Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

Restriction enzyme improves the efficiency of genetic transformations in Moniliophthora perniciosa, the causal agent of witches’ broom disease in Theobroma cacao

The presence of restriction enzymes in the transformation mixture improved the efficiency of transformation in Moniliophthora perniciosa. The influence of the vector shape (linear or circular), the patterns of plasmid integration in genomic sites and the influence of the promoter used to express the gene marker were also analyzed. The addition of BamHI or NotI increased the number of transformants by 3-10-fold and 3-fold, respectively, over the control without added enzyme. The use of pre-linearized plasmid did not increase the transformation efficiency in comparison with the circular plasmid. However, the frequency of multi-copy transformants increased significantly. The transformation procedure here reported resulted in better production of protoplasts and transformation efficiency. In addition, the time necessary for the detection of the first transformants and the number of insertions were reduced.

A presença de enzima de restrição na mistura de transformação aumentou a eficiência da transformação em Moniliophthora perniciosa. A influência da forma do vetor (linear ou circular), o padrão de integração do plasmídeo nos sítios genômicos e a influência do promotor usado para expressar o gene marcador foram também analisados. A adição de BamHI ou NotI aumentou o número de transformantes 3-10 vezes e 3 vezes, respectivamente, em relação ao controle sem a adição da enzima. O uso de plasmídeos pré-linearizados não aumentou a eficiência da transformação quando comparado à eficiência obtida com plasmídeos circulares. No entanto, a freqüência de transformantes multi-cópias aumentou significativamente. Juntos os procedimentos reportados aqui resultaram em processos mais eficientes de produção de protoplastos e transformação, onde o tempo necessário para o aparecimento dos transformantes e o número de inserções múltiplas foi reduzido.