Genetic aspects of targeted insertion mutagenesis in yeasts

Klinner, Ulrich; Schäfer, Bernd

doi:10.1016/j.femsre.2003.10.002

Cited by 45 publications

(35 citation statements)

References 217 publications

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“…In Saccharomyces cerevisiae, transformation efficiency is usually much higher with ends-in vectors than with circular and ends-out vectors (Hastings et al, 1993); the last two have comparable transformation efficiencies. Very often ends-in vectors have been demonstrated to integrate in multiple tandem repeats not typical for circular and ends-out vectors (Klinner & Schäfer 2004). Mitotic stability is highest for ends-out vectors, because their integration is not accompanied by the formation of duplicated homologous regions.…”

Section: Different Dna Topologies In Gene Targetingmentioning

confidence: 99%

“…The most popular explanation of the mechanism of gene conversion that is supposed to take place in such events is synthesis dependent strand annealing (SDSA), in which the newly synthesized strand is displaced as a single strand from the migrating replication bubble and the complementary strand can be synthesized. Integration of all kinds of vectors is very often indicated by drawing crossing-over symbols, although a number of results support the assumption that crossing-over essentially does not occur during integration, particularly in the case of ends-out vectors (Klinner & Schäfer 2004).…”

Section: Different Dna Topologies In Gene Targetingmentioning

confidence: 99%

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Is it possible to improve homologous recombination in Chlamydomonas reinhardtii?

et al. 2008

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Targeted modification of the genome has long been an aim of many geneticists and biotechnologists. Gene targeting is a main molecular tool to examine biological effects of genes in a controlled environment. Effective gene targeting depends on the frequency of homologous recombination that is indispensable for the insertion of foreign DNA into a specific sequence of the genome. The main problem associated with the development of an optimal procedure for gene targeting in a particular organism is the variability of homologous recombination (HR) in different species. Chlamydomonas reinhardtii is an attractive model system for the study of many cellular processes and is also an interesting object for the biotechnology industry. In spite of many advantages of this model system, C. reinhardtii does not readily express heterologous genes and does not allow targeted integration of foreign DNA into its genome easily. This paper compares data obtained from several different experiments designed for improving gene targeting in different organisms and reviews the suitability of particular techniques in C. reinhardtii cells.

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Section: Different Dna Topologies In Gene Targetingmentioning

confidence: 99%

Section: Different Dna Topologies In Gene Targetingmentioning

confidence: 99%

Is it possible to improve homologous recombination in Chlamydomonas reinhardtii?

et al. 2008

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“…Furthermore, many specialized expression vectors, including episomal ones (267), and numerous other useful tools such as reporter genes, immunotags, and genetically selectable markers (107,108,135,312) have been available. In addition, the extraordinarily high efficiency of homologous recombination in this species has facilitated targeted manipulations within chromosomes (172). The popular-ity of S. cerevisiae in basic and applied research is undoubtedly also influenced by its classification as GRAS (generally regarded as safe) by the U.S. Food and Drug Administration (FDA).…”

Section: Introductionmentioning

confidence: 99%

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Nevoigt

2008

Microbiol Mol Biol Rev

526

333

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SUMMARY The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

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“…The homologous recombination rate of P. guilliermondii could be as low as 1/1,000 (17). In the present study, the insertion mutagenesis strategy with ends-in vector was used, which was reported to yield higher homologous recombinant rate in yeast (22), and resulted in a homologous integration rate of 1/150.…”

Section: Resultsmentioning

confidence: 92%

Triggering Respirofermentative Metabolism in the Crabtree-Negative Yeast Pichia guilliermondii by Disrupting the CAT8 Gene

Zhong

Xia

2014

Appl Environ Microbiol

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bPichia guilliermondii is a Crabtree-negative yeast that does not normally exhibit respirofermentative metabolism under aerobic conditions, and methods to trigger this metabolism may have applications for physiological study and industrial applications. In the present study, CAT8, which encodes a putative global transcriptional activator, was disrupted in P. guilliermondii. This yeast's ethanol titer increased by >20-fold compared to the wild type (WT) during aerobic fermentation using glucose. A comparative transcriptional analysis indicated that the expression of genes in the tricarboxylic acid cycle and respiratory chain was repressed in the CAT8-disrupted (⌬CAT8) strain, while the fermentative pathway genes were significantly upregulated. The respiratory activities in the ⌬CAT8 strain, indicated by the specific oxygen uptake rate and respiratory state value, decreased to one-half and one-third of the WT values, respectively. In addition, the expression of HAP4, a transcriptional respiratory activator, was significantly repressed in the ⌬CAT8 strain. Through disruption of HAP4, the ethanol production of P. guilliermondii was also increased, but the yield and titer were lower than that in the ⌬CAT8 strain. A further transcriptional comparison between ⌬CAT8 and ⌬HAP4 strains suggested a more comprehensive reprogramming function of Cat8 in the central metabolic pathways. These results indicated the important role of CAT8 in regulating the glucose metabolism of P. guilliermondii and that the regulation was partially mediated by repressing HAP4. The strategy proposed here might be applicable to improve the aerobic fermentation capacity of other Crabtree-negative yeasts. P ichia guilliermondii is a Crabtree-negative yeast that is famous for the production of a wide variety of products, ranging from vitamins to biofuels, and is potentially a "new model yeast" (1). The current trend toward white biotechnology appears to favor the use of P. guilliermondii due to its extraordinary capacity to metabolize both C 5 and C 6 sugars from lignocellulosic hydrolysates and tolerate the harsh conditions of nondetoxified hydrolysate (1-4). Previously, a self-screened P. guilliermondii was used to produce ethanol from lignocellulosic hydrolysate in our lab; an ethanol titer of 56 g liter Ϫ1 and productivity higher than 1 g liter Ϫ1 h Ϫ1 were obtained in this experiment (4). In contrast to Crabtreepositive yeasts, such as Saccharomyces cerevisiae, the onset of fermentation in P. guilliermondii does not depend on the sugar concentration but is regulated by a decrease in the oxygen levels. Under fully aerobic conditions, P. guilliermondii does not prefer to undergo a fermentative metabolism, which may limit its spectra of products (5). Specifically, the production of several important cytosolic acetyl-coenzyme A related products in yeast, such as isoprenoids and polyhydroxyalkanoates, require a respirofermentative metabolism under aerobic conditions (6, 7). Therefore, studying the triggering of the fermentative metabolism of the Crab...

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Genetic aspects of targeted insertion mutagenesis in yeasts

Cited by 45 publications

References 217 publications

Is it possible to improve homologous recombination in Chlamydomonas reinhardtii?

Is it possible to improve homologous recombination in Chlamydomonas reinhardtii?

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Triggering Respirofermentative Metabolism in the Crabtree-Negative Yeast Pichia guilliermondii by Disrupting the CAT8 Gene

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