Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061

ABSTRACT: BACKGROUND: The ketoisovalerate reductase (EC 1.2.7.7 ) is required for the formation of beauvericin via the nonribosomal peptide synthetase biosynthetic pathway. It catalyzes the NADPH-specific reduction of ketoisovaleric acid to hydroxyisovalerate. However, little is known about the bioinformatics' data about the 2-Kiv reductase in Fusarium. To date, heterologous production of the gene KivRFp from Fusarium has not been achieved. RESULTS: The KivRFp gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene KivRFp contained a 1,359 bp open reading frame (ORF) encoding a polypeptide of 452 amino acids with a molecular mass of 52 kDa. Sequence analysis indicated that it showed 61% and 52% amino acid identities to ketoisovalerate reductase from Beauveria bassiana ATCC 7159 (ACI30654) and Metarhizium acridum CQMa 102 (EFY89891), respectively; and several conserved regions were identified, including the putative nucleotide-binding signature site, GXGXXG, a catalytic triad (Glu405, Asn184, and Lys285). The KivRFp exhibited the highest activity at 35[DEGREE SIGN]C and pH 7.5 respectively, by reduction of ketoisovalerate. It also exhibited the high level of stability over wide temperature and pH spectra and in the presence of metal ions or detergents. CONCLUSIONS: A new ketoisovalerate reductase KivRFp was identified and characterized from the depsipeptide-producing fungus F. proliferatum. KivRFp has been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering and directed evolution, towards the goal of developing efficient enzyme for downstream biotechnological applications.     

Cloning and characterization of the gene cluster required for beauvericin biosynthesis in Fusarium proliferatum

Beauvericin, a cyclohexadepsipeptide-possessing natural product with synergistic antifungal, insecticidal, and cytotoxic activities. We isolated and characterized the fpBeas gene cluster, devoted to beauvericin biosynthesis, from the filamentous fungus Fusarium proliferatum LF061. Targeted inactivation of the F. proliferatum genomic copy of fpBeas abolished the production of beauvericin. Comparative sequence analysis of the FpBEAS showed 74% similarity with the BbBEAS that synthesizes the cyclic trimeric ester beauvericin in Beauveria bassiana, which assembles N-methyl-dipeptidol monomer intermediates by the programmed iterative use of the nonribosomal peptide synthetase modules. Differences between the organization of the beauvericin loci in F. proliferaturm and B. bassiana revealed the mechanism for high production of beauvericin in F. proliferatum. Our work provides new insights into beauvericin biosynthesis, and may lead to beauvericin overproduction and creation of new analogs via synthetic biology approaches.     

Purification and characterization of nitrilase from Fusarium solani IMI196840

Nitrilase activity in Fusarium solani IMI196840 (approx. 1500 U l⁻¹ of culture broth) was induced by 2-cyanopyridine. The enzyme was purified by a factor of 20.3 at a yield of 26.9%. According to gel filtration, the holoenzyme was an approx. 550-kDa homooligomer consisting of subunits with a molecular weight of approximately 40 kDa, as determined by SDS-PAGE. Mass spectrometry analysis of the tryptic fragments suggested a high similarity of this enzyme to the hypothetical CN hydrolases from Aspergillus oryzae, Gibberella zeae, Gibberella moniliformis and Nectria haematococca. Circular dichroism and fluorescence spectra indicated that secondary structure content and overall tertiary structure, respectively, were almost identical in nitrilases from F. solani IMI196840 and F. solani O1. The melting temperatures of the enzymes were 49.3 °C and 47.8 °C, respectively. The best substrates for the purified nitrilase from F. solani IMI196840 were benzonitrile and 4-cyanopyridine, which were hydrolyzed at the rates of 144 and 312 U mg⁻¹ protein, respectively, under the optimum conditions of pH 8 and 45 °C. The enzyme was highly chemoselective, producing ≤2% amides as by-products.     

Arthrobacter nitroguajacolicus腈水解酶基因的克隆和表达

腈水解酶是一类能将腈类化合物催化生成酸的氰基水解酶。目前已有多个菌种的腈水解酶基因序列被报道,如敏捷食酸菌Acidovoraxfacilis,粪产碱菌Alcaligenesfaecalis,睾丸丛毛单胞菌Comamonastestoteroni,肺炎克雷伯菌Klebsiellapneumoniae,假单胞菌Pseudomonas属菌株,红球菌Rhodococcus属菌株,但节杆菌属菌株Arthrobacternitroguajacolicus的腈水解酶基因序列尚未见报道。经由野生型酶的分离纯化,基因文库筛选及侧翼序列扩增等步骤,克隆得到该菌株的腈水解酶基因,从而为进一步研究该酶的特性及构建用于工业生产的重组菌打下基础。     

Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum

Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization–ion trap–mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60°C, thermal stability for 2 h at 40°C, optimum pH 3.5 (km=62 μM) measured on 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4–8 (75% stability at pH levels 2.2 and 9) for 2 h at 25°C.     

Characterization of a new mitochondrial plasmid from Fusarium proliferatum

A 10.3kb linear mitochondrial DNA plasmid designated pFP1 was isolated from Fusarium proliferatum. The DNA sequence of the plasmid consists of 10,336bp with perfect terminal inverted repeats of 400bp. Two major, non-overlapping ORFs were identified on opposite strands, encoding a phage-type RNA polymerase and a family B type DNA polymerase, respectively. One additional minor ORF encoding a putative highly basic protein was also identified. The copy number of pFP1, as determined by RT-PCR, ranged between 1.8 and 3.1 per mtDNA copies depending on the host strain. Real-time PCR analysis of a total of 400 cultures surviving ethidium bromide curing indicated that no plasmid-free strains could be obtained by this treatment. Further single spore selections of the survivors with reduced plasmid content were needed to obtain plasmid-free clones. No phenotypic differences were found between the wild-type strains and their plasmid-free progenies.     

Restoration of gibberellin production in Fusarium proliferatum by functional complementation of enzymatic blocks

Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5' noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs.     

Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.     

Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles.

A novel nitrilase that preferentially catalyzes the hydrolysis of aliphatic nitriles to the corresponding carboxylic acids and ammonia was found in the cells of a facultative crotononitrile-utilizing actinomycete isolated from soil. The strain was taxonomically studied and identified as Rhodococcus rhodochrous. The nitrilase was purified, with 9.08% overall recovery, through five steps from a cell extract of the stain. After the last step, the purified enzyme appeared to be homogeneous, as judged by polyacrylamide gel electrophoresis, analytical centrifugation, and double immunodiffusion in agarose. The relative molecular weight values for the native enzyme, estimated from the ultracentrifugal equilibrium and by high-performance liquid chromatography, were approximately 604,000 +/- 30,000 and 650,000, respectively, and the enzyme consisted of 15 to 16 subunits identical in molecular weight (41,000). The enzyme acted on aliphatic olefinic nitriles such as crotononitrile and acrylonitrile as the most suitable substrates. The apparent Km values for crotononitrile and acrylonitrile were 18.9 and 1.14 mM, respectively. The nitrilase also catalyzed the direct hydrolysis of saturated aliphatic nitriles, such as valeronitrile, 4-chlorobutyronitrile, and glutaronitrile, to the corresponding acids without the formation of amide intermediates. Hence, the R. rhodochrous K22 nitrilase is a new type distinct from all other nitrilases that act on aromatic and related nitriles.     

Cloning and functional characterization of the HRASLS2 gene

The HRAS-like suppressor 2 (HRASLS2) gene belongs to the H-REV107 gene family involved in the regulation of cell growth and differentiation. HRASLS2 is expressed at high levels in normal tissues of the small intestine, kidney, and trachea. We cloned HRASLS2 cDNA from human SW480 colon cancer cells. Most wild-type, and some N- and C-terminal truncated HRASLS2 (HRASLS2DeltaNDeltaC) were expressed as a granular pattern located at perinuclear region in HtTA cervical cancer cells, while truncation at the C-terminus only (HRASLS2DeltaC) resulted in a diffuse pattern. Wild-type HRASLS2 significantly suppressed colony formation of HeLa and HCT116 cells. HRASLS2DeltaNDeltaC significantly inhibited colony formation of HCT116 cells, but HRASLS2DeltaC did not affect cell growth. HRASLS2 suppressed the RAS-GTP levels and total RAS protein by 44% and 25%, respectively in HtTA cells; however, the suppression was not observed in truncated HRASLS2 variants. In conclusion, the HRASLS2 protein suppressed growth and RAS activities of cancer cells, and the C-terminal hydrophobic domain appeared to be indispensable for both activities.     

Asymmetric hydrolysis of chiral nitriles byRhodococcus rhodochrous NCIMB 11216 nitrilase

Summary The enantioselective potential ofRhodococcus rhodochrous NCIMB 11216 nitrilase has been measured, using a range of chiral nitriles with various C-2 group substitutions. The highest selectivity was achieved during the biotransformation of (+/–) 2-methylhexanitrile where the reaction appears enantiospecific for the (+) enantiomer.     

Purification and characterization of a nitrilase from Brassica napus

In germinating seedlings of Brassica napus glucosinolate levels decrease and are potentially degraded to nitriles by a myrosinase. Little is known about the metabolism of glucosinolate aglycone products and the objective of this work was to investigate nitrilase activity and carry out a purification of the enzyme from seedlings of B. napus . A nitrilase capable of converting phenylpropionitrile to phenylpropionic acid was purified to apparent homogeneity from seedlings of B. napus . The protein has a molecular mass of approximately 420 kDa made up of 38 kDa subunits. The pI of the native protein was found to be 4.6. Under denaturing conditions on an isoelectric focusing (IEF) gel a major and minor protein was observed with pI in the range of 5.4-5.9, suggesting the presence of isoforms. Apart from the potential role of the nitrilase in indole-3-acetic acid (IAA) synthesis a developmental study with seedlings indicates that the increase in activity observed may be linked to the in vivo degradation of glucosinolates.     

Purification and properties of an extracellular beta-xylosidase from a newly isolated Fusarium proliferatum

An extracellular beta-xylosidase from a newly isolated Fusarium proliferatum (NRRL 26517) capable of utilizing corn fiber xylan as growth substrate was purified to homogeneity from the culture supernatant by DEAE-Sepharose CL-6B batch adsorption chromatography, CM Bio-Gel A column chromatography, Bio-Gel A-0.5 m gel filtration and Bio-Gel HTP Hydroxyapatite column chromatography. The purified beta-xylosidase (specific activity, 53 U/mg protein) had a molecular weight of 91,200 as estimated by SDS-PAGE. The optimum temperature and pH for the action of the enzyme were 60 degrees C and 4.5, respectively. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It had a Km value of 0.77 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a Ki value of 5 mM. The enzyme did not require any metal ion for activity and stability. Comparative properties of this enzyme with other fungal beta-xylosidases are presented.     

Mild hydrolysis of nitriles by the immobilized nitrilase from Aspergillus niger K10

The cell free extract from the nitrile-hydrolyzing strain Aspergillus niger K10 (0.25 mg of protein) was adsorped onto a 1 mL HiTrap Butyl Sepharose column. The benzonitrile-hydrolyzing activity of the immobilized enzyme (about 1.6 U/mg of protein) was stable at pH 8 and 35 °C within the examined period (4 h). The enzyme load on the above column was increased 18 times in order to achieve high nitrile conversion. This enzyme preparation was used for the conversion of 3-cyanopyridine and 4-cyanopyridine under the above conditions. The initial substrate conversion was nearly quantitative. The activity was fairly stable; the conversion of 3-cyanopyridine decreased to 70% after 15 h, while the conversion of 4-cyanopyridine was 60% of the initial value after 39 h. The former substrate was converted into nicotinic acid and nicotinamide (molar ratio approximately 16:1) and the latter one into isonicotinic acid and isonicotinamide (molar ratio approximately 3:1).     

Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.     

Fdb1 and Fdb2, Fusarium verticillioides loci necessary for detoxification of preformed antimicrobials from corn

Fusarium verticillioides is a fungus of significant economic importance because of its deleterious effects on plant and animal health and on the quality of their products. Corn (Zea mays) is the primary host for F. verticillioides, and we have investigated the impact of the plant's antimicrobial compounds (DIMBOA, DIBOA, MBOA, and BOA) on fungal virulence and systemic colonization. F. verticillioides is able to metabolize these antimicrobials, and genetic analyses indicated two loci, Fdb1 and Fdb2, were involved in detoxification. Mutation at either locus caused sensitivity and no detoxification. In vitro physiological complementation assays resulted in detoxification of BOA and suggested that an unknown intermediate compound was produced. Production of the intermediate compound involved Fdbl, and a lesion in fdb2 preventing complete metabolism of BOA resulted in transformation of the intermediate into an unidentified metabolite. Based on genetic and physiological data, a branched detoxification pathway is proposed. Use of genetically characterized detoxifying and nondetoxifying strains indicated that detoxification of the corn antimicrobials was not a major virulence factor, since detoxification was not necessary for development of severe seedling blight or for infection and endophytic colonization of seedlings. Production of the antimicrobials does not appear to be a highly effective resistance mechanism against F. verticillioides.     

Cloning and functional characterization of a homoglutathione synthetase from pea nodules

The thiol tripeptide glutathione (GSH; γ Glu-Cys-Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; γ Glu-Cys- β Ala), in addition to or instead of GSH. We have isolated from a pea ( Pisum sativum L.) nodule library a cDNA, GSHS2 , that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min⁻¹ mg⁻¹ protein with a standard substrate concentration (5 m M β -alanine) and K_m values of 1.9 m M for β -alanine and 104 m M for glycine. The specificity constant (V_max/K_m) shows that the pure enzyme is 57.3-fold more specific for β -alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity.     

Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.