ArticleBMC biotechnology2013
Functional expression of a blood tolerant laccase in Pichia pastoris.
Article in BMC biotechnology, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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13 citing papers in PubMed, 44 citations in OpenAlex.
- Article
- Yeast Midas' touch: recent advances in the valorisation of methanol by yeasts.World journal of microbiology & biotechnology · 2025Review
- Fungal Laccases: Fundamentals, Engineering and Classification Update.Biomolecules · 2023Review
- Tailor-made alkaliphilic and thermostable fungal laccases for industrial wood processing.Biotechnology for biofuels and bioproducts · 2022Article
- Genome-based engineering of ligninolytic enzymes in fungi.Microbial cell factories · 2021Review
- Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent.Molecular biotechnology · 2021Article
- The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization.AMB Express · 2019Article
- A simple route to highly active single-enzyme nanogels.Chemical science · 2018Article
- Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells.Scientific reports · 2017Article
- The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris.BMC biotechnology · 2016Article
- Yeast Hosts for the Production of Recombinant Laccases: A Review.Molecular biotechnology · 2016Review
- Scaling-up Fermentation of Pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement.Scientific reports · 2016Article
- High Level Secretion of Laccase (LccH) from a Newly Isolated White-Rot Basidiomycete, Hexagonia hirta MSF2.Frontiers in microbiology · 2016Article
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Authors and funding
5 authors at 2 institutions in 3 countries.
Funding
Abstract
backgroundBasidiomycete 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.
resultsA blood-active laccase (ChU-B mutant) fused to the native/evolved α-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 α-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.
conclusionsThe 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.
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