Publications of Patrick Fickers
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See detailEngineering Yarrowia lipolytica for the Synthesis of Glutathione from Organic By-Products
Do Thi Hoang, Diem ULiege; Fickers, Patrick ULiege

in Microorganisms (2020), 8(4),

Tripeptide glutathione, which plays important roles in many cellular mechanisms, is also a biotechnology-oriented molecule with applications in medicine, food and cosmetic. Here, the engineering of the ... [more ▼]

Tripeptide glutathione, which plays important roles in many cellular mechanisms, is also a biotechnology-oriented molecule with applications in medicine, food and cosmetic. Here, the engineering of the yeast Yarrowia lipolytica for the production of this metabolite at high titer values from various agro-industrial by-products is reported. The constitutive overexpression of the glutathione biosynthetic genes GSH1 and GSH2 encoding respectively γ-glutamylcysteine synthetase and glutathione synthetase, together with the INU1 gene from Kluyveromyces marxianus encoding inulinase yielded a glutathione titer value and a productivity of 644 nmol/mg protein and 510 µmol/gDCW, respectively. These values were obtained during bioreactor batch cultures in a medium exclusively comprising an extract of Jerusalem artichoke tuber, used as a source of inulin, and ammonium sulfate, used as a nitrogen source. [less ▲]

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See detailThe quest for a cell factory for the production of recombinant proteins: Pichia pastoris vs Yarrowia lipolytica
Vandermies, Marie ULiege; Theron, Chrispian ULiege; Fickers, Patrick ULiege

Conference (2019, September 17)

1. Introduction Mastering microbial recombinant protein production is critical for application fields ranging from low-value agribusiness processes to high-value pharmaceutical products ... [more ▼]

1. Introduction Mastering microbial recombinant protein production is critical for application fields ranging from low-value agribusiness processes to high-value pharmaceutical products. Hyperglycosylation and alcoholic fermentation issues of model yeast Saccharomyces cerevisiae stimulated the development of non-conventional yeasts as alternative hosts for recombinant expression. In this regard, Pichia pastoris has been preferentially employed, despite a lack of comparative basis with other non-conventional yeasts. Here we report on the direct comparison of P. pastoris with another non-conventional yeast, Yarrowia lipolytica, for the production of industrial lipase B from Candida antartica (CalB) in small-scale bioreactors. 2. Methods In Y. lipolytica, CalB gene was expressed under the control of the erythritol-inducible promoter pEYK1-3AB, in a strain deleted for the gene EYK1 (allowing to use erythritol as a free inducer, no longer metabolized by the cells). Glycerol was employed as main carbon source. In P. pastoris, CalB gene was expressed under the control of the methanol-inducible promoter pAOX1, in a MutS strain (metabolizing methanol slower due to main alcohol oxidase deletion). Sorbitol was employed as main carbon source. The same CalB sequence was cloned in both strains, since no rare codon was detected and no significant difference in codon usage was observed between Y. lipolytica and P. pastoris. Batch cultures were realized in duplicate in Eppendorf DASbox® Mini Bioreactor System (120 mL working volume) over 72h. Temperature, pH, agitation rate, and aeration were adapted to suit both yeasts. Culture samples were analyzed for CalB gene expression, lipase activity and carbon source concentration (using HPLC). 3. Results and discussion For the present comparison, Y. lipolytica and P. pastoris were cultivated under conditions considered as the most efficient for each species. Under the specified conditions, Y. lipolytica growth rate and final biomass (µ = 0.26 and final DCW = 9 g/L, respectively) largely outperformed those of P. pastoris (µ = 0.07 and final DCW = 5 g/L, respectively). Moreover, 5-fold higher CalB production levels were observed with Y. lipolytica as a host than with P. pastoris. Maximal specific lipase activity was reached earlier in the culture with Y. lipolytica, i.e. 5700 U/gDCW after 28h, versus 1200 U/gDCW after 56h for P. pastoris. In contrast, a 7-fold higher CalB expression level was observed in P. pastoris than in Y. lipolytica. Additional experiments with CalB, EGFP, and fusion protein EGFP-CalB in P. pastoris ruled out the hypotheses of processing or secretion issues, as well as CalB inactivation in P. pastoris supernatant. In fact, CalB suffers from degradation within P. pastoris cells due to the activation of unfolded protein response, endoplasmic-reticulum associated degradation, and proteasomal degradation in response to high intracellular levels of recombinant protein. 4. Conclusions In the present study, Y. lipolytica outperformed P. pastoris in terms of growth performances, recombinant enzyme activity and promoter induction easiness (erythritol being much more convenient to handle than methanol). Such elements tend to point out Y. lipolytica as an advantageous host for recombinant protein production compared to P. pastoris. Further studies focusing on other recombinant proteins shall refine these conclusions. [less ▲]

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See detailStrategies at Bioreactor Scale for the Production of Recombinant Proteins in Yarrowia lipolytica
Vandermies, Marie ULiege; Fickers, Patrick ULiege

in Sibirny, Andriy (Ed.) Non-conventional Yeasts: from Basic Research to Application (2019)

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as cell factory presents ... [more ▼]

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and possibility of posttranslational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at high level, and to secrete protein in large amounts. In the recent years, several reviews were dedicated to genetic tools developed for this purpose. Although the construction of efficient cell factory for recombinant protein synthesis is important, the development of an efficient process for protein production constitutes an equally vital aspect. Indeed, a sports car could not drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering. [less ▲]

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See detailCharacterization of a non-toxic pyomelanin pigment produced by the yeast Yarrowia lipolytica
Ben Tahar, Imen ULiege; Kus-Liśkiewicz, Małgorzata; Lara, Yannick ULiege et al

in Biotechnology Progress (2019)

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See detailThe quest for the best cell factory for recombinant protein production: Yarrowia lipolytica vs Pichia pastoris
Vandermies, Marie ULiege; Theron, Chrispian ULiege; Fickers, Patrick ULiege

Poster (2019, June 04)

In the present study, the performances of the emerging cell factories Y. lipolytica and P. pastoris were compared for their ability to synthetize and secrete recombinant proteins in bioreactors. As a case ... [more ▼]

In the present study, the performances of the emerging cell factories Y. lipolytica and P. pastoris were compared for their ability to synthetize and secrete recombinant proteins in bioreactors. As a case study, the lipase CalB from Candida antarctica was cloned under the control of the strong inducible promoters pEYK300A3B and pAOX1 and expressed in Y. lipolytica EYK1ko and P. pastoris MutS recipient strains, respectively. Surprisingly, Y. lipolytica performances were far superior in terms of cell growth, extracellular lipase activity, although P. pastoris showed a significantly higher level of CalB gene expression. According to our results, neither of codon usage bias, protein processing and secretion, or CalB lipase inactivation could be incriminated. It is therefore hypothesized that the observed difference lies in post-translational mechanisms activated by the overexpression of recombinant proteins, namely the unfolded protein response (UPR) and the endoplasmic reticulum (ER) associated degradation (ERAD). Here indeed, the proteasome was shown activated in P. pastoris following recombinant protein expression. In conclusion, keeping specific process constraints in mind, the selection of the adequate cell factory can dramatically improve the production of a given recombinant protein. [less ▲]

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See detailAstin C production by the endophytic fungus Cyanodermella asteris in planktonic and immobilized culture conditions
Vassaux, Antoine ULiege; Tarayre, Cédric; Arguelles Arias, Anthony ULiege et al

in Biotechnology Journal (2019)

The fungal endophyte Cyanodermella asteris was recently isolated from the medicinal plant Aster tataricus. This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory ... [more ▼]

The fungal endophyte Cyanodermella asteris was recently isolated from the medicinal plant Aster tataricus. This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite was compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth was used as a support. In these conditions, the fungus exclusively grew on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism was different according to the culture conditions in terms of substrate consumption rate, cell-growth, and astin C production. Immobilized-cell cultures yielded a 30% increase of astin C production associate to a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turned out to be beneficial both for astin C production and fungus development onto the support. Finally, influence of culture parameters such as pH and medium composition, on astin C production was evaluated. With optimized culture conditions, astin C yield was further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from Aster tataricus (0.89 and 0.16 mg/g respectively). [less ▲]

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See detailBy-products show potential in glutathione production by engineered non-conventional yeast Yarrowia lipolytica
Do Thi Hoang Diem, ULiege; Steels, Sébastien ULiege; Denies, Olivia ULiege et al

Poster (2019, May 08)

Strong constitutive promoter pTEF were used to overexpress genes encoding the two enzymes (L-cysteine gamma-ligase and glutathione synthase) which catalyze the synthesis of GSH in Yarrowia lipolytica ... [more ▼]

Strong constitutive promoter pTEF were used to overexpress genes encoding the two enzymes (L-cysteine gamma-ligase and glutathione synthase) which catalyze the synthesis of GSH in Yarrowia lipolytica. Several strains which can overexpress GSH1 or GSH2 or both of them have been successfully obtained. The newly constructed strains for glutathione overproduction show different compared to control. [less ▲]

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See detailNon-Conventional Yeast Species for Recombinant Protein and Metabolite Production
Do Thi Hoang Diem, ULiege; Vandermies, Marie ULiege; Fickers, Patrick ULiege et al

in Reference module in Life Sciences (2019)

Humans have been using microorganisms for our benefit for millennia, initially by harvesting microbial metabolites and more recently by using microbes to produce recombinant proteins. The continual ... [more ▼]

Humans have been using microorganisms for our benefit for millennia, initially by harvesting microbial metabolites and more recently by using microbes to produce recombinant proteins. The continual improvements of technologies for researching and editing genes and genomes has seen vast advances in the fields of recombinant protein production and metabolic engineering. Recombinant proteins have an abundant applications, including chemical catalysis and pharmaceutical uses, while metabolic engineering can allow improved yields of recombinant proteins and metabolites, or the introduction of new synthetic routes for metabolites. Yeasts are unicellular eukaryotes, and therefore are promising organisms for protein and metabolite production as they offer a favourable middle ground between simpler prokaryotes and more complex eukaryotes. Furthermore, many species are generally regarded as safe to use even in applications for food and pharmaceutical industries. This module presents some of the most commonly used tools and approaches for manipulating yeast species toward the production of proteins and metabolites of interest, aiming to provide up-to-date information along with some historical context. Research on two of the most generally applied species are described in more detail, before notable other species showing promising characteristics are briefly described. Finally, yeast elements common between species, that can be used as tools for the exploration or further development of lesser-studied species, are also mentioned. [less ▲]

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See detailUn édulcorant dans votre café ?
Vancsok, Catherine ULiege; Fickers, Patrick ULiege

Learning material (2019)

Découvrir les activités de collègues de l’université actifs dans d’autres disciplines sur le temps de la pause-déjeuner ? C’est l’objectif de ce set de table, qui présentait le contenu d’un article sous ... [more ▼]

Découvrir les activités de collègues de l’université actifs dans d’autres disciplines sur le temps de la pause-déjeuner ? C’est l’objectif de ce set de table, qui présentait le contenu d’un article sous forme vulgarisée. La publication sélectionnée porte sur une recherche menée au TERRA (Faculté de Gembloux Agro-Bio Tech) avec le Pr. Fickers. [less ▲]

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See detailBioreactor-Scale Strategies for the Production of Recombinant Protein in the Yeast Yarrowia lipolytica
Vandermies, Marie ULiege; Fickers, Patrick ULiege

in Microorganisms (2019), 7(2), 40

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents ... [more ▼]

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as a cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and the possibility of post-translational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at a high level, and to secrete protein in large amounts. In recent years, several reviews have been dedicated to genetic tools developed for this purpose. Though the construction of efficient cell factories for recombinant protein synthesis is important, the development of an efficient process for recombinant protein production in a bioreactor constitutes an equally vital aspect. Indeed, a sports car cannot drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering. [less ▲]

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See detailNonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production
Vassaux, A.; Meunier, Loïc ULiege; Vandenbol, Micheline ULiege et al

in Biotechnology Advances (2019)

Fungi are notoriously prolific producers of secondary metabolites including nonribosomal peptides (NRPs). The structural complexity of NRPs grants them interesting activities such as antibiotic, anti ... [more ▼]

Fungi are notoriously prolific producers of secondary metabolites including nonribosomal peptides (NRPs). The structural complexity of NRPs grants them interesting activities such as antibiotic, anti-cancer, and anti-inflammatory properties. The discovery of these compounds with attractive activities can be achieved by using two approaches: either by screening samples originating from various environments for their biological activities, or by identifying the related clusters in genomic sequences thanks to bioinformatics tools. This genome mining approach has grown tremendously due to recent advances in genome sequencing, which have provided an incredible amount of genomic data from hundreds of microbial species. Regarding fungal organisms, the genomic data have revealed the presence of an unexpected number of putative NRP-related gene clusters. This highlights fungi as a goldmine for the discovery of putative novel bioactive compounds. Recent development of NRP dedicated bioinformatics tools have increased the capacity to identify these gene clusters and to deduce NRPs structures, speeding-up the screening process for novel metabolites discovery. Unfortunately, the newly identified compound is frequently not or poorly produced by native producers due to a lack of expression of the related genes cluster. A frequently employed strategy to increase production rates consists in transferring the related biosynthetic pathway in heterologous hosts. This review aims to provide a comprehensive overview about the topic of NRPs discovery, from gene cluster identification by genome mining to the heterologous production in fungal hosts. The main computational tools and methods for genome mining are herein presented with an emphasis on the particularities of the fungal systems. The different steps of the reconstitution of NRP biosynthetic pathway in heterologous fungal cell factories will be discussed, as well as the key factors to consider for maximizing productivity. Several examples will be developed to illustrate the potential of heterologous production to both discover uncharacterized novel compounds predicted in silico by genome mining, and to enhance the productivity of interesting bio-active natural products. © 2019 Elsevier Inc. [less ▲]

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See detailInducible promoter for gene expression and synthetic biology
Nicaud, Jean-Marc; Trassaert, Marion; Thomas, Stéphane et al

Patent (2018)

The invention is related to an inducible promoter for improved and regulated gene expression, useful in synthetic biology and metabolic engineering. In particular, the present invention relates to a ... [more ▼]

The invention is related to an inducible promoter for improved and regulated gene expression, useful in synthetic biology and metabolic engineering. In particular, the present invention relates to a nucleotide sequence comprising the regulatory regions of an erythritol-and erythrulose-inducible promoter in yeast and uses thereof in an expression system thus allowing an improved and regulated gene expression and production of gene product. [less ▲]

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See detailMetabolic engineering of Yarrowia lipolytica for taurine synthesis
Vandermies, Marie ULiege; Fickers, Patrick ULiege

Conference (2018, September 10)

Detailed reference viewed: 46 (6 ULiège)
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See detailpH level has a strong impact on population dynamics of the yeast Yarrowia lipolytica and oil micro-droplets in multiphasic bioreactor
Bouchedja D. N.; Danthine, Sabine ULiege; Kar T. et al

in FEMS Microbiology Letters (2018), 365(6),

The oleaginous yeast Yarrowia lipolytica has the ability to use oils and fats as carbon source, making it a promising cell factory for the design of alternative bioprocesses based on renewable substrates ... [more ▼]

The oleaginous yeast Yarrowia lipolytica has the ability to use oils and fats as carbon source, making it a promising cell factory for the design of alternative bioprocesses based on renewable substrates. However, such a multiphasic bioreactor design is rather complex and leads to several constraints when considering emulsification of the oil-in-water mixture, foaming and cell growth/physiology on hydrophobic substrate. This study aims to shed light on the effect of pH changes on the physico-chemical properties of the cultivation medium and on cell physiology. It was indeed observed that at a pH value of 6, cell growth rate and intracellular lipid accumulation were optimized. Additionally, foaming was significantly reduced. In order to avoid over foaming in bioreactor, without impairing cell physiology, the use of alternative processes that can only act on the physical structure of culture medium, seems to be an effective alternative to usual chemical anti-foam agents. [less ▲]

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See detailYarrowia lipolytica morphological mutant enables lasting in situ immobilization in bioreactor
Vandermies, Marie ULiege; Kar, Tambi ULiege; Carly, Frédéric et al

in Applied Microbiology and Biotechnology (2018)

In the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype ... [more ▼]

In the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the metallic structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica. [less ▲]

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See detailLa levure comme usine cellulaire
Vandermies, Marie ULiege; Denies, Olivia ULiege; Do, HoangDiem et al

Diverse speeche and writing (2018)

Présentation des travaux du laboratoire levures du MiPI lors de l'inauguration du bâtiment TERRA

Detailed reference viewed: 28 (7 ULiège)
See detailNew filamentous mutant of Yarrowia lipolytica and its use in biofilm bioreactors
Vandermies, Marie ULiege; Kar, Tambi; Carly et al

Poster (2018, May 17)

The non-conventional yeast Yarrowia lipolytica is regarded as a promising cell factory for the production of recombinant proteins as well as added value chemicals. This organism possesses interesting ... [more ▼]

The non-conventional yeast Yarrowia lipolytica is regarded as a promising cell factory for the production of recombinant proteins as well as added value chemicals. This organism possesses interesting metabolic properties, such as high capacity of biomolecule synthesis and secretion, substrate flexibility and adequacy with culture at high-cell density. Another characteristic trait of Y. lipolytica lies in its ability to adopt an ovoid or filamentous morphology (hyphae and pseudohyphae) according to environmental conditions. To date, several effectors of the dimorphic transition have been identified and characterized at the molecular level. However, the dimorphic transition remains difficult to master during cultures in bioreactor, thus negatively affecting the process reproducibility. Yet, filamentous organisms are particularly well suited for the development of immobilized-cell processes. In the present study, we have isolated and characterized a Y. lipolytica morphological mutant growing exclusively in the pseudohyphal morphology by means of insertion mutagenesis and high throughput screening methods. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the stainless steel structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica. [less ▲]

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See detailCatabolic selectable marker eases genome editing in Yarrowia lipolytica
Vandermies, Marie ULiege; Denies, Olivia ULiege; Nicaud, Jean-Marc et al

Conference (2018, February 08)

Selectable markers are a central component of genome edition technologies. In the yeast Yarrowia lipolytica, these markers are traditionally based on antibiotic resistance (hygromycin B) or auxotrophy (e ... [more ▼]

Selectable markers are a central component of genome edition technologies. In the yeast Yarrowia lipolytica, these markers are traditionally based on antibiotic resistance (hygromycin B) or auxotrophy (e.g., leucine, uracil). However, the use of the former is impaired by a high level of spontaneous resistance, and the use of the latter by continuous complementation of the culture medium or restoration of prototrophy to the strains. As an alternative, genes related to the catabolism of carbon sources, or “catabolic selectable markers”, present the advantage of not being involved in essential metabolic pathways. The recently identified EYK1, encoding erythrulose kinase, can serve as an efficient catabolic selectable marker for genome editing in Y. lipolytica. Compared to auxotrophic markers such as URA3 and LEU2, EYK1 increases the growth rate of transformants on selective medium and the efficiency of genome edition. The utility of the marker EYK1 in a replicative vector was also demonstrated. Besides, the cloning-free strategy developed here simplifies the construction of disruption cassettes for genome editing in Y. lipolytica. [less ▲]

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See detailVersatile regulated promoter for Yarrowia lipolytica
Vandermies, Marie ULiege; Trassaert, Marion; Carly, Frédéric et al

Poster (2018, February 08)

The non-conventional yeast Yarrowia lipolytica is an emerging cell factory for the production of recombinant protein and metabolites. Still, there is a lack of versatile inducible promoters in this yeast ... [more ▼]

The non-conventional yeast Yarrowia lipolytica is an emerging cell factory for the production of recombinant protein and metabolites. Still, there is a lack of versatile inducible promoters in this yeast. In the present study, we isolated and characterised the promoter of the recently discovered gene EYK1 (encoding an erythrulose kinase). The promoter pEYK1 shows high induction levels in presence of erythritol and erythrulose, while its induction is impaired in presence of glycerol and glucose. Based on pEYK1 upstream activating sequence, we developed a series of promoters with enhanced induction levels. The induction levels of pEYK1 were also improved in strain eyk1Δ, allowing to use erythritol and erythrulose as free inducers. [less ▲]

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