Comparison of promoters suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeastYarrowia lipolytica

Juretzek, Thomas; Wang, H.; Nicaud, Jean‐Marc; Mauersberger, Stephan; Barth, Gerold

doi:10.1007/bf02942206

Cited by 64 publications

(48 citation statements)

References 28 publications

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“…7c). The same localization pattern was observed with transformed cells overexpressing GPR1-GFP under control of the strong heterologous POT1 promoter (Juretzek et al, 2000) and in cells expressing GPR1-GFP under a weak promoter (data not shown).…”

Section: Intracellular Localizationsupporting

confidence: 77%

Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica

et al. 2003

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Adaptation of cells to acetic acid requires a hitherto unknown number of proteins. Studies on the GPR1 gene and its encoded protein in the ascomycetous fungus Yarrowia lipolytica have revealed an involvement of this protein in the molecular processes of adaptation to acetic acid. Gpr1p belongs to a novel family of conserved proteins in prokaryotic and eukaryotic organisms that is characterized by the two motifs (A/G)NPAPLGL and SYG(X)FW (GPR1_FUN34_YaaH protein family). Analysis of four trans-dominant mutations and N-terminal deletion analysis of Gpr1p identified the amino acid sequence FGGTLN important for function of this protein in Y. lipolytica. Deletion of GPR1 slowed down adaptation to acetic acid, but had no effect on growth in the presence of acetic acid. Expression of GPR1 is induced by acetic acid and moderately repressed by glucose. It was shown by subcellular fractionation that Gpr1p is an integral membrane protein, which is also suggested by the presence of five to six putative transmembrane spanning regions. Fluorescence microscopy confirmed a localization to the plasma membrane. A model is presented describing a hypothetical function of Gpr1p during adaptation to acetic acid.

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Section: Intracellular Localizationsupporting

confidence: 77%

Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica

et al. 2003

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“…These comparisons are difficult due to the slight differences in the restriction sites used to create these hybrid promoters, the use of rich media in previous reports, and the use of replicative plasmids here compared with the integrated plasmids used in other studies. Even with these differences and discrepancies, this work still presents up to a 4-fold increase in performance compared with the best reported endogenous promoters or previously constructed hybrid promoters (22,28). This illustrates that multiple tandem repeats of the UAS1B enhancer element activate transcription to levels far higher than those on May 9, 2018 by guest http://aem.asm.org/ previously described and that this enhancer activation can occur at regions more than 2,000 nucleotides upstream of the start codon (for UAS1B [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] -Leum).…”

Section: Discussionmentioning

confidence: 77%

Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

Blazeck

Liu

Redden

et al. 2011

Appl Environ Microbiol

283

278

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The development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeast Yarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported for Y. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters in Y. lipolytica are enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.

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“…These results clearly show that the exclusive exchange of the SDH2 promoter is insufficient and an additional limitation of oxygen is essential for the production of succinic acid with the genetically modified strain Y. lipolytica H222-AZ1. Nevertheless, the presence of malate indicates that the activity of the ICL1 promoter is still too high so another strain (Y. lipolytica H222-AZ2) was generated where the natural SDH2 promoter was substituted by the POT1 promoter, which has less activity than the ICL1 promoter on glycerol as carbon source (Juretzek et al 2000). Y. lipolytica H222-AZ2 was tested in the same way as Y. lipolytica H222 and H222-AZ1 in a 1-l bioreactor.…”

Section: Cultivation In 1-l Stirred Tank Reactormentioning

confidence: 99%

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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Comparison of promoters suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeastYarrowia lipolytica

Cited by 64 publications

References 28 publications

Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica

Characterization, localization and functional analysis of Gpr1p, a protein affecting sensitivity to acetic acid in the yeast Yarrowia lipolytica

Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach

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

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