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Enzymology and Protein Engineering

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Thomas Bulter, Miguel Alcalde, Volker Sieber, Peter Meinhold, Christian Schlachtbauer, Frances H. Arnold
Thomas Bulter
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
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Miguel Alcalde
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
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Volker Sieber
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
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Peter Meinhold
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
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Christian Schlachtbauer
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
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Frances H. Arnold
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
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  • For correspondence: frances@cheme.caltech.edu
DOI: 10.1128/AEM.69.2.987-995.2003
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  • Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution
    - August 05, 2003

ABSTRACT

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in kcat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.

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Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution
Thomas Bulter, Miguel Alcalde, Volker Sieber, Peter Meinhold, Christian Schlachtbauer, Frances H. Arnold
Appl. Environ. Microbiol. Feb 2003, 69 (2) 987-995; DOI: 10.1128/AEM.69.2.987-995.2003

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Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution
Thomas Bulter, Miguel Alcalde, Volker Sieber, Peter Meinhold, Christian Schlachtbauer, Frances H. Arnold
Appl. Environ. Microbiol. Feb 2003, 69 (2) 987-995; DOI: 10.1128/AEM.69.2.987-995.2003
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