title: Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution.
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 k(cat), 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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125,USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bulter
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
authors.forename: Peter
authors.initials: P
authors.lastname: Meinhold
authors.forename: Christian
authors.initials: C
authors.lastname: Schlachtbauer
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D052160:Benzothiazoles; D019020:Directed Molecular Evolution; D006031:Glycosylation; D006835:Hydrazones / Q000378:metabolism; D042845:Laccase; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D020037:Sordariales / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D006835:Hydrazones; D013451:Sulfonic Acids; C009370:syringaldazine; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D010088:Oxidoreductases; D042845:Laccase