Homologous expression of Phanerochaete chrysosporium manganese peroxidase, using bialaphos resistance as a dominant selectable marker

AbstractEnzyme production by immobilized Phanerochaete chrysosporium was evaluated in airlift bioreactor and agitated cultures. Free mycelium and immobilized mycelium on alginate beads were tested in the decolourization of 50 and 500 mg/L of Remazol Brilliant Blue R. Dye concentration did not inhibit the fungi development in all tests. In addition, high decolourization percentage of dye was found with free mycelium (99%) in agitated flasks and with immobilized mycelium in airlift (98%). However, decolourization period by immobilized mycelium (120 h) was greater than that by the free mycelium (14 h). Important manganese peroxidase, lignine peroxidase and laccase activities were identified in decolourization process. Manganese peroxidase appeared to be promoted by high dye concentrations during the treatment with immobilized mycelium, but this enzyme was not detected with free mycelium in airlift. Bioreactor prompted also laccase and lignine peroxidase actions in both tests; free mycelium registered a maximum laccase action of 31.569 × 103 U/L in 70 h, whereas immobilized mycelium registered 1.680 × 103 U/L in 170 h, while lignine peroxidase secretion by free P. chrysosporium was higher (1.300 × 103 U/L) than immobilized mycelium (1.250 × 103 U/L). Maximum laccase activity coincided with the maximum percentage of decolourization, however, high peroxidase activity was identified from the start of dye treatment.

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Insertional mutagenesis in Coprinus cinereus : use of a dominant selectable marker to generate tagged, sporulation-defective mutants

Current Genetics ◽

10.1007/s002940050512 ◽

1999 ◽

Vol 36 (6) ◽

pp. 371-382 ◽

Cited By ~ 59

Author(s):

W. Jason Cummings ◽

Martina Celerin ◽

Jennifer Crodian ◽

Linda K. Brunick ◽

Miriam E. Zolan

Keyword(s):

Insertional Mutagenesis ◽

Selectable Marker ◽

Coprinus Cinereus ◽

Dominant Selectable Marker

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The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.4011-4019.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 4011-4019

Author(s):

J A Nelson ◽

P B Savereide ◽

P A Lefebvre

Keyword(s):

Chlamydomonas Reinhardtii ◽

Selectable Marker ◽

Marker Gene ◽

Small Subunit ◽

Direct Selection ◽

Nuclear Transformation ◽

Ribulose Bisphosphate Carboxylase ◽

Bisphosphate Carboxylase ◽

Dominant Selectable Marker ◽

Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

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