RESUMO
Multiple copies of expression cassettes driven by the Trichoderma reesei xylanase 2 (xyn2) and cellobiohydrolase 2 (cbh2) promoters were introduced into the recombinant T. reesei EC-21 generated to express a thermostable Dictyoglomus thermophilum xylanase (XynB) under the egl2 promoter for further improvement of the enzyme yield. The transformants were screened based on increased XynB activity only. Multiple promoter transformant MPP-4 expressing the xynB gene under all the three promoters was found to be the highest producer of XynB, giving a 65% increase in yield compared to the parental single-promoter recombinant EC-21. The multiple-promoter transformant strains harboured six to nine copies of the xynB gene. Amongst the three promoters, egl2 seemed to have the strongest effect on XynB expression. The shotgun approach we used proved to be effective for rapid enhancement of protein expression using three promoters active at the near-neutral pH of the cultivation medium.
Assuntos
Bactérias/genética , Proteínas de Bactérias/biossíntese , Endo-1,4-beta-Xilanases/biossíntese , Expressão Gênica , Regiões Promotoras Genéticas , Trichoderma/metabolismo , beta-Glucosidase/biossíntese , Bactérias/enzimologia , Proteínas de Bactérias/genética , Endo-1,4-beta-Xilanases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Trichoderma/genética , beta-Glucosidase/genéticaRESUMO
We successfully isolated a lipase gene (designated lipPA) directly from the genomic DNA of an Antarctic isolate of Penicillium allii using PCR and a suite of degenerate primers specifically designed to target two conserved regions of fungal lipase genes. We applied the biolistic transformation system to successfully integrate the lipPA gene into a heterologous fungal host, Trichoderma reesei, one of the most powerful secretors of extracellular proteins, and induced the transformant to secrete an active lipase into the growth medium. The recombinant lipase had a temperature optimum of 25 degrees C at pH 7.9 and retained greater than 50% of the maximum activity from 10 degrees C to 35 degrees C and over a pH range from 4.0 to 8.5.
Assuntos
Expressão Gênica , Lipase/genética , Penicillium/genética , Sequência de Aminoácidos , Regiões Antárticas , Sequência de Bases , Biolística , Primers do DNA , Componentes do Gene , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA , Temperatura , Trichoderma/genéticaRESUMO
Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.
