Escherichia coli as a production host for novel enzymes from basidiomycota

Zelena, Katerina; Eisele, Nadine; Berger, Ralf G.

doi:10.1016/j.biotechadv.2014.08.006

Cited by 15 publications

(15 citation statements)

References 204 publications

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“…A large‐scale utilization of fungal peroxidases is often hampered by their low expression in native and heterologous expression hosts (e.g., Escherichia coli , S. cerevisiae or P. pastoris ) . Many research groups use E. coli as an expression host, but the peroxidases mostly aggregate in inclusion bodies and have to be tediously refolded . Peroxidase expression levels in heterologous hosts have not yet exceeded 100 mg L −1 in the culture medium, but are mostly in the range of 1–20 mg L −1 .…”

Section: Resultsmentioning

confidence: 99%

Redesign of a New Manganese Peroxidase Highly Expressed in Pichia pastoris towards a Lignin‐Degrading Versatile Peroxidase

2018

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Manganese peroxidases, lignin peroxidases, and versatile peroxidases secreted by white rot fungi are supposed to play an essential role in lignin degradation. Thus, these enzymes have attracted significant attention as potential biocatalysts. Versatile peroxidases are the most interesting ones, since they comprise activities of manganese and lignin peroxidases. Herein, we demonstrate how the properties of a new manganese peroxidase from Moniliophthora roreri, designated MrMnP1, were shifted towards those of a versatile peroxidase. MrMnP1 was cloned in Pichia pastoris X‐33 and highly expressed in a fed‐batch fermentation, yielding 132 mg L−1 of active enzyme. To extend the substrate spectrum of MrMnP1, a catalytically active tryptophan present in lignin and versatile peroxidases was first introduced. Additionally, the role of five amino acids at positions adjacent to the catalytic tryptophan was elucidated through their replacement by those found in a versatile peroxidase from Pleurotus eryngii. The resulting mutants demonstrated new activities towards high‐redox‐potential substrates, such as lignin dimers, veratryl alcohol, and the azo dye Reactive Black 5.

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Section: Resultsmentioning

confidence: 99%

Redesign of a New Manganese Peroxidase Highly Expressed in Pichia pastoris towards a Lignin‐Degrading Versatile Peroxidase

2018

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“…Laccases have great potential for industrial applications due to their wide variety of oxidation substrates; therefore, many researchers have attempted to clone novel laccase genes and improve their activity and stability. The main disadvantage of using E. coli as a heterologous host, is the production of catalytically inactive protein aggregates, the so‐called inclusion bodies . The highly reductive environment of the bacterial cytosol and the inability of E. coli to perform eukaryotic post‐translational modifications required for folding and activity, typically results in the formation of insoluble proteins .…”

Section: Discussionmentioning

confidence: 99%

Expression, purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli

Liu

Jia

et al. 2017

J Basic Microbiol

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Laccases are multicopper oxidases (E.C. 1.10.3.2) that catalyze the oxidation of many phenolic compounds. In this study, a novel laccase, Stlac4, from Setosphaeria turcica was cloned and expressed in Escherichia coli by insertion into the pET-30a expression plasmid. The recombinant laccase was purified and visualized on SDS-PAGE as a single band with an apparent molecular weight of 71.5 KDa, and confirmed by Western blot. The maximum activity of the purified laccase was 127.78 U · mg −1 , the optimum temperature and pH value were 60°C and 4. Laccases are wide-spread in nature and over 220 species of laccases have been isolated from many sources,including plants, fungi, bacteria, and insects [3][4][5][6][7]. Fungal laccases are the most extensively studied multicopper oxidases, and while primarily considered to be involved in lignin degradation [8], recent studies have shown that fungal laccases participate in fruiting body formation [9], pigment biosynthesis [10], pathogenesis, and stress defense [11]. Laccases are also very important and valuable enzymes for various biotechnological and industrial applications, such as biodegradation of lignin without polluting the environment, degradation of recalcitrant compounds, environmental protection and bioremediation, biological bleaching in the paper industry, and textile dye decolorization [12,13]. Several laccase genes have been cloned from different fungal sources and heterologously expressed with the specific purpose of using laccases more efficiently in biotechnology. Like other enzymes, laccases often face harsh conditions in industrial processes, such as high temperature, high salt concentration, and extremely acidic or alkaline pH [14,15]. Identification of laccase enzymes robust to harsh conditions could improve economic viability of this process. The majority of fungal laccases operate in the range of 30-55°C, and their optimal pH range is limited to mildly acidic conditions [16]. Many strategies Shuangxin Ma and Ning Liu contributed equally to this work.68 |

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“…The heterologous production of active class II peroxidases, LiPs, VPs and MnPs, which are heme proteins, is often difficult and has resulted in low yields and only the expression of apoprotein without heme group in both bacterial (e.g. Escherichia coli) [61] and eukaryotic (e.g. Aspergillus spp.)…”

Section: Efficiency Of Automatic Screeningmentioning

confidence: 99%

Improved Efficiency in Screening for Lignin-Modifying Peroxidases and Laccases of Basidiomycetes

Kinnunen

Maijala

Järvinen

et al. 2017

CBIOT

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Abstract:Background: Wood rotting white-rot and litter-decomposing basidiomycetes form a huge reservoir of oxidative enzymes, needed for applications in the pulp and paper and textile industries and for bioremediation.Objective: The aim was (i) to achieve higher throughput in enzyme screening through miniaturization and automatization of the activity assays, and (ii) to discover fungi which produce efficient oxidoreductases for industrial purposes.Methods: Miniaturized activity assays mostly using dyes as substrate were carried out for lignin peroxidase, versatile peroxidase, manganese peroxidase and laccase. Methods were validated and 53 species of basidiomycetes were screened for lignin modifying enzymes when cultivated in liquid mineral, soy, peptone and solid state oat husk medium.Results: Manganese peroxidases were the most common enzymes produced by 96% of the species. They typically had acidic pH optima, although Hyphodontia sp., Pleurotus pulmonarius and Trametes ochracea produced enzymes highly active at pH 7. Versatile peroxidase was produced by 66% of the fungi with efficient production from Phlebia radiata, P. pulmonarius and Galerina marginata. Novel lignin peroxidase producing fungi Cylindrobasidium evolvens and Daedaleopsis septentrionalis were found among the 26% of the species showing here lignin peroxidase production. Laccase was shown in 92% of the species. Several fungi produced laccase active at pH 7, which is noteworthy because usually laccases of white-rot fungi are efficient and relevant for many industrial applications. Conclusion:Automated screening allowed us to monitor many specific enzyme activities and extend the range of assay conditions from relatively small fungal cultivation sample volumes.

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Escherichia coli as a production host for novel enzymes from basidiomycota

Cited by 15 publications

References 204 publications

Redesign of a New Manganese Peroxidase Highly Expressed in Pichia pastoris towards a Lignin‐Degrading Versatile Peroxidase

Redesign of a New Manganese Peroxidase Highly Expressed in Pichia pastoris towards a Lignin‐Degrading Versatile Peroxidase

Expression, purification, and characterization of a novel laccase from Setosphaeria turcica in Eschericha coli

Improved Efficiency in Screening for Lignin-Modifying Peroxidases and Laccases of Basidiomycetes

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