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Applied and Environmental Microbiology, February 2003, p. 987-995, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.987-995.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Thomas Bulter, Miguel Alcalde, Volker Sieber,{dagger} Peter Meinhold, Christian Schlachtbauer,{ddagger} and Frances H. Arnold*

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125

Received 19 July 2002/ Accepted 7 November 2002

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.

* Corresponding author. Mailing address: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125. Phone: (626) 395-4162. Fax: (626) 568-8743. E-mail: frances{at}cheme.caltech.edu.

{dagger} Present address: Degussa Texturant Systems GmbH & Co. KG, D-85354 Freising, Germany.

{ddagger} Present address: International University Bremen, D-28725 Bremen, Germany.

Applied and Environmental Microbiology, February 2003, p. 987-995, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.987-995.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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