Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica
© The Author(s) 2017
Received: 26 August 2016
Accepted: 10 February 2017
Published: 17 February 2017
The yeast Yarrowia lipolytica is an increasingly common biofactory. To enhance protein expression, several promoters have been developed, including the constitutive TEF promoter, the inducible POX2 promotor, and the hybrid hp4d promoter. Recently, new hp4d-inspired promoters have been created that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter. Three different protein-secretion signaling sequences can be used: preLip2, preXpr2, and preSuc2.
To our knowledge, our study is the first to use a set of vectors with promoters of variable strength to produce proteins of industrial interest. We used the more conventional TEF and hp4d promoters along with five new hybrid promoters: 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, 8UAS1-pTEF, and hp8d. We compared the production of RedStar2, glucoamylase, and xylanase C when strains were grown on three media. As expected, levels of RedStar2 and glucoamylase were greatest in the strain with the 8UAS1-pTEF promoter, which was stronger. However, surprisingly, the 2UAS1-pTEF promoter was associated with the greatest xylanase C production and activity. This finding underscored that stronger promoters are not always better when it comes to protein production. We therefore developed a method for easily identifying the best promoter for a given protein of interest. In this gateway method, genes for YFP and α-amylase were transferred into a pool of vectors containing different promoters and gene expression was then analyzed. We observed that, in most cases, protein production and activity were correlated with promoter strength, although this pattern was protein dependent.
Protein expression depends on more than just promoter strength. Indeed, promoter suitability appears to be protein dependent; in some cases, optimal expression and activity was obtained using a weaker promoter. We showed that using a vector pool containing promoters of variable strength can be a powerful tool for rapidly identifying the best producer for a given protein of interest.
KeywordsYarrowia lipolytica Protein production RedStar2 Glucoamylase Xylanase Hybrid promoters
Increasing the efficiency of heterologous gene expression is a major goal for the agrifood, bioconversion, and pharmaceutical industries as they have a growing need for recombinant proteins. Expression systems using yeasts present several advantages: yeasts are easy to manipulate, they are unicellular organisms with rapid growth rates, and they are eukaryotes that can incorporate post-translational modifications. In addition to the more conventional Saccharomyces cerevisiae , alternative model species are also used as biofactories, including Pichia pastoris, Hansanula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus [2–5], and Yarrowia lipolytica [6, 7].
Production systems exploiting Y. lipolytica have several advantages [7, 8]. First, Y. lipolytica is a non-pathogenic organism that can grow on a diversity of substrates. Second, the products of several Y. lipolytica-based processes have received the “generally recognized as safe” (GRAS) designation from the FDA. Third, Y. lipolytica has a naturally strong secretory ability [7, 8] and demonstrates weak protein glycosylation .
Several genetic tools are available to enhance protein expression in Y. lipolytica. Indeed, integrative expression cassettes containing different markers, such as LEU2, URA3, ADE2, and LYS5, have been constructed. They can be used to transform competent auxotrophic strains of Y. lipolytica. Moreover, several promoters are also available, including the constitutive TEF promoter, the constitutive and hybrid hp4d promoter, and the inducible POX2 and LIP2 promoters [10–14]. In addition, several transformation methods have been developed to optimize the transformation rate [15–17]. Currently, the lithium-acetate method is the most common, whether the goal is to inactivate endogenous genes or to transform expression cassettes . All of these tools have been successfully used in Y. lipolytica to produce such proteins as xylanase, lipase, leucine aminopeptidase II, human interferon, α2b endoglucanase II, and cellobiohydrolase II [6, 9, 14, 19, 20]. Past studies have also identified at least three sequences that can be used to optimize protein secretion in Y. lipolytica: preLip2, preXpr2, and preSuc2 [6, 14, 21–24].
Several studies have suggested that Y. lipolytica is better than P. pastoris at producing heterologous proteins [20, 25]. Indeed, Nars and colleagues  found that, as opposed to P. pastoris, E. coli, or simple free cells, Y. lipolytica was the best candidate for generating extracellular Lip2 because it can form a stable isotope-labeled version of the protein. Boonvitthya and colleagues  compared endoglucanase II and cellobiohydrolase II production in Y. lipolytica and P. pastoris. In YT medium, Y. lipolytica produced up to 15 mg/L of endoglucanase and 50 mg/L of cellobiohydrolase. Furthermore, the enzymes produced by Y. lipolytica had higher levels of specific activity than did their counterparts in P. pastoris. Finally, it has been found that Y. lipolytica has weaker protein glycosylation than does P. pastoris .
One of the first strong constitutive promoters was developed by Novo, using the TEF1 gene, which encodes the translation elongation factor-1α . Later, Madzak and colleagues  identified the upstream activating sequence UAS1 in the XPR2 gene (which encodes the secreted alkaline extracellular protease). This discovery led to the development of the hp4d promoter, which is based on the minimal LEU2 promoter and contains four UAS1 tandem elements; with this promoter, expression increases as the number of UAS1 tandem elements increases. More recently, several research groups have used this basic model (i.e., multiple UAS tandem elements associated with a core promoter) to develop improved promoters [27–29]. It has been found that the core promoter and the upstream activating sequence (i.e., the UAS1 tandem elements) act independently and that, as previously noted, promoter strength increases with the number of UAS1 tandem elements. Shabbir Hussain and colleagues  showed that promoter strength can be fine-tuned by engineering the sequences of the TATA box, the core promoter, or the upstream activating region. To quantify promoter strength, they used fluorescent proteins and β-galactosidase assays.
However, to our knowledge, no study to date has used these UAS1-based promoters to produce proteins of industrial interest. Here, we used two conventional promoters, pTEF and hp4d, as well as five new hybrid promoters of our own construction. To create the latter, we added two, three, four, or eight UAS1 tandem elements to pTEF; we also added four tandem elements to hp4d. Promoter strength in transformed Y. lipolytica strains was quantified using RedStar2, a fluorescent protein, as a reporter; we also analyzed the production of secreted Aspergillus niger glucoamylase (GA) and xylanase C (XlnC). GA is a glucan 1,4-alpha-glucosidase that belongs to the glycosyl hydrolase family. It catalyzes the degradation of starch and other complex sugars, releasing d-glucose. GA is largely used to produce biolipids and bioethanol from starch or lignocellulosic materials [30, 31]. XlnC is a beta-1,4-beta-xylanase that breaks down hemicellulose, a component of plant cell walls, releasing xylose. The paper, textile, and pet-food industries are major consumers of xylanase.
Our results revealed that optimal protein expression, secretion, and activity are not always correlated with promoter strength. Consequently, we developed a simple method for improving protein expression that involves the use of a pool of vectors containing promoters of variable strength.
Yeast strains, growth media, and culture conditions
The Y. lipolytica wild-type strain W29 (ATCC20460) was used as the basis for all the Y. lipolytica strains built in this study (see Additional file 1: Table S1 for the full list). The auxotrophic strain Po1d (Leu− Ura−) has previously been described by Barth and Gaillardin . Escherichia coli strain DH5α was used to construct the plasmids, except in the case of vectors containing ccdB, for which E. coli strain DB3.1 was used. E. coli growth media and culture conditions have been previously described by Sambrook and colleagues , and those for Y. lipolytica have been described by Barth and Gaillardin . Rich medium (YPD) and minimal glucose medium (YNB) were prepared as described elsewhere . The YPD medium contained 10 g/L of yeast extract (Difco, Paris, France), 10 g/L of Bacto Peptone (Difco, Paris, France), and 10 g/L of glucose (Sigma Aldrich, Saint-Quentin Fallavier, France). The YNB medium contained 1.7 g/L of yeast nitrogen base without amino acids and ammonium sulfate (YNBww; Difco, Paris, France), 10 g/L of glucose (Sigma), 5.3 g/L of NH4Cl, and 50 mM phosphate buffer (pH 6.8). This minimal medium was supplemented with uracil (0.1 g/L) and/or leucine (0.1 g/L) as necessary. YP2D4 medium contained 10 g/L of yeast extract (Difco, Paris, France), 20 g/L of Bacto Peptone (Difco, Paris, France), and 40 g/L of glucose (Sigma Aldrich, Saint-Quentin Fallavier, France). Solid media were created by adding 1.6% agar.
Plasmid and strain construction
List of primers used in this study
Hybrid promoter construction
Verification of plasmid construction and insertion into the Y. lipolytica genome
Insertion of YFP gene into pENTR™/D-TOPO®
Insertion of α-amylase gene into pENTR™/D-TOPO®
Addition of a BamHI site close to the promoter in JMP1529 to obtain JMP3030
qPCR related to the XlnC gene
qPCR related to the actin gene
GA was cloned into the JMP2482, JMP2484, JMP2397, JMP2607, JMP2471, and JMP2473 plasmids at the BamHI and AvrII restriction sites, yielding JMP3781 (LEU2ex 2UAS1-pTEF-GA), JMP3782 (LEU2ex 3UAS1-pTEF-GA), JMP3783 (LEU2ex 4UAS1-pTEF-GA), JMP3784 (LEU2ex 8UAS1-pTEF-GA), JMP3785 (LEU2ex hp4d-GA), and JMP3786 (LEU2ex hp8d-GA), respectively.
XlnC was cloned into the JMP2482, JMP2484, JMP2397, JMP2607, JMP2471, and JMP2473 plasmids at the BamHI and AvrII restriction sites, yielding the JMP3096 (LEU2ex 2UAS1-pTEF-XlnC), JMP3097 (LEU2ex 3UAS1-pTEF-XlnC), JMP3098 (LEU2ex 4UAS1-pTEF-XlnC), JMP3099 (LEU2ex 8UAS1-pTEF-XlnC), JMP3100 (LEU2ex hp4d-XlnC), and JMP3101 (LEU2ex hp8d-XlnC) plasmids, respectively.
The sequences of the genes encoding YFP and α-amylase are provided in Additional file 3: Data S1. These genes were inserted into pENTR™/D-TOPO® in accordance with the manufacturer’s instructions using the primers listed in Table 1.
The overexpression cassettes, obtained by digesting the plasmids with NotI, were used to transform individual strains via the lithium-acetate method . Transformants were selected utilizing YNB Ura, YNB Leu, or YNB medium, depending on their genotype, and their genomic DNA was prepared as described by Querol and colleagues . The primers used to verify expression cassette insertion are given in Table 1.
Restriction enzymes were obtained from OZYME (Saint-Quentin-en-Yvelines, France). PCR was performed using an Eppendorf 2720 thermal cycler; GoTaq DNA polymerases (Promega, Madison, WI, USA) were employed to verify the results and PyroBest DNA polymerases (Takara, Saint-Germain-en-Laye, France) were employed to carry out cloning. PCR and DNA fragment purification were performed as previously described . The amounts of DNA obtained were measured using MySpec (VWR, Fontenay-sous-Bois, France). All the reactions were performed in accordance with the manufacturer’s instructions. The sequencing of the cloned fragments was performed by GATC Biotech (Konstanz, Germany). Clone Manager software was used for the gene sequence analysis (Sci-Ed Software, Morrisville, NC, USA).
Forty ng/µL of each of the recipient plasmids was mixed with pENTR™/D-TOPO® containing the YFP or α-amylase gene. The transfer of the genes of interest was performed using LR Clonase® in accordance with the manufacturer’s instructions (Invitrogen, Saint-Aubin, France). The mixture was used to transform E. coli strain DB3.1. The resulting transformants were then pooled, and their DNA was extracted and digested before Y. lipolytica was transformed in turn.
Supernatant was obtained from cultures grown for 72 h in YNB, YPD, or YP2D4 media and was concentrated tenfold in 30 mM Tris (pH 8.0) and 50 mM NaCl using Amicon Ultra-0.5 10 K centrifugal filters (Merck Millipore Ltd, Ireland). Protein production was analyzed via polyacrylamide gel electrophoresis (SDS-PAGE); 4–12% Tris–Glycine gels and an XCell SureLock™ Mini-Cell electrophoresis system (Novex, Life Technologies, Saint-Aubin, France) were used. Prism (MW1; 19–130 kDa) and wide-range (MW2; 14–212 kDa) protein molecular weight markers were used as standards (VWR Chemicals, Fontenay-sous-Bois, France). The gels were stained with 0.2% Coomassie Brilliant Blue R dye (Thermo Fisher Scientific, Villebon-sur-Yvette, France).
Twenty-μL samples were analyzed for protein content using the Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, Villebon-sur-Yvette, France) in accordance with the manufacturer’s instructions.
GA activity was measured as previously described , with the following modifications. Samples containing 40 μL of supernatant were incubated for 2–10 min with 1.8 mL of a 0.2% soluble cornstarch solution (30 °C, pH 5). The resulting glucose concentration was determined via high-performance liquid chromatography: an UltiMate® 3000 system (Dionex-Thermo Fisher Scientific, UK) with an Aminex HPX87H column coupled to an RI detector was used. The column was eluted with 0.01 N H2SO4 at room temperature and a flow rate of 0.6 mL/min. Identification and quantification were achieved via comparison to standards. Enzyme activity was expressed in U mL/L of supernatant, where one unit of GA activity (1 U) was defined as the amount of GA required to release 1 μmol of glucose per minute.
XlnC activity was determined using the EnzChek® Ultra Xylanase Assay Kit (Molecular Probes Invitrogen Ltd., Paisley, UK) in 30 mM Tris (pH 8.0) and 50 mM NaCl at 25 °C in a BioLector® (Biotek, Colmar, France). Prior to the assays, supernatant from cultures grown in YNB medium was diluted 50- and 100-fold, and supernatant from cultures grown in YPD or YP2D4 was diluted 500- and 1000-fold. As in the case of GA, one unit of XlnC activity (1 U) was defined as the amount of XlnC required to release 1 µmol of xylose per minute.
The growth of the Y. lipolytica strains was analyzed using a microtiter plate reader, as previously described . RedStar2 fluorescence and YFP fluorescence were analyzed at emission wavelength settings of 558 and 586 nm, respectively; the reception wavelength settings were 505 and 530 nm, respectively.
Images were acquired using a Zeiss Axio Imager M2 microscope (Zeiss, Le Pecq, France) and Axiovision v. 4.8 software (Zeiss, Le Pecq, France).
RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) followed by DNA digestion with DNase I (RNase-free; New England BioLabs, Evry, France). cDNA synthesis was performed with the Maxima First Strand cDNA Synthesis Kit with dsDNase (Thermofisher Scientific, Courtaboeuf, France). PCR quantification was performed with CFX Connect™ Real-Time PCR Detection System (Bio-Rad, Marnes-la-Coquette, France) using the SsoAdvanced™ Universal SYBR® Green Supermix Kit (Bio-Rad, Marnes-la-Coquette, France). The number of XlnC mRNA copies was determined using the cycle threshold (Ct) values, which were standardized using results for the expression of the actin gene (YALI0D08272g); the number of XlnC mRNA copies found in the strain containing pTEF-XlnC was employed as a reference.
Results and discussion
RedStar2 expression varies with promoter strength
To examine how protein expression varied with promoter strength, we constructed seven promoters (see diagram in Fig. 1b). Two were conventional promoters: pTEF and hp4d. Four new hybrid promoters were generated by combining two, three, four, or eight UAS1 tandem elements taken from hp4d with the TEF promoter, yielding 2UAS1-pTEF, 3UAS1-pTEF, 4UAS1-pTEF, and 8UAS1-pTEF, respectively (Fig. 1b). We also created a derivative of the hp4d promoter by adding four supplementary UAS1 tandem elements, thus generating hp8d (Fig. 1b).
Promoter strength affects xylanase C production but not glucoamylase production
As expected, GA production varied with promoter strength and increased with medium richness (Additional file 4: Figure S2a–d). However, high production levels may or may not translate into high activity levels. To determine if there was a correlation between the two variables, GA activity was estimated by measuring the disappearance of starch and the appearance of glucose. Activity was found to be positively associated with production (Additional file 4: Figure S2e).
As for GA, we examined the correlation between XlnC production and activity (Fig. 4b). As expected, the WT strain, JMY2900, demonstrated no XlnC activity. Surprisingly, activity levels were not always associated with production levels, which could suggest that there was co-secretion of non-active or less-active forms of the enzyme. Although the two variables were correlated when the strains were grown in YNB, the correlation was weak or completely absent when the strains were grown in YP2D4 or YPD, respectively (Fig. 4a, b). For instance, the strain containing 3UAS1-pTEF had a production level similar to that of the strain containing 4UAS1-pTEF, but the former’s activity level was much lower. Indeed, its activity level resembled that of the strain containing pTEF. Interestingly, activity levels were 1.5–2 times higher than expected for the strains containing 2UAS1-pTEF and hp8d (Fig. 4b). Oddly, although these promoters increased protein production two to fourfold, compared to the strain containing pTEF, activity increased three to sixfold (Fig. 4a, b; Additional file 5: Table S2, Additional file 6: Table S3). These results underscore that enzyme expression, production, and activity are not always linearly related to promoter strength. Indeed, these relationships may vary and depend on the specific enzyme and growth medium used.
A gateway vector pool for selecting the best protein producer
Since promoter strength was not necessarily correlated with heterologous protein production, we decided to develop a method for rapidly identifying transformants with optimized production; we used a pool of vectors containing promoters that varied in strength. To simplify the approach, we employed a gateway system that allowed in vitro cloning and the counter-selection of the correct clone using CcdB toxicity. We constructed a derivative of the gateway plasmid JMP1529 described in Leplat et al. : JMP3030 (gateway-ClaI-pTEF-BamHI). Derivatives were constructed using ClaI-BamHI-based promoter exchange (Additional file 1: Table S1).
Promoters upstream of the α-amylase gene and YFP gene in the different clones
3UAS1-pTEF or 4UAS1-pTEF
4UAS1-pTEF or hp8d
Relative results for the experiments examining RedStar2, glucoamylase, and xylanase C expression under the seven different promoters studied
yellow fluorescent protein
polymerase chain reaction
RD: wrote the project proposal, designed the experiments, built some of the plasmids and strains, performed the RedStar2 experiment, performed the vector-pool experiment, analyzed the results, and wrote the manuscript; FB: built the different promoters as well as some of the plasmids and strains and performed the RedStar2 experiment; TD: wrote the project proposal; RLA: performed the test for GA activity; JV: built some of the strains and performed the vector-pool experiment; MT: quantified protein production and ran the related gels; ST: built some of the plasmids and strains; JM: wrote the project proposal and designed the experiments; CL: wrote the project proposal, designed the experiments, built some of the plasmids and strains, performed the RedStar2 experiment, quantified protein production, ran the related gels, performed the test for XlnC activity, carried out quantitative PCR, analyzed the results, and wrote the manuscript. All authors read and approved the final manuscript.
We thank the French government for giving us IDEX funding (Grant No 2015-0445I). R. Ledesma-Amaro received financial support from the European Union (Marie-Curie FP7 COFUND People Program: AgreenSkills Fellowship). We thank Jessica Pearce and Lindsay Higgins for their language editing services.
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing is not relevant as no datasets were generated or analyzed during the current study.
C. Leplat received IDEX funding (Grant No 2015-0445I), which paid for all the experiments and J. Vion’s salary. R. Ledesma-Amaro received financial support from the European Union (Marie-Curie FP7 COFUND People Program: AgreenSkills Fellowship).
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- Domínguez A, Fermiñán E, Sánchez M, González FJ, Pérez-Campo FM, García S, Herrero AB, San Vicente A, Cabello J, Prado M, Iglesias FJ, Choupina A, Burguillo FJ, Fernández-Lago L, López MC. Non-conventional yeasts as hosts for heterologous protein production. Int Microbiol. 1998;1:131–42.Google Scholar
- Madhavan A, Sukumaran RK. Promoter and signal sequence from filamentous fungus can drive recombinant protein production in the yeast Kluyveromyces lactis. Bioresour Technol. 2014;165:302–8.View ArticleGoogle Scholar
- Bragança CR, Colombo LT, Roberti AS, Alvim MC, Cardoso SA, Reis KC, de Paula SO, da Silveira WB, Passos FM. Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1). Appl Microbiol Biotechnol. 2015;99:1191–203.View ArticleGoogle Scholar
- Spohner SC, Schaum V, Quitmann H, Czermak P. Kluyveromyces lactis: an emerging tool in biotechnology. J Biotechnol. 2016;222:104–16.View ArticleGoogle Scholar
- Wagner JM, Alper HS. Synthetic biology and molecular genetics in non-conventional yeasts: current tools and future advances. Fungal Genet Biol. 2016;89:126–36.View ArticleGoogle Scholar
- Nicaud JM, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, Gaillardin C. Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res. 2002;2:371–9.Google Scholar
- Madzak C, Gaillardin C, Beckerich JM. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol. 2004;109:63–81.View ArticleGoogle Scholar
- Nicaud JM. Yarrowia lipolytica. Yeast. 2012;29:409–18.View ArticleGoogle Scholar
- Madzak C, Beckerich JM. Heterologous protein expression and secretion in Yarrowia lipolytica. In: Yarrowia lipolytica, vol. 25. Berlin: Springer; 2013. p. 1–76.
- Müller S, Sandal T, Kamp-Hansen P, Dalbøge H. Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast. 1998;14:1267–83.View ArticleGoogle Scholar
- Madzak C, Treton B, Blanchin-Roland S. Strong hybrid promoters and integrative expression/secretion vectors for quasiconstitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. Mol Microbiol Biotechnol. 2000;2:207–16.Google Scholar
- Nthangeni MB, Urban P, Pompon D, Smit MS, Nicaud JM. The use of Yarrowia lipolytica for the expression of human cytochrome P450 CYP1A1. Yeast. 2004;21:583–92.View ArticleGoogle Scholar
- Sassi H, Delvigne F, Kar T, Nicaud JM, Coq AM, Steels S, Fickers P. Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica. Microb Cell Fact. 2016;15:159.View ArticleGoogle Scholar
- Gasmi N, Fudalej F, Kallel H, Nicaud JM. A molecular approach to optimize hIFN α2b expression and secretion in Yarrowia lipolytica. Appl Microbiol Biotechnol. 2011;89:109–19.View ArticleGoogle Scholar
- Barth G, Gaillardin C. Yarrowia lipolytica. In: Wolf K, editor. Non conventional yeasts in biotechnology, vol. 1. Springer: Germany; 1996. p. 313–88.View ArticleGoogle Scholar
- Chen DC, Beckerich JM, Gaillardin C. One-step transformation of the dimorphic yeast Yarrowia lipolytica. Appl Microbiol Biotechnol. 1997;48:232–5.View ArticleGoogle Scholar
- Xuan JW, Fournier P, Gaillardin C. Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration. Curr Genet. 1988;14:15–21.View ArticleGoogle Scholar
- Le Dall MT, Nicaud JM, Gaillardin C. Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet. 1994;26:38–44.View ArticleGoogle Scholar
- Boonvitthya N, Bozonnet S, Burapatana V, O’Donohue MJ, Chulalaksananukul W. Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica. Mol Biotechnol. 2013;54:158–69.View ArticleGoogle Scholar
- Wang W, Wei H, Alahuhta M, Chen X, Hyman D, Johnson DK, Zhang M, Himmel ME. Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica. PLoS ONE. 2014;9:e111443.View ArticleGoogle Scholar
- Hong SP, Seip J, Walters-Pollak D, Rupert R, Jackson R, Xue Z, Zhu Q. Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1 IN promoter. Yeast. 2012;29:59–72.View ArticleGoogle Scholar
- De Pourcq K, Vervecken W, Dewerte I, Valevska A, Van Hecke A, Callewaert N. Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man 8 GlcNAc 2 and Man5 GlcNAc2. Microb Cell Fact. 2012;11:53.View ArticleGoogle Scholar
- Lazar Z, Rossignol T, Verbeke J, Crutz-Le Coq AM, Nicaud JM, Robak M. Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications. J Ind Microbiol Biotechnol. 2013;40:1273–83.View ArticleGoogle Scholar
- Moon HY, Van TL, Cheon SA, Choo J, Kim JY, Kang HA. Cell-surface expression of Aspergillus saitoi-derived functional α-1,2-mannosidase on Yarrowia lipolytica for glycan remodeling. J Microbiol. 2013;51:506–14.View ArticleGoogle Scholar
- Nars G, Saurel O, Bordes F, Saves I, Remaud-Siméon M, André I, Milon A, Marty A. Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems. Protein Expr Purif. 2014;101:14–20.View ArticleGoogle Scholar
- Madzak C, Blanchin-Roland S, Cordero Otero RR, Gaillardin C. Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter. Microbiology. 1999;145:75–87.View ArticleGoogle Scholar
- Blazeck J, Liu L, Redden H, Alper H. Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl Environ Microbiol. 2011;77:7905–14.View ArticleGoogle Scholar
- Blazeck J, Reed B, Garg R, Gerstner R, Pan A, Agarwala V, Alper HS. Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica. Appl Microbiol Biotechnol. 2013;97:3037–52.View ArticleGoogle Scholar
- Shabbir Hussain M, Gambill L, Smith S, Blenner MA. Engineering promoter architecture in oleaginous yeast Yarrowia lipolytica. ACS Synth Biol. 2016;5:213–23.View ArticleGoogle Scholar
- Ledesma-Amaro R, Dulermo T, Nicaud JM. Engineering Yarrowia lipolytica to produce biodiesel from raw starch. Biotechnol Biofuels. 2015;8:148.View ArticleGoogle Scholar
- Mehmood N, Husson E, Jacquard C, Wewetzer S, Büchs J, Sarazin C, Gosselin I. Impact of two ionic liquids, 1-ethyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium methylphosphonate, on Saccharomyces cerevisiae: metabolic, physiologic, and morphological investigations. Biotechnol Biofuels. 2015;8:17.View ArticleGoogle Scholar
- Sambrook J, Maniatis T, Fritsch EF. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Mlícková K, Roux E, Athenstaedt K, d’Andrea S, Daum G, Chardot T, Nicaud JM. Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica. Appl Environ Microbiol. 2004;70:3918–24.View ArticleGoogle Scholar
- Dulermo R, Gamboa-Meléndez H, Michely S, Thevenieau F, Neuvéglise C, Nicaud JM. The evolution of Jen3 proteins and their role in dicarboxylic acid transport in Yarrowia. Microbiologyopen. 2015;4:100–20.View ArticleGoogle Scholar
- Querol A, Barrio E, Huerta T, Ramón D. Molecular monitoring of wine fermentations conducted by active dry yeast strains. Appl Environ Microbiol. 1992;58:2948–53.Google Scholar
- Dulermo R, Gamboa-Meléndez H, Ledesma-Amaro R, Thévenieau F, Nicaud JM. Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica. Biochim Biophys Acta. 2015;1851:1202–17.View ArticleGoogle Scholar
- Viktor MJ, Rose SH, van Zyl WH, Viljoen-Bloom M. Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases. Biotechnol Biofuels. 2013;6:167.View ArticleGoogle Scholar
- Pignède G, Wang H, Fudalej F, Gaillardin C, Seman M, Nicaud JM. Autocloning vectors for gene expression and amplification for the yeast Y. lipolytica. Appl Environ Microbiol. 2000;66:3283–9.View ArticleGoogle Scholar
- Leplat C, Nicaud JM, Rossignol T. High-throughput transformation method for Yarrowia lipolytica mutant library screening. FEMS Yeast Res. 2015;15:fov052.View ArticleGoogle Scholar
- Dulermo R, Gamboa-Meléndez H, Dulermo T, Thevenieau F, Nicaud JM. The fatty acid transport protein Fat1p is involved in the export of fatty acids from lipid bodies in Yarrowia lipolytica. FEMS Yeast Res. 2014;14:883–96.View ArticleGoogle Scholar
- Idiris A, Tohda H, Kumagai H, Takegawa K. Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol. 2010;86:403–17.View ArticleGoogle Scholar
- Li W, Zhou X, Lu P. Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res Microbiol. 2004;155:605–10.View ArticleGoogle Scholar
- Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol. 2014;98:5301–17.View ArticleGoogle Scholar
- Liu Z, Tyo KE, Martínez JL, Petranovic D, Nielsen J. Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae. Biotechnol Bioeng. 2012;109:1259–68.View ArticleGoogle Scholar
- Greenbaum D, Colangelo C, Williams K, Gerstein M. Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol. 2003;4:117.View ArticleGoogle Scholar
- Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet. 2012;13:227–32.Google Scholar