Abstract: 【Objective】 The aim of this study was to recombine and express laccase gene of Ganoderma lucidum L strain in Aspergillus niger (A.niger), and optimize its fermentation conditions, so as to obtain a genetic engineering strain with high yield of G.lucidum laccase.【Method】 Lac-L was cloned from G.lucidum L and optimized according to codon preference of A.niger.The promoter, signal peptide and terminator of A.niger glucoamylase (glaA) were selected as the expression source, and the pyrG gene of Aspergillus oryzae was selected as the screening marker.The laccase expression cassette was constructed and transformed into A.niger X1 (ΔpyrG) protoplast by polyethylene glycol-CaCl₂ method.PCR verification and enzyme activity determination of the transformants were performed to screen recombinant strains with high laccase production, and the carbon source, the ratio of inorganic and organic nitrogen sources, the concentration of Cu²⁺ and fermentation time of the solid-state fermentation medium for positive transformants were optimized.【Result】 Twenty-five recombinant laccase transformants were screened by uracil deficiency and 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt)(ABTS) medium, and the heterologous expression strain 6 of G.lucidum laccase was obtained by enzyme activity rescreening.The enzyme activity reached 3 532.6 U/kg after 72 h fermentation, while it was only 58.37 U/kg in the host strain.The optimal solid-state fermentation conditions for recombinant strain 6 were as follows:Wheat bran was used as the basic medium, adding 2% maltose, 2% (NH4)₂SO₄ and 0.25 g/L CuSO₄, and the laccase activity of the recombinant strain reached 6 255.47 U/kg after 60 h of fermentation.【Conclusion】 The heterologous expression of laccase gene Lac-L from G.lucidum L strain was successfully realized in A.niger.

Key words: laccase; Aspergillus niger; heterologous expression; fermentation conditions