A Post-genomic Study of Lentinula edodes Laccases: From Basic Sciences to Potential Applications and Molecular Engineering

Laccases from basidiomycetes, and their laccase-mediator systems (LMSs), are versatile biocatalysts for "green" applications including bioremediation and biorefinery. Scarcity of sources of enzymes, lack of simple expression platforms, inconsistent enzymology methods, and blurred directions of molecular evolution are major challenges to exploitations of laccases. The present study addressed these challenges using our genome sequence of Lentinula edodes. A laccase family of ten isozymes plus two allelic forms was cloned from L. edodes mycelia. The yeast Pichia pastoris expression platform was then established to achieve robust heterologous expression of five laccases (Lcc1A, Lcc1B, Lcc4A, Lcc5 and Lcc7). Pilot study on Lcc1A and Lcc1B provided standardised methodology for enzymological and application comparisons among the recombinant laccases. Lcc1A and its LMSs were the most efficient in biodegradation of synthetic dyes and polyaromatic hydrocarbons, and could improve the enzymatic saccharification of steam-pretreated softwoods by 37% to 46%. On the other hand, Lcc7 had the highest activity (∼7-fold higher than that of Lcc1A) on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) which is the most commonly employed benchmark substrate for laccase screening. The inconsistent performance of recombinant laccases on ABTS and applications showed the need for a more indicative benchmark substrate for application-oriented engineering of fungal laccases. A correlation analysis revealed that the laccase activity on guaiacol associated much better with the enzyme performance on decolourisation of structurally different dyes than commonly employed ABTS and 2,6-dimethoxyphenol. Sequence analyses also suggested potential amino acid residues in conserved motifs and substrate binding loops that could be responsible for variations of enzymatic properties among the recombinant laccases. This study reported not only a robust yeast expression platform of L. edodes laccases and novel enzymes, but also important sequence-function relationship and an indicative substrate for engineering of laccases for efficient industrial applications.