Efficient gene targeting in <i>Kluyveromyces lactis</i>

Kooistra, Rolf; Hooykaas, Paul J. J.; Steensma, H. Yde

doi:10.1002/yea.1131

Cited by 146 publications

(145 citation statements)

References 29 publications

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“…Following the reports of Ninomiya et al 1) and Kooistra et al, 2) further improvements were reported in an nkuAÁ strain of A. nidulans by Nayak et al 5) In our experiments, no transformant was obtained by illegitimate integration in a strain that has the same nkuAÁ allele (A1149), indicating that it was exclusively catalyzed by the Ku70 protein under the conditions imposed. By adding excess amounts of non-homologous fragments, illegitimate integration was suppressed (Fig.…”

supporting

confidence: 80%

“…Ninomiya et al 1) and Kooistra et al 2) found that inactivation of the non-homologous end joining (NHEJ) pathway resulted in increased gene targeting efficiency in Neurospora crassa and Kluyveromyces lactis, respectively. These discoveries were expanded not only to many fungal species, but also to plants and human cells.…”

mentioning

confidence: 99%

“…A different approach to transient inhibition of NHEJ was developed in Kooistra et al: 2) gene targeting was significantly improved by the addition of a large amount of AluIdigested salmon sperm DNA (size range 100-500 bp).…”

mentioning

confidence: 99%

“…Improvement of gene targeting by the addition of non-homologous fragments was reported by Kooistra et al 2) for K. lactis, and the authors hypothesized that homologous recombination (HR) was activated by recognition of excess DNA ends as a signal for DSB repair. Our results are consistent with the proposal that the activated HR was responsible, at least in part, for improved gene targeting, rather than inactivation of NHEJ as sole and exclusive cause.…”

mentioning

confidence: 99%

“…Disruption of KU orthologs, resulting in inactivation of NHEJ, is a proven approach to effective gene targeting in a wide range of species. 3,4) Based on the research of Ninomiya et al 1) and Kooistra et al, 2) transient interference of NHEJ was a logical next step to extend practical applications, 6,7) and this was achieved by the addition of excess amounts of non-homologous DNA fragments. Therefore, in conclusion, this approach was practical in terms of the fate of the fragments, and it produced successful targeting events and is a good candidate for the improvement of gene targeting by transiently suppression of NHEJ.…”

mentioning

confidence: 99%

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Improvement of Gene Targeting inAspergillus nidulanswith Excess Non-Homologous Fragments

Matsuhira

Itoh

2012

Bioscience, Biotechnology, and Biochemistry

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Efficiency of gene targeting was increased more than 10-fold when an excess molar ratio of non-homologous fragments was added in the transformation of Aspergillus nidulans. In the targeted transformants, integration of such fragments into the host chromosome was a rare event. Hence, our approach proved practical in terms of producing successful targeting events without disturbing the host chromosomes.

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supporting

confidence: 80%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Improvement of Gene Targeting inAspergillus nidulanswith Excess Non-Homologous Fragments

Matsuhira

Itoh

2012

Bioscience, Biotechnology, and Biochemistry

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Development of new laccases by directed evolution: Functional and computational analyses

Festa¹,

Autore

Fraternali

et al. 2008

Proteins

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Laccases are blue multicopper oxidases that couple the four-electron reduction of oxygen with the oxidation of a broad range of aromatic substrates. These fungal enzymes can be used for many applications such as bleaching, organic synthesis, bioremediation, and in laundry detergents. Laccases from Pleurotus ostreatus have been successfully heterologously expressed in yeasts. The availability of established recombinant expression systems has allowed the construction of mutated, "better performing" enzymes through molecular evolution techniques. In the present work, random mutagenesis experiments on poxc and poxa1b cDNAs, using error prone PCR (EP-PCR) have been performed. By screening a library of 1100 clones the mutant 1M9B was selected, it shows a single mutation (L112F) leading to an enzyme more active but less stable with respect to the wild-type enzyme (POXA1b) in all the analyzed conditions. This mutant has been used as a template for a second round of EP-PCR. From this second generation library of 1200 clones, three mutants have been selected. Properties of the four mutants, 1M9B screened from the first library, and 1L2B, 1M10B, and 3M7C from the second library, were analyzed. The better performing mutant 3M7C presents, besides L112F, only one substitution (P494T) responsible both for the significantly increased stability and for the exhibited higher activity of this mutant. Molecular dynamics simulations have been performed on three-dimensional models of POXA1b, 1M9B, and 3M7C, and hypotheses on the structure-function relationships of these proteins have been formulated.

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Current awareness on yeast

2004

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (3 weeks journals ‐ search completed 15th. Sept. 2004)

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Efficient gene targeting in Kluyveromyces lactis

Cited by 146 publications

References 29 publications

Improvement of Gene Targeting inAspergillus nidulanswith Excess Non-Homologous Fragments

Improvement of Gene Targeting inAspergillus nidulanswith Excess Non-Homologous Fragments

Development of new laccases by directed evolution: Functional and computational analyses

Current awareness on yeast

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