Molecular Cloning And Regulatory Expression Mechanism Of Novel Fungal Laccase Genes

The PCR amplification using the degenerate primers, based on the Cu-binding conserved sequences of laccase, and the long distance-inverse PCR technique were used to clone novel fungal laccase genes. Three novel laccase isozyme genes (lacA, lacB and lacC) as well as a laccase pseudo-gene (lcD) have been identified from basidiomycete Trametes sp. AH28-2. These genes display a high similarity with other basidiomycete laccases at the amino acid level. An inferred TATA box and several putative CAAT, MRE, XRE and CreA consensus sequences were identified in the 5'-noncoding regions of lacA, lacB and lacC. For the three promoters of lacA - lacC, the average G+C contents are 59%, 60% and 52% respectively. Different from the TATA boxes of lacA and lacB at about -100 upstream of the initial translation codon, the TATA box of lacC is located at -172. On the other hand, copper ion is necessary for the synthesis of all laccase isozymes in Trametes sp. AH28-2. Moreover, copper at different concentrations has an effect on the expression of different isozyme components, with lower Cu~2+ concentration (< 0.5 mmol/L) inducing LacA and a novel laccase while the latter will disappear when increasing Cu~2+ concentration up to 1-2 mmol/L. More interestingly, different aromatic compounds can induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of LacA while 3,5-dihydrotoluene mainly stimulating the production of LacB. Upon induction by 3,5-dihydrotoluene, the ratio of LacA to LacB decreased in the later phase of induction. It was also observed the glucose-repression effect on the laccase expression in Tramete sp. AH28-2, with the laccase activity in cultures increasing rapidly when the glucose was displaced with cellobiose. Furthermore, quantitative RT-PCR showed that the accumulation of laccase mRNA transcripts is accompanied with the increase of corresponding enzyme activity in cultures.The lacA cDNA from Trametes sp. AH28-2 was cloned into the expression vector pPIC9K, using the leader sequence of a-factor as the secretion signal, and heterologously expressed in Pichia pastoris strain GS115. The highest laccase production of 4.0 mg/L (1360 U/mg) was obtained in shaken-flask "fermentation at 20°C in BMM medium containing 0.3 mmol/L Cu2+ and 0.2% (w/v) alanine, with the addition amount of 0.5% (v/v) methanol being most suitable for laccase synthesis. The apparent Km for ABTS (24.6 umol/L) and the carbohydrate content of the recombinant LacA (8.5%) are approximately identical to that of native LacA. However, the optimal pHs of recombinant LacA to substrates ABTS and guaiacol have a trend of shift to acidity in comparison with native LacA. In addition, the pHs optimum for enzyme stability are respectively 5.5 and 8.5 for the recombinant and native laccases, and recombinant LacA is more stable than the latter in pH2.2-6.0. Thermal stability was also investigated. Furthermore, mutagenesis of a transformant was performed by low-energy nitrogen ion implantation and one mutant (MAI) of excellent laccase producer was isolated with laccase production of 7.7 mg/L (1085 U/mg) in cultures being 92.5% more than the control (4.0 mg/L). Compared to the non-mutant recombinant LacA, five amino acid residues have been detected to mutate in the peptide sequence of the mutant laccase (MAI-LacA) when deduced from the corresponding cDNA sequence where seven nucleotides have been transformed. Several characters are identical for the two recombinant laccases while the catalytic ability of them has a slight variation. The apparent Km of MAI-LacA (28.8 umol/L) has an increase of 17.7% and the relative activity has a decrease of 20.2% in comparison with the non-mutant recombinant LacA. Moreover, the thermal stability of MAI-LacA is superior to the latter.