Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining

Kretzschmar, Anne; Otto, Christina; Holz, Martina; Werner, Severine; Hübner, Linda; Barth, Gerold

doi:10.1007/s00294-013-0389-7

Cited by 110 publications

(91 citation statements)

References 47 publications

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“…To explore this possibility, we tried to obtain independent clones lacking the YlMHB1 gene. To increase the frequency of homologous recombinants, we took advantage of the Ku-deficient mutant of Y. lipolytica constructed in the Barth laboratory (87). The decreased level of the nonhomologous endjoining pathway in this strain leads to an increase in the frequency (up to 85%) of homologous recombinants.…”

Section: Discussionmentioning

confidence: 99%

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

et al. 2014

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bMitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a ⌬mhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the ⌬mhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. Individual eukaryotic cells contain a population of mitochondrial DNA (mtDNA) molecules, the number of which may reach several thousand copies. For example, aerobically grown diploid cells of the yeast Saccharomyces cerevisiae contain, on average, 100 molecules of 85-kbp mtDNA. With a distance of 0.34 nm between base pairs in DNA, the total length of mtDNA reaches almost 3 mm per cell, while the diameter of the cell does not exceed 3 to 4 m. Analogously to its nuclear counterpart, mtDNA must be packaged into condensed nucleoprotein structures termed mitochondrial nucleoids (mt-nucleoids) (1-6). The size of mt-nucleoids in S. cerevisiae ranges from 0.2 to 0.9 m, meaning that mtDNA in yeasts undergoes compaction of roughly 3 orders of magnitude. Although it is known that the size and shape (oval versus globular) of mt-nucleoids, the number of mt-nucleoids (ranging from 50 to 70) per diploid cell (2), and the number of copies (up to 9) of mtDNA per mt-nucleoid (3) in S. cerevisiae depend on physiological conditions, the molecular mechanisms mediating nucleoid maintenance, dynamics, and roles in mtDNA distribution/segregation during cell division are largely not understood. This is also true for mammalian mt-nucleoids, which, in contrast to their yeast counterparts, contain a relatively small number of mtDNA molecules and are thus more solitary in nature (7,8). Description of these intra-and interspecific differences in the organization of mt-nucleoids would greatly facilitate our understanding of the m...

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Section: Discussionmentioning

confidence: 99%

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

et al. 2014

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“…Several transformants showed high stability under the conditions tested. This high stability may be explained by the long sequence homology that is necessary for homologous recombination in Y. lipolytica (39). In contrast, S. cerevisiae needs much shorter homologous sequences for recombination events (40).…”

Section: Discussionmentioning

confidence: 99%

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

Matthäus

Ketelhot

Gatter

et al. 2014

Appl Environ Microbiol

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183

162

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The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g ؊1 (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.

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“…The Y. lipolytica strain H222 (MATA, wild type; (Barth and Gaillardin 1996); DSM 27185), the recipient strain H222-SW2 (MATA ura3-302 ku70Δ-1572; Kretzschmar et al (2013)), and the newly constructed strains H222-AZ1 (MATA ura3-302 ku70Δ-1572 pICL1-SDH2 URA3) and H222-AZ2 (MATA ura3-302 ku70Δ-1572 pPOT1-SDH2 URA3) as well as the E. coli strain DH5αc for cloning were used in this study. Strains were maintained on YPD agar at 4°C.…”

Section: Strainsmentioning

confidence: 99%

“…Up to 0.5 g succinic acid per gram of cells was detected for all tested Y. lipolytica strains. Additionally, succinic acid production was detected in both glycerol-(up to 5.1 g l −1 ) and glucose-containing media (up to 1.4 g l −1 ) (Kretschmar 2010;Yuzbashev et al 2010). Furthermore, first approaches for an increase in succinic acid production were presented in Kamzolova et al (2009b) and Yuzbashev et al (2010).…”

Section: Introductionmentioning

confidence: 95%

The influence of oxygen limitation for the production of succinic acid with recombinant strains of Yarrowia lipolytica

Jost

Holz

Aurich

et al. 2014

Appl Microbiol Biotechnol

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The yeast Yarrowia lipolytica is able to produce high amounts of several organic acids such as pyruvic, citric, isocitric, alpha-ketoglutaric, and succinic acid. Here we report on the influence of the reduced activity of succinate dehydrogenase in Y. lipolytica on its ability to produce succinate. The recombinant strains Y. lipolytica H222-AZ1 and H222-AZ2 were created by exchange of the native promoter of the succinate dehydrogenase subunit 2 encoding gene by inducible promoters. During the cultivation of the strain Y. lipolytica H222-AZ1 in shaking flask experiments, it was found that the promoter exchange resulted in an increase in succinic acid (SA) production. Moreover, it was found that the production of SA depends on an additional limitation of oxygen. Fed-batch cultivations in 1-l bioreactors confirmed this fundamental finding. Y. lipolytica H222-AZ1 produced 2 g l(-1) of SA with oxygen supply and 9.2 g l(-1) under the limitation of oxygen after 165 h. By using a less active promoter in Y. lipolytica H222-AZ2, the production of SA was increased to 25 g l(-1) with a productivity of 0.152 g (l*h)(-1) and a selectivity of 67 % after 165 h. Yields of 2.39 g SA per gram biomass and 0.26 g SA per gram glycerol were found.

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Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining

Cited by 110 publications

References 47 publications

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

The influence of oxygen limitation for the production of succinic acid with recombinant strains of Yarrowia lipolytica

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