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Expression of a new laccase from Moniliophthora roreri at high levels in Pichia pastoris and its potential application in micropollutant degradation.

ABSTRACT: Laccases have gained significant attention due to their emerging applications including bioremediation, biomass degradation and biofuel cells. One of the prerequisites for the industrial application of laccases is their sufficient availability. However, expression levels of recombinantly expressed laccases are often low. In this study Mrl2, a new laccase from the basidiomycete Moniliophthora roreri, was cloned in Pichia pastoris and produced in an optimized fed-batch process at an exceptionally high yield of 1.05 g l-1. With a redox potential of 0.58 V, Mrl2 belongs to mid-redox potential laccases. However, Mrl2 demonstrated high kcat values of 316, 20, 74, and 36 s-1 towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol, respectively. Mrl2 remained stable above pH 6 and in the presence of many metal ions, which is important for application in bioremediation. Mrl2 was investigated for the ability to degrade endocrine disrupting chemicals (EDCs) and non-steroidal anti-inflammatory drugs (NSDAIs) at neutral pH value. The enzyme accepted and converted estrone, 17?-estradiol, estriol, the synthetic contraceptive 17?-ethinyl estradiol and bisphenol A at pH 7 faster than high-potential laccases from Trametes versicolor. For example, within 30 min Mrl2 removed more than 90% bisphenol A, 17ß-estradiol, 17?-ethinyl estradiol and estriol, respectively. The concentration of the recalcitrant drug diclofenac dropped by 56% after 20 h incubation with Mrl2.

SUBMITTER: Bronikowski A

PROVIDER: S-EPMC5371579 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Expression of a new laccase from Moniliophthora roreri at high levels in Pichia pastoris and its potential application in micropollutant degradation.

Bronikowski Agathe A Hagedoorn Peter-Leon PL Koschorreck Katja K Urlacher Vlada B VB

AMB Express 20170329 1

Laccases have gained significant attention due to their emerging applications including bioremediation, biomass degradation and biofuel cells. One of the prerequisites for the industrial application of laccases is their sufficient availability. However, expression levels of recombinantly expressed laccases are often low. In this study Mrl2, a new laccase from the basidiomycete Moniliophthora roreri, was cloned in Pichia pastoris and produced in an optimized fed-batch process at an exceptionally ...[more]

PMID: 28357784

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Project description:BackgroundThe hemibiotrophic pathogens Moniliophthora perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot disease) are among the most important pathogens of cacao. Moniliophthora perniciosa has a broad host range and infects a variety of meristematic tissues in cacao plants, whereas M. roreri infects only pods of Theobroma and Herrania genera. Comparative pathogenomics of these fungi is essential to understand Moniliophthora infection strategies, therefore the detection and in silico functional characterization of effector candidates are important steps to gain insight on their pathogenicity.ResultsCandidate secreted effector proteins repertoire were predicted using the genomes of five representative isolates of M. perniciosa subpopulations (three from cacao and two from solanaceous hosts), and one representative isolate of M. roreri from Peru. Many putative effectors candidates were identified in M. perniciosa: 157 and 134 in cacao isolates from Bahia, Brazil; 109 in cacao isolate from Ecuador, 92 and 80 in wild solanaceous isolates from Minas Gerais (Lobeira) and Bahia (Caiçara), Brazil; respectively. Moniliophthora roreri showed the highest number of effector candidates, a total of 243. A set of eight core effectors were shared among all Moniliophthora isolates, while others were shared either between the wild solanaceous isolates or among cacao isolates. Mostly, candidate effectors of M. perniciosa were shared among the isolates, whereas in M. roreri nearly 50% were exclusive to the specie. In addition, a large number of cell wall-degrading enzymes characteristic of hemibiotrophic fungi were found. From these, we highlighted the proteins involved in cell wall modification, an enzymatic arsenal that allows the plant pathogens to inhabit environments with oxidative stress, which promotes degradation of plant compounds and facilitates infection.ConclusionsThe present work reports six genomes and provides a database of the putative effectorome of Moniliophthora, a first step towards the understanding of the functional basis of fungal pathogenicity.

Project description:Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory.A blood-active laccase (ChU-B mutant) fused to the native/evolved ?-factor prepro-leader was cloned under the control of two different promoters (P(AOX1) and P(GAP)) and expressed in Pichia pastoris. The most active construct, which contained the P(AOX1) and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the ?-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers.The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.

Project description:BACKGROUND: Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. RESULTS: In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg(-1) protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. CONCLUSION: Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L(-1) in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation.

Project description:Taxonomy: Moniliophthora roreri (Cif.) H.C. Evans et al. ; Phylum Basidiomycota; Class Agaricomycetes; Order Agaricales; Family Marasmiaceae; Genus Moniliophthora. Biology: Moniliophthora roreri attacks Theobroma and Herrania species causing frosty pod rot. Theobroma cacao (cacao) is the host of major economic concern. Moniliophthora roreri is a hemibiotroph with a long biotrophic phase (45-90 days). Spore masses, of apparent asexual origin, are produced on the pod surface after initiation of the necrotrophic phase. Spores are spread by wind, rain and human activity. Symptoms of the biotrophic phase can include necrotic flecks and, in some cases, pod malformation, but pods otherwise remain asymptomatic. Relationship to Moniliophthora perniciosa: Moniliophthora roreri and Moniliophthora perniciosa, causal agent of witches' broom disease of cacao, are closely related. Their genomes are similar, including many of the genes they carry which are considered to be important in the disease process. Moniliophthora perniciosa, also a hemibiotroph, has a typical basidiomycete lifestyle and morphology, forming clamp connections and producing mushrooms. Basidiospores infect meristematic tissues including flower cushions, stem tips and pods. Moniliophthora roreri does not form clamp connections or mushrooms and infects pods only. Both pathogens are limited to the Western Hemisphere and are a threat to cacao production around the world. Agronomic importance: Disease losses caused by frosty pod rot can reach 90% and result in field abandonment. Moniliophthora roreri remains in the invasive phase in the Western Hemisphere, not having reached Brazil, some islands within the Caribbean and a few specific regions within otherwise invaded countries.Disease managementThe disease can be managed by a combination of cultural (for example, maintenance of tree height and removal of infected pods) and chemical methods. These methods benefit from regional application, but can be cost prohibitive. Breeding for disease resistance offers the greatest potential for frosty pod rot management and new tolerant materials are becoming available.

Project description:Cutinases are enzymes produced by phytopathogenic fungi like Moniliophthora roreri. The three genome-located cutinase genes of M. roreri were amplified from cDNA of fungi growing in different induction culture media for cutinase production. The mrcut1 gene was expressed in the presence of a cacao cuticle, while the mrcut2 and mrcut3 genes were expressed when an apple cuticle was used as the inducer. The sequences of all genes were obtained and analyzed by bioinformatics tools to determine the presence of signal peptides, introns, glycosylation, and regulatory sequences. Also, the theoretical molecular weight and pI were obtained and experimentally confirmed. Finally, cutinase 1 from M. roreri (MRCUT1) was selected for heterologous expression in Escherichia coli. Successful overexpression of MRCUT1 was observed with the highest enzyme activity of 34,036 U/mg under the assay conditions at 40°C and pH 8. Furthermore, the degradation of different synthetic polyesters was evaluated; after 21 days, 59% of polyethylene succinate (PES), 43% of polycaprolactone (PCL), and 31% of polyethylene terephthalate (PET) from plastic residues were degraded. IMPORTANCE Plastic pollution is exponentially increasing; even the G20 has recognized an urgent need to implement actions to reduce it. In recent years, searching for enzymes that can degrade plastics, especially those based on polyesters such as PET, has been increasing as they can be a green alternative to the actual plastic degradation process. A promising option in recent years refers to biological tools such as enzymes involved in stages of partial and even total degradation of some plastics. In this context, the MRCUT1 enzyme can degrade polyesters contained in plastic residues in a short time. Besides, there is limited knowledge about the biochemical properties of cutinases from M. roreri. Commonly, fungal enzymes are expressed as inclusion bodies in E. coli with reduced activity. Interestingly, the successful expression of one cutinase of M. roreri in E. coli with enhanced activity is described.

Dataset Information

Expression of a new laccase from Moniliophthora roreri at high levels in Pichia pastoris and its potential application in micropollutant degradation.

Publications

Expression of a new laccase from Moniliophthora roreri at high levels in Pichia pastoris and its potential application in micropollutant degradation.

Similar Datasets

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