Summary: APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2003, p. 987995 Vol. 69, No. 2
0099-2240/03/$08.00 0 DOI: 10.1128/AEM.69.2.987995.2003
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, Peter Meinhold,
Christian Schlachtbauer, 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 sub-
strate 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 heter-