Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus

Abstract

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

FIG. 1.

Accumulation of hph mRNA in P. cinnabarinus transformants containing pHYM1:1 (lanes 1 and 2) and pHYM1:2 (lanes 3 and 4) as analyzed by RT-PCR. Samples were treated with RNase (lanes 1 and 3) or DNase (lanes 2 and 4). M represents a 100-bp ladder.

FIG. 2.

Northern analysis of SC3 mRNA in 3-day-old cultures of strains of P. cinnabarinus transformed with the SC3 gene of S. commune under control of its own promoter (T1 to T6) or under control of the GPD promoter of S. commune (GP1 to GP6). An mRNA of S. commune 4-40 served as a control. The blots were probed with a 300-bp fragment of the coding sequence of the SC3 gene.

FIG. 3.

Temporal accumulation of SC3 mRNA in 1- to 6-day-old liquid shaken cultures of the recombinant P. cinnabarinus strains T3 (A) and GP2 (B). These strains express the SC3 gene of S. commune behind its own promoter or that of GPD, respectively. The Northern blot was probed with an internal fragment of the SC3 gene. An RNA from a 3-day-old culture of S. commune 4-40 served as a control. Lanes 1 to 6 represent samples from days 1 to 6, respectively.

FIG. 4.

Consumption of maltose (triangles) and evolution of laccase activity (circles) in liquid shaken cultures of strains S1 (A), G14 (B), and L12-7 (C) containing the lac1 gene under regulation of the SC3 promoter or those of GPD and lac1, respectively. Cultures were grown in MM in the absence (closed symbols) or presence (open symbols) of ethanol.

FIG. 5.

Accumulation of lac1 mRNA in liquid shaken cultures of strains S1 (A), G14 (B), and L12-7 (C) containing the lac1 gene under regulation of the SC3 promoter or those of GPD and lac1, respectively. RNAs were hybridized with a 1,557-bp fragment of the coding sequence of lac1 and with 18S ribosomal DNA to quantify the amount of RNA loaded in each lane. Cultures were grown in the absence or presence of ethanol.