Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.     

Mineralization of 14C-Ring-Labeled Synthetic Lignin Correlates with the Production of Lignin Peroxidase, not of Manganese Peroxidase or Laccase

Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12- to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.     

Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora

A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I–P–X–P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5 kb contained the complete sequence of the Cs-mnp1 gene, including 162 bp and 770 bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT–AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9 kb was amplified using inverse PCR. A putative TATAA element was identified 5′ of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.     

Studies on the Production of Fungal Peroxidases in Aspergillus niger

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.     

The white-rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>: conditions for the production of lignin-degrading enzymes

Investigating optimal conditions for lignin-degrading peroxidases production by Phanerochaete chrysosporium (P. chrysosporium) has been a topic for numerous researches. The capability of P. chrysosporium for producing lignin peroxidases (LiPs) and manganese peroxidases (MnPs) makes it a model organism of lignin-degrading enzymes production. Focusing on compiling and identifying the factors that affect LiP and MnP production by P. chrysosporium, this critical review summarized the main findings of about 200 related research articles. The major difficulty in using this organism for enzyme production is the instability of its productivity. This is largely due to the poor understanding of the regulatory mechanisms of P. chrysosporium responding to different nutrient sources in the culture medium, such as metal elements, detergents, lignin materials, etc. In addition to presenting the major conclusions and gaps of the current knowledge on lignin-degrading peroxidases production by P. chrysosporium, this review has also suggested further work, such as correlating the overexpression of the intra and extracellular proteins to the nutrients and other culture conditions to discover the regulatory cascade in the lignin-degrading peroxidases production process, which may contribute to the creation of improved P. chrysosporium strains leading to stable enzyme production.     

Calnexin Overexpression Increases Manganese Peroxidase Production in Aspergillus niger

Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.     

Lipid Peroxidation by the Manganese Peroxidase of Phanerochaete chrysosporium Is the Basis for Phenanthrene Oxidation by the Intact Fungus

The manganese peroxidase (MnP) of Phanerochaete chrysosporium supported Mn(II)-dependent, H₂O₂-independent lipid peroxidation, as shown by two findings: linolenic acid was peroxidized to give products that reacted with thiobarbituric acid, and linoleic acid was peroxidized to give hexanal. MnP also supported the slow oxidation of phenanthrene to 2,2′-diphenic acid in a reaction that required Mn(II), oxygen, and unsaturated lipids. Phenanthrene oxidation to diphenic acid by intact cultures of P. chrysosporium occurred to the same extent that oxidation in vitro did and was stimulated by Mn. These results support a role for MnP-mediated lipid peroxidation in phenanthrene oxidation by P. chrysosporium.     

Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris

A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C.     

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae.

A manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3' untranslated region of the glucoamylase gene of Aspergillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

Characterization of manganese peroxidases from the hyperlignolytic fungus IZU-154.

Four isozymes of manganese peroxidase (MnP) were identified in the culture fluid of the hyperlignolytic fungus IZU-154 under nitrogen starvation conditions. One of them was purified and characterized kinetically. The specific activity and Kcat/K(m) value of the MnP from IZU-154 were 1.6 times higher than those of the MnP from a typical lignin-degrading fungus, Phanerochaete chrysosporium. Two cDNAs encoding MnP isozymes from IZU-154 were isolated. The coding sequence of the two cDNAs, IZ-MnP1 cDNA and IZ-MnP2 cDNA, were 1,152 (384 amino acids) and 1,155 (385 amino acids) bp in length, respectively. They exhibit 96.2% identity at the nucleotide level and 95.1% identity at the amino acid level. Southern blot analysis indicated that two MnP isozyme genes exist in IZU-154 genomic DNA. The primary structures of two MnPs from IZU-154 were similar to those of MnPs from P. chrysosporium. The amino acid sequences including the important residues identified in MnPs from P. chrysosporium, such as the manganese-binding residues, the calcium-binding residues, the disulfide bonds, and the N-glycosylation site, were conserved in the two deduced IZ-MnPs. However, several discrepancies were found in the context around the distal histidine residue between MnP from IZU-154 and MnP from P. chrysosporium, which likely led to the difference in the kinetic parameters for MnP function.     

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment.     

Increasing manganese peroxidase productivity of Phanerochaete chrysosporium by optimizing carbon sources and supplementing small molecules

Aims: To overproduce manganese peroxidase (MnP) using Phanerochaete chrysosporium. Effects of carbon sources, agricultural by‐products and small molecules were tested to enhance the MnP productivity. Methods and Results: Various carbon sources and agricultural by‐products were compared as the basal medium. A MnP activity of 4·7 ± 0·31 U ml^?1 was obtained using mannose as a carbon source. The enzyme productivity further reached 7·36 ± 0·05 and 8·77 ± 0·23 U ml^?1 when the mannose medium was supplemented with cyclic adenosine monophosphate (cAMP) and S‐adenosylmethionine, respectively. Conclusions: This study revealed the highest MnP productivity obtained by optimizing the carbon sources and supplementing small molecules. It can provide insight for: (i) making high quantities of enzymes by optimizing media resources and (ii) engineering the global regulatory genes in P. chrysosporium for the onsite production of MnP. Significance and Impact of the Study: As MnP is an important enzyme to hydrolyse lignin polymers which protects the plant cell wall from exposing of cellulose fibres, the production of the enzyme is a current demand for biofuel biotechnology. The knowledge generated from this study can help to advance the technology for stable production of MnP.     

Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts

Summary Lignin (LiP) and manganese peroxidase (MnP) excretion by Phanerochaete chrysosporium INA-12 was significantly increased in response to fungal extract supplementation. LiP and MnP production was increased 1.7- and 1.8-fold, respectively, with fungal extracts from agitated pellet cultures of strain INA-12, namely fungal extracts P₆ and P₄. In cultures supplemented with a fungal extract harvested from static cultures of strain INA-12 (fungal extract S₄), LiP and MnP production was increased 1.8- and 1.6-fold, respectively. Succinate dehydrogenase activity, a mitochondrial marker, was significantly enhanced (2.7-fold) in cultures with the addition of fungal extracts. Correspondence to: M. Asther     

Molecular characterization of manganese peroxidases from white-rot fungus Polyporus brumalis

The cDNAs of six manganese-dependent peroxidases (MnPs) were isolated from white-rot fungus Polyporus brumalis. The MnP proteins shared similar properties with each other in terms of size (approximately 360–365 amino acids) and primary structure, showing 62–96 % amino acid sequence identity. RT-PCR analysis indicated that these six genes were predominantly expressed in shallow stationary culture (SSC) in a liquid medium. Gene expression was induced by treatment with dibutyl phthalate (DBP) and wood chips. Expression of pbmnp4 was strongly induced by both treatments, whereas that of pbmnp5 was induced only by DBP, while pbmnp6 was induced by wood chips only. Then, we overexpressed pbmnp4 in P. brumalis under the control of the GPD promoter. Overexpression of pbmnp4 effectively increased MnP activity; the transformant that had the highest MnP activity also demonstrated the most effective decolorization of Remazol Brilliant Blue R dye. Identification of MnP cDNAs can contribute to the efficient production of lignin-degradation enzymes and may lead to utilization of basidiomycetous fungi for degradation of lignin and numerous recalcitrant xenobiotics.     

Manganoporphyrins and ascorbate enhance gemcitabine cytotoxicity in pancreatic cancer

Pharmacological ascorbate (AscH⁻) selectively induces cytotoxicity in pancreatic cancer cells vs normal cells via the generation of extracellular hydrogen peroxide (H₂O₂), producing double-stranded DNA breaks and ultimately cell death. Catalytic manganoporphyrins (MnPs) can enhance ascorbate-induced cytotoxicity by increasing the rate of AscH⁻ oxidation and therefore the rate of generation of H₂O₂. We hypothesized that combining MnPs and AscH⁻ with the chemotherapeutic agent gemcitabine would further enhance pancreatic cancer cell cytotoxicity without increasing toxicity in normal pancreatic cells or other organs. Redox-active MnPs were combined with AscH⁻ and administered with or without gemcitabine to human pancreatic cancer cell lines, as well as immortalized normal pancreatic ductal epithelial cells. The MnPs MnT2EPyP (Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin pentachloride) and MnT4MPyP (Mn(III)tetrakis(N-methylpyridinium-4-yl) porphyrin pentachloride) were investigated. Clonogenic survival was significantly decreased in all pancreatic cancer cell lines studied when treated with MnP + AscH⁻ + gemcitabine, whereas nontumorigenic cells were resistant. The concentration of ascorbate radical (Asc^•−, an indicator of oxidative flux) was significantly increased in treatment groups containing MnP and AscH⁻. Furthermore, MnP + AscH⁻ increased double-stranded DNA breaks in gemcitabine-treated cells. These results were abrogated by extracellular catalase, further supporting the role of the flux of H₂O₂. In vivo growth was inhibited and survival increased in mice treated with MnT2EPyP, AscH⁻, and gemcitabine without a concomitant increase in systemic oxidative stress. These data suggest a promising role for the use of MnPs in combination with pharmacologic AscH⁻ and chemotherapeutics in pancreatic cancer.     

Genetic analysis of cysteine-poor prolamin polypeptides reduced in the endosperm of the rice esp1 mutant

The esp1 mutant CM21 specifically exhibits reduced levels of cysteine-poor (CysP) prolamin bands with pIs of 6.65, 6.95, 7.10, and 7.35 in rice seed. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis demonstrated that the bands with pIs 6.65, 6.95, and 7.35 are encoded by different structural genes. These results suggest that the Esp1 locus encodes a regulatory factor involved in the synthesis and/or accumulation of CysP prolamin molecules. Isoelectric focusing (IEF) analysis of CysP prolamins in chromosome substitution lines showed that structural genes for bands with pI values of 6.95, 7.10, and 7.35, which are reduced in esp1 mutant lines, are located as a gene cluster in the 44.2 cM region on chromosome 5.     

Improvement of Manganese Peroxidase Production by the Hyper Lignin-Degrading Fungus Phanerochaete sordida YK-624 by Recombinant Expression of the 5-Aminolevulinic Acid Synthase Gene

The manganese peroxidase (MnP) gene (mnp4) promoter of Phanerochaete sordida YK-624 was used to drive expression of 5-aminolevulinic acid synthase (als), which is a key heme biosynthesis enzyme. The expression plasmid pMnP4pro-als was transformed into P. sordida YK-624 uracil auxotrophic mutant UV-64, and 14 recombinant als expressing-transformants were generated. Average cumulative MnP activities in the transformants were 1.18-fold higher than that of control transformants. In particular, transformants A-14 and A-61 showed significantly higher MnP activity (approximately 2.8-fold) than wild type. RT-PCR analysis indicated that the increased MnP activity was caused by elevated recombinant als expression. These results suggest that the production of MnP is improved by high expression of als.