Heterologous expression of Melanocarpus albomyces laccase in Saccharomyces cerevisiae and Trichoderma reesei

The thermophilic fungus Melanocarpus albomyces produces a laccase, which has several interesting properties considering both industrial applications and the structure-function studies of laccases. This presentation describes the recent result on heterologous expression of M. albomyces laccase in Saccharomyces cerevisiae and Trichoderma reesei. M. albomyces laccase was expressed in S. cerevisiae in order to enable directed evolution studies. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The production levels were significantly enhanced by replacing them with the prepro-sequence of the S. cerevisiae alfa-factor gene. The role of a C-terminal extension in production of M. albomyces laccase in S. cerevisiae was also studied. Laccase production was increased six-fold when the region encoding the C-terminal extension was removed from the eDNA. The highest laccase production levels obtained were about 3 mg l-1 which is very suitable for protein engineering purposes. M. albomyces lac1 gene was also expressed in T. reesei in order to obtain a reliable and efficient source for the laccase. The laccase gene was expressed under the strong cbh1 promoter in two different expression constructs: in an expression cassette coding for a non-fused laccase and in a cassette coding for a hydrophobin-laccase fusion protein. Both proteins were expressed efficiently in T. reesei, and significantly higher activities were obtained with the non-fused laccase in shake flask cultures (about 230 mg l-1). Northern and Western blotting analyses indicated that the main reason for lower production with the fusion construct was at the post-translational level. The possible induction of unfolded protein response (UPR) pathway by laccase expression in T. reesei was studied by Northern hybridisations with genes involved in UPR. The results indicated that laccase expression did not cause severe secretion stress in T. reesei. The T. reesei transformant producing highest levels of M. albomyces laccase was also grown in a fermenter in batch and fed-batch modes. The highest laccase production levels were 920 mg 1-1 in the fed-batch fermentation. Characterization of the recombinant laccase showed that T. reesei was able to process the laccase correctly both at its N- and C-termini. The specific activities, pH optima, thermostabilities, and kinetic parameters with four different substrates were also similar for the recombinant and native laccases.