements of GFP fluorescence as well as quantitative RT-PCR revealed that expression of the phe genes is induced by phenol at the transcriptional level. The transcription of pheA2A1 and pheR was repressed by succinate, whereas no repression by glucose or glycerol was observed. Activation of the R. erythropolis CCM2595 pheA2 promoter by PheR, an AraC-type transcriptional regulator, was demonstrated by overexpression of the pheR gene. Analysis of the transcriptional regulation of two similar phe clusters from R. jostii RHA1 by various substrates showed that the type of carbon catabolite repression and the temporal transcriptional pattern during cultivation are different in each of the three phe clusters analyzed.

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.