Promoter-tagged restriction enzyme-mediated insertion (PT-REMI) mutagenesis in

Shuster, J. R.; Connelley, M. Bindel

doi:10.1007/s004380051056

Cited by 5 publications

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“…The ability to introduce or delete genes remains difficult, although some advances in transformation have been reported, e.g., restriction enzyme-mediated integration [101] and Agrobacterium tumefaciens -Ti plasmid-mediated transformation [102]. Levels of production of non-fungal proteins have been low compared to those of homologous proteins.…”

Section: Production Of Recombinant Proteins In Microbial Hostsmentioning

confidence: 99%

Microbial Enzymes: Tools for Biotechnological Processes

Adrio¹,

2014

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Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi.

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Section: Production Of Recombinant Proteins In Microbial Hostsmentioning

confidence: 99%

Microbial Enzymes: Tools for Biotechnological Processes

Adrio¹,

2014

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Genomics of Protein Secretion and Hyphal Growth in Aspergillus

Archer

Turner

The Mycota

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a These sites provide an entry point to the sequence data but access requires agreement being reached with the respective company b Genome sequence data for A. oryzae are not yet available. A summary of the A. oryzae genome sequencing project and related topics has been published (Machida 2002) Table. 5.1. Websites for Aspergillus genome information (modified after Archer and Dyer 2004) Table. 5.2. Summary of information derived from the genome sequences of Aspergillus species a A. fumigatus b A. nidulans c A. niger d A. oryzae e Genome size (Mb) f 29 30 36 37 Predicted genes f 10,000 10,000 14,000 14,000 Genes with Pfam hits f 4,400 4,500 5,300 5,300 a This table was published in extended form elsewhere (Archer and Dyer 2004), and the data were obtained from the websites and contributions from William Nierman (TIGR, USA), James Galagan (Broad Institute, USA), Masa Machida (Tsukuba, Japan), and Gert Groot and Noel van Peij (DSM, The Netherlands) b

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Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae

et al. 2003

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We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

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Promoter-tagged restriction enzyme-mediated insertion (PT-REMI) mutagenesis in

Cited by 5 publications

References 0 publications

Microbial Enzymes: Tools for Biotechnological Processes

Microbial Enzymes: Tools for Biotechnological Processes

Genomics of Protein Secretion and Hyphal Growth in Aspergillus

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae

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