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.

FIG. 1.

Mutations in the selected MtL mutants during the evolution of laccase total activity in S. cerevisiae. +, mutation; ⧫, new mutation. Grey indicates synonymous mutations; subscript numbers on codons indicate codon usage. Footnote symbols: a, error-prone PCR; b, in vivo gap repair; c, in vivo recombination; d, StEP.

FIG. 2.

Positions of the mutations in the most active laccase (T2), relative to the processing sites of the MtL protein. The mutations in T2 are A(n20)P, S3I, and H(c2)R. The open triangles mark the positions of the mutations. The arrows mark the protease cleavage sites. For mutant H(c2)R and the wild type, sequences are given. The | indicates the cleavage position.

FIG. 3.

Thermostability of wild-type MtL expressed in the yeast S. cerevisiae and in A. oryzae, and of two mutants found in generation 9. Mutant 8H9 was discovered with the stability screen, and 3A6 was discovered with the activity screen. Tubes containing 50 μl of B&R buffer (10 mM; pH 6) containing 0.3 U of enzyme activity/ml were incubated at various temperatures and assayed for residual activity after 0, 1, 7, and 24 h at 23°C with ABTS as substrate. The activities were normalized to the initial activity at 23°C before the incubation.

FIG. 4.

SDS-PAGE of two mutants from generation 9. 3E3 is identical to 3A6 except for mutation N396S. The purified enzymes were deglycosylated using the N-glycosidase PNGaseF. Samples were analyzed before and after deglycosylation. The gel was stained with Coomassie brilliant blue.

FIG. 5.

(A) pH-activity profiles of wild-type (wt) MtL expressed in S. cerevisiae, wt expressed in A. oryzae, and of mutants 37A7 (generation 7), 8H9 (stability mutant, generation 9), 3A6 (activity mutant, generation 9), and T2 (generation 10). Activities were measured in B&R buffer at different pHs with ABTS as substrate. Laccase activity is normalized to the optimum activity value of the enzymes. (B) Stability of MtL wild type and mutants at pH 3. Enzyme samples (0.3 U/ml) were incubated in B&R buffer, pH 3, and residual activity at pH 6 was measured with ABTS as substrate. The activities were normalized to the initial activity at pH 6 before the incubation.

FIG. 6.

Model of MtL mutant T2 derived from the structure of MaL. Mutations are in black ball and stick display. Residues presumably participating in substrate binding (26) are in grey ball and stick display. Copper atoms are shown as grey spheres. The model was created using DeepView and the Swiss-Model server.