Two chromosome-integrating vectors, pLC1 and pLC2, were used. The former is the pUC19-based vector carrying the Lentinus edodes ras gene promoter and priA gene terminator, and the latter is the pBR322-based vector carrying the promoter and terminator of the priA gene. The manganese (II) peroxidase (MnP) cDNA (mnpc) derived from Pleurotus ostreatus was fused between the promoter and terminator of pLC1 and pLC2, yielding the recombinant plasmids pLC1-mnp and pLC2-mnp. These plasmids were introduced into protoplasts of the Coprinus cinereus trp1 strain with the C. cinereus TRP1-containing plasmid pCc1001 by co-transformation. Two Trp+ transformants for each plasmid, showing clearly higher lignin-decolorization activities, were obtained through introduction of pLC1-mnp and pLC2-mnp. Southern-blot analysis revealed that the four transformants all possess the mnpc sequence on their chromosomes. One Trp+ MnP+ transformant (named TF2-7), which was derived from the introduction of pLC2-mnp and carried the highest number of copies (approx. 10) of mnpc, showed remarkably high lignin-decolorization and -degradation activities; at the time of cultivation when only 35%-40% of the lignin was decolored and degraded by the control Trp+ transformant obtained by the introduction of pCc1001 alone, almost all of the lignin was decolored and degraded by TF2-7.
Molecular-bred Coprinus cinereus monokaryotic strains with high lignin- and xylan-degrading activities were mixed-cultured at 27 degrees C in the liquid medium containing 0.5% (w/v) cut rice straw and 0.025% MnCl2. After 3 weeks, the culture supernatant was extensively treated with crude cellulase, showing the presence in it of 9.3% of the total cellulose of rice straw. When rice straw treated with 0.1 N NaOH or cultured with Ganoderma applanatum were used, the recoveries of the cellulose increased up to 29%. The same experiments were done by using a non-bred control strain, showing the recoveries of the cellulose from the treated or cultured rice straw to be 8%.
The Bacillus subtilis endo (β‐1,4‐) D‐xylanase structural gene (xyn) was trimmed away from its signal sequence and then fused after the signal sequence of the basidiomycete Pleurotus ostreatus manganese(II) peroxidase cDNA. The resulting modified gene (xyn′) was inserted between the promoter and terminator of two chromosome‐integrating, heterologous protein expression vectors. These recombinant plasmids were introduced into protoplasts of the monokaryotic Coprinus cinereus trp1 strain with the C. cinereus TRP1‐containing plasmid. One Trp+ Xyn+ transformant for each of the recombinant plasmids was obtained, which showed a markedly high xylan‐degrading activity as compared with the control Trp+ transformant.
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