Patent ID: 12203078

EXAMPLES—MATERIALS AND METHODS

Strain and Medium Composition

All the strains used in this study are listed in Table 1. TheY. lipolyticastrains derived from the wild-typeY. lipolyticaW29 strain (ATCC20460). The auxotrophic derivative Po1d (Leu−Ura−) was previously described by Barth and Gaillardin (1996). Strain JMY1212 (MatA ura3-302 xpr2-322, LEU2, zeta platform, derived from Po1d) was used as the basis for promoter study in this study, the derivative strain JMY7126 (MatA ura3-302 xpr2-322, Δlys5, Δeyk1, LEU2, zeta platform, derived from JMY1212) carrying a deletion of EYK1 was also used to see inducible expression of promoter in strain unable to use erythritol as a carbon source.Escherichia colistrain DH5α was used for hosting and amplification of recombinant plasmid DNA. The media and growth conditions forE. coliwere as described in Sambrook et al. (1989). YPD and YNB medium together with growth conditions forY. lipolyticahave been previously described by Barth and Gaillardin (1996). To meet auxothrophic requirement, uracil (0.1 g/L), lysine (0.8 g/L), and leucine (0.1 g/L) were added in culture medium when necessary. Casamino acids, (0.2% Bacto Casamino Acids, Difco, Paris, France), were added for bioreactors culture for faster growth rate. Growth ofY. lipolyticawere performed in baffled 250 mL flask and incubated at 28° C. at 160 rpm. YNB medium was supplemented with carbon source as follows: 10 g/L glucose (YNBD); 10 g/L glycerol (YNBG); 10 g/L erythritol (YNBOL); 10 g/L erythrulose (YNBOSE); 1 g/L glycerol, 10 g/L erythritol, 0.5 g/L yeast extract and 0.5 g/L peptone (YNBE). Growth of Δeyk1 strains in microplates were performed in YNB medium with 2.5 g/L glucose or 2.5 g/L glycerol as carbon source and 2.5 g/L erythritol or 2.5 g/L erythrulose as inducer. YNBDETo is an YNB medium with 10 g/L glucose, 10 g/L erythritol, 5 g/L tributyrin and 1.5% agar.

Culture in Fed-Batch Bioreactor

Bioreactor cultures were performed in duplicate in a 2-L Biostat B-Twin fermentor (Sartorius) containing 1 L of medium and kept at 28° C. Stirrer speed was set to 800 RPM, and the aeration rate was 1 L/min. The pH was set at 6.8 and automatically maintained by the addition of 20% (w/v) NaOH or 40% (w/v) H3PO4when necessary. Glycerol (56.9 g/L solution) was fed for 24 h at a flow rate of 0.4 g/L·h, then at a flow rate of 0.8 g/L·h for an additional 24 h. Yeast cultures were inoculated at an initial optical density at 600 nm of 0.5. Cell growth was monitored by optical density at 600 nm (00600). Cell dry weight (CDW) was determined by using the relation 00600=0.29 gCDW correlation.

TABLE 1List of strains and plasmids usedStrain orGenotype or other relevantplasmid*characteristicsSource or referenceE. coliDH5αϕ80dlacZΔm15, recA1, endA1, gyrA96, thi-Promega1, hsdR17 (rk−, mk+), supE44, relA1, deoR,Δ(lacZYA-argF)U169pCR4Blunt-Cloning vector, kanamycinInvitrogenTOPO ®pJET 1.2Cloning vector, ampicilineThermo scientificpUC57GeneScript Biotech donor vectorGeneScript BiotechGGE020pCR4Blunt-TOPO-T1-3Lip2(Celińska et al., 2017)GGE077pCR4Blunt-TOPO-G1-RedstarII(Celińska et al., 2017)GGE085pCR4Blunt-TOPO-pTEF1(Celińska et al., 2017)GGE104pCR4Blunt-TOPO-pEYK1-3ABGGE114pSB1A3-ZetaUP-URA3-RFP-ZetaDOWN(Celińska et al., 2017)GGE0130pCR4Blunt-TOPO-pEYK1-2ABThis workGGE0132pCR4Blunt-TOPO-pEYK1-4ABThis workGGE140pCR4Blunt-TOPO-pEYD1ABThis workGGE172pCR4Blunt-TOPO-pEYD1A*BThis workGGE174pCR4Blunt-TOPO-pEYD1AB*This workGGE238pCR4Blunt-TOPO-pEYK1This workGGE250pCR4Blunt-TOPO-pEYK1-5ABThis workRIE132Cre-EYK1 (2.2 kb pTEF-EYK1 fragment inThis work(RIP132)pRRQ2)FCP007pJET 1.2-pEYK300; ClaI-BamHIThis workJME461pRRQ2 (Cre ARS68 LEU2)Richard et al. (2001)JME507JMP113 (URA3ex marker)Fickers et al. (2003)JME508JMP114 (LEU2ex marker)Fickers et al. (2003)JME547pUB4-CREFickers et al. (2003)JME803JMP62-pPOX2-URA3exHaddouche et al. (2010)JME1046pTEF-URA3; JMP62 type vector with pTEFThis workpromoterJME1427JMP62-pTEF-YFP-LEU2exB Treton, unpublishedJMP62-php4d-YFP-URA3exB Treton, unpublishedJME2027pCR4Blunt-TOPO - ClaI-4UAS1xpr2-BstBIDulermo et al., (2017)JME3265JMP62-LYS5exunpublishedJME3267PUT-lys5, PLYS5-UR43ex-TLYS5This workJME3739pTEF-CalBop-URA3ex, CalB expressedThis workunder the pTEF promoter(15ACCYRP_1762990_pJME1046-CalB)JME3934JMP62-pEYK300-YFP-URA3exThis work(FCP013)JME3994JMP62-pEYK450-YFR-UR43exThis work(JMP3994)JME3988JMP62-pEYK300aB-YFP-URA3exThis work(JMP3988)JME3991JMP62-pEYK300Ba-YFP-URA3exThis work(JMP3991)JME3998JMP62-pHU4-EYK300-YFP-URA3exThis work(JMP3998)JME4056pGEM6-easy-cre-EYK1Vandermies et al., 2017JME4123PUC57-pEYK300A3BGenScript, Hong-KongJME4124PUC57-pEYK300A3bGenScript, Hong-KongJME4137JMP62-pEYK300A3B-YFP-URA3exThis work(JMP4137)JME4139JMP62-pEYK300A3b-YFP-URA3exThis work(JMP4139)JME4265Cre-ARS68-pTEF-EYK1Vandermies et al. 2017(RIE132)JME4266pEYK3AB-URA3ex; JMP62 type vector withThis workpEYK3AB promoterJME4230pHu8-URA3ex; JMP62 type vector withThis workpHu8 promoterJME4365pEYK3AB-CalBop-URA3ex; JMP62 typeThis workvector with pEYK3AB promoterJME4384pEYK3AB-CalBop-LYS5ex; JMP62 typeThis workvector with pEYK3AB promoterJME4417pUC57-EYK1-4AB-coreTEFThis studyY. lipolyticaW29MatA, CLIB89, ATCC20460, Wild-typeBarth & GaillardinFrench strain(1996)Po1dMatA ura3-302 leu2-270 xpr2-322 URA3exBarth & Gaillardin(JMY195)LEU2 deleted for the extracellular protease(1996)AEP encoded by XPR2 geneRIY146Po1d eyk1::LEU2, Ura+This workRIY176Po1d Δeyk1, Ura+Leu+This workRIY180RIY176 + pEYK300-YFP-LEU2ex (Ura+Leu+)This work(JMY6637)RIY201Po1d lip2::URA3, Leu+This workRIY203Po1d Δlip2This workRIY132ARS68/CEN php4d-Cre pTEF-EYK1This workRIY212Po1d eyd1::URA3exThis workRIY225Po1d Δeyd1This workJMY330Po1d, Ura+Haddouche et al, (2010)JMY1212MatA, leu2-270, ura3-LEU2-ZETA, xpr2-322,Bordes et al., (2003)Δlip2, Δlip7, Δlip8, deleted for the threelipases Lip2, Lip7 and Lip8 and theextracellular lipase AEP, zeta-LEU2 platformat the URA3 locusJMY2101Po1d, Leu+Dulermo et al. (2017)JMY2876JMY330 + pTEF-YFP-LEU2ex (Ura+ Leu+)B. Treton unpublishedJMY2900Po1d, Ura+Leu+Dulermo et al. (2017)JMY5207JMY1212 lys5::URA3ex,This workJMY6245JMY2101 + pEYK300-YFP-URA3ex (Ura+This work(FCY003)Leu+)JMY6369JMY2101 + pEYK300aB-YFP-URA3ex (Ura+This workLeu+)JMY6372JMY2101 + pEYK300Ab-YFP-URA3ex (Ura+This workLeu+)JMY6375JMY2101 + pEYK450-YFP-URA3ex (Ura+This workLeu+)JMY6380JMY2101, + pHU4-EYK300-YFP-URA3exThis work(Ura+Leu+)JMY6681JMY2101 + pEYK300A3B-YFP-URA3exThis work(Ura+Leu+)JMY6684JMY2101 + pEYK300A3b-YFP-URA3exThis work(Ura+Leu+)JMY7121JMY1212 Δlys5This workJMY7123JMY1212 Δlys5, eyk1::URA3exThis workJMY7126JMY1212 Δlys5, Δeyk1This workJMY7240JMY7126 + pEYK3AB-CalB-URA3ex +This workpEYK3AB-CalB-LYS5ex, (multicopieintegration of CalB)*JME for theE. colistrain, JMP for the plasmid.
Growth in Microplate and Fluorescence Analysis

Y. lipolyticaprecultures were grown overnight in YNBD, before being centrifuged, washed with an equal volume of YNB medium without carbon source and resuspended in 1 mL of the same medium. 96-well microplates containing 200 μL of the appropriated medium (final volume) were inoculated with washed cells at an OD600 nmof 0.1. Growth was performed in a microtiter plate reader Synergy Mx (Biotek, Colmar, France), following the manufacturer's instructions at 28° C. and 110 rpm. OD600 nmand fluorescence were measured every 20 min for 72 h. YFP fluorescence was analyzed with the wavelength settings ex: 505 nm/em: 530 nm. Fluorescence was expressed as specific fluorescence unit (SFU, normalized to biomass value) or mean specific fluorescence value (mSFU, mean value of SFU for the different sampling times). In all case, the SFU value of the wild-type strain JMY2900 (i.e. cell intrinsic fluorescence) was deduced from that of the YFP reporter strain in the same experimental conditions (sampling time and medium). Cultures were performed in replicates.

Growth and RedStarII Florescence Analysis

YNB medium supplemented with glucose (10 g/L) or erythritol (10 g/L) was used for growth and florescence analysis. Growth of Δeyk1 strains were performed in YNB Lysine medium with 0.25% glucose as carbon source and 0.25% erythritol as inducer as described previously (Trassaert et al. 2017). EachY. lipolyticaclone from the plate was grown in YNBD for 24 hours. Cells were then transferred to fresh medium (final volume 200 μL) in 96-well microplates. Growth was performed in a microtiter plate reader Synergy Mix (Biotek, Colmar, France) following the manufacturer's instructions at 28° C. and 110 rpm. OD600 nm and red fluorescence were measured every two hours for 60 hrs. Red fluorescence was analyzed with the wavelength settings ex: 558 nm/em: 586 nm. Fluorescence was expressed as mean specific fluorescence value per hour (SFU/h, mean value of SFU per hours). RedStarII fluorescence was expressed as specific fluorescence unit per hour. For RedStarII measurement, no intrinsic fluorescence was detected. Cultures were performed at least in duplicates.

Growth in Chemostat and Monitoring of Promoter Induction by Flow Cytometry

Y. lipolyticaprecultures were performed in YNBD for 12 h and washed cells were used for bioreactor inoculation at an OD600 nmof 0.5. Chemostat were performed in 200 ml (150 ml working volume) DASGIP® DASbox Mini Bioreactors SR0250ODLS (Eppendorf, Hamburg, Germany). They were first run for 7 h in batch mode before being shifted in continuous mode with dilution rates as stipulated in the text. Feeding of fresh medium was ensured by a Watson Marlow 323S peristaltic pump (Watson Marlow, Falmouth Cornwall, UK), and removal of spent medium was ensured by a Watson Marlow 120 U/DM3 peristaltic pump (Watson Marlow, Falmouth Cornwall, UK). Culture parameters were set as follows: temperature, 30° C.; agitation rate, 800 rpm; aeration rate at 1 vvm. Carbon source pulses (CSP) in the reactors were at fixed volume (4.2 ml), regardless of the pulse concentration. After each CSP, biomass, YFP fluorescence and carbon source concentrations were monitored for 8 hours with a sampling frequency of one hour. CSP were performed at steady state. Chemostat cultures were performed in duplicate.

YFP fluorescence was monitored using a BD Accuri™ C6 Flow Cytometer (BD Biosciences, NJ, USA). Flow rate was fixed to 14 μl/min, and samples were diluted with phosphate saline buffer (PBS) to reach a cell density ranging between 500 and 2500 cells/μl. For each sample, 40,000 cells were analyzed using the FL1-A channel to identify fluorescence associated with the YFP (excitation was performed with a 20-mW, 488-nm solid-state blue laser; the emission wavelength was 533/30 nm). Additionally, data from the forward scatter channel (FSC-A) were collected to get information on the size dispersion among the cell population. The flow cytometry dotplots (FL1-A/FSC-A) were analyzed using CFlowPlus software (Accuri, BD Bioscience). For further processing, the raw data were exported as .fcs files and loaded in MatLab using the fca_readfsc function (downloaded from the MatLab File Exchange file server; http://www.mathworks.com). Background noise (cell intrinsic fluorescence) was fixed at 4,000 fluorescence units. This value encompasses the fluorescence level of at least 99.3% of the wild-type cells (strain JMY2900) grown in YNBG (glycerol), YNBOL (erythritol) and of JMY6245 (pEYK300-YFP) grown in YNBG (glycerol). Relative fluorescence (RFU) was defined as the sample median fluorescence value minus the intrinsic fluorescence value. Proportion of induced cells refers to the number of cells showing a fluorescence signal higher than 4,000 fluorescence units, relative to the total number of analyzed cells in the sample (i.e. 40,000). Gate Q1-UR of FSC-A/FL1-A cytograms encompasses induced cells.

Sequence Analysis

Genome sequences ofYarrowiaspecies were assembled and annotated by Cécile Neuvéglise, Hugo Devillers and coworkers (to be published). Homologues of EYD1 and EYK1 genes inYarrowiaspecies were identified by Blast on the private site of GRYC (Genome Resources for Yeast Chromosomes; http://gryc.inra.fr) using EYD1 and EYK1 genes as template as described previously (Carly et al. 2017). Promoter regions were retrieved using the download functionality developed by H. Devillers. Multiple alignment of nucleotide sequence of EYK1 and EYD1 genes promoters among theYarrowiaclade:Y. lipolytica(YALI),Yarrowia phangngensis(YAPH),Yarrowia yakushimensis(YAYA),Yarrowia alimentaria(YAAL), andYarrowia galli(YAGA) were then performed using the program Clustal Omega (Sievers, et al. 2011) available at http://www.ebi.ac.uk/Tools/msa/clustalo/. From those alignments, the motifs conserved through evolution and thus, more likely to carry a regulatory function, were identified. The conserved motifs were named Box A and Box B. To test their function as Upstream Activating Sequence or Upstream Activation Sequence (UAS), Region containing these conserved motifs+5 to 17 bases encompassing the conserved motifs were selected.

Plasmid and Yeast Strain Construction

Plasmid Construction

Restriction enzymes, DNA polymerases, and ligases were used in accordance with the manufacturer's recommendations. Restriction enzymes were obtained from OZYME (Saint-Quentin-en-Yvelines, France) except I-SceI that was obtained from New Englands Biolab. PCR amplifications were performed using an Eppendorf 2720 thermal cycler with PyroBest DNA polymerase (Takara) for cloning purpose and with GoTaq DNA polymerase (Promega) for deletion/overexpression verification. PCR fragments were purified using a QIAgen Purification Kit (Qiagen, Hilden, Germany), and DNA fragments were recovered from agarose gels using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). DNA sequencing was performed by GATC Biotech and primers were synthetized by Eurogentec (Seraing, Belgium). The Clone Manager software package (Sci-Ed Software) was used for gene sequence analysis and primer design. Disruption and expression cassettes were transformed with the lithium acetate method (Le Dall et al., 1994). Transformants were selected on YNBcasa, YNBura, or YNB depending on their genotype (Barth and Gaillardin, 1996). The genomic DNA from yeast transformants was prepared as described by Querol et al. (1992). The plasmids used in this study are summarized in Table 1 and primers are listed in Table 2. Primers MT-URA3-for, MT-YFP-rev, pTEF-start, 61stop were used to verify successful insertion of the expression cassette and the promoter sequences. For each transformation, at least three independent transformants carrying the correct integration were analysed. The representative clones were used for this study. The vectors carrying the yellow fluorescent protein (YFP) under the control of the pTEF and php4d have been previously described (Table 1). The pEYK1 promoters and its derivatives (mutated and hybrid promoters) were introduced by exchange of the ClaI-BamH1 region or the ClaI-SpeI region of YFP encoding plasmid as described below.Y. lipolyticastrains were transformed by the lithium acetate method as described previously (Le Dall et al., 1994). Expression vectors (400 ng) were digested with NotI and subjected to electrophoresis. The bands corresponding to the expression cassettes were extracted from the gel and used for transformation. Cre-mediated marker rescue and curing of the replicative cre expression plasmid were performed as described previously (Fickers et al., 2003).

Plasmid Construction by Golden Gate Assembly

Most amplicons of promoters were cloned in the donor vectors (pCR Blunt II TOPO vectors; Thermo Fisher Scientific, Villebon sur Yvette, France), verified by BsaI digestions and sequencing. Some of the promoters were synthesized and cloned in the donor vector (pUC57) by GeneScript Biotech (New Jersey, US) (See Table 1). All the primers used to amplify the promoter were designed to have the upstream overhang “ACGG” and the downstream overhang “AATG” (See Table 3) to be applied to Golden Gate assembly. Other building blocks of Golden Gate assembly (destination vector, RedStarII, and Lip2 terminator) were prepared by purification of plasmids from our own GGE collection (Golden GateE. colicollection). The destination vector GGE114, pSB1A3-ZetaUP-URA3-RFP-ZetaDOWN (Table 1) contains the following part: zeta UP, URA3, RFP (Red fluorescent protein giving redE. colicolony), and zeta DOWN. Promoter name, primer pairs, and template used for PCR are described in Table 4. The Golden Gate Assembly Strategy (GGAS) is presented inFIG.17). The Golden Gate reaction conditions were performed following the previously published protocol (Celińska et al. 2017). The reaction mixture contained pre-calculated equimolar amount of each Golden Gate bio-bricks and the destination vector (50 pmoles of ends), 1 μl of T4 DNA ligase buffer (NEB), 5 U of BsaI, 200 U of T4 and ddH2O up to 10 μl. The following thermal profile was applied: [37° C. for 5 min, 16° C. for 5 min]*60 cycles, 55° C. for 5 min, 80° C. for 5 min, 15° C. ∞. Subsequently, the reaction mixture was used forE. coliDH5α transformation. White colonies were screened for identification of complete assemblies, afterwards PCR and restriction enzyme digestion of plasmids were conducted for verification. All bio-bricks were verified by sequencing.

TABLE 2List of primers usedSEQIDGene/NO:namePrimersSequences *14EYK1EYK-P-LGTTGTGTGATGAGACCTTGGDeletion of EYK1TGCverification of EYK115EYK1EYK-P-R-SfiIAAAGGCCATTTAGGCCGCAGdeletionCTCCTCCGACAATCTTG16EYK1EYK-T-L-SfiITAAGGCCTTGATGGCCACAAGTAGAGGGAGGAGAAGC17EYK1EYK-T-RGTTTAGGTGCCTGAAGACGGTG18EYK1EYK-V1CGTACCCGAGATTGTACTGTVerification of EYK1TGTCdeletion19EYK1EYK-V2CATAACCGCCTACCCTTGTAGC20EYK1EYK1-AFTTCTTGGGCCCGGCCTAAATCatabolic marker EYK1.GGCCCTGTTATCCCTAGATCSitesGATATAGAGSfi1; Apa1 and21EYK1EYK1-KRAATAAGGTACCGGCCATCAASfi1*; Kpn1GGCCATTCGATTTGTCTTAGAGGAACGC22EYD1EYD-P-FoAAGCGTCCGAGACTGTCGGADeletion of EYD1 and23EYD1EYD-P-sfi-AAAGGCCATTTAGGCCACTGverification of EYD1RevACGTCTGTCTTGACGCdeletion24EYD1EYD-T-Sfi-TATGGCCTTGATGGCCCAGCFoATTGAGTCCAACGAGC25EYD1EYD-T-RevAGATCGAAGTTGGAATGAGA26EYD1EYD-VCGAGTTTCTAAGATGTACAT27EYD1URA3-P-R1GTTGCCAATATCTGCGAACTTTCTG28LPR-FATAGGCCTAAATGGCCTGCAMarkers amplificationTCGATCTAGGGATAACAGGwith Sfi1 and Sfi1*29LPR-RATAGGCCATCAAGGCCGCTAGATAGAGTCGAGAATTACCCTG30LIP2LIP2-PFCGGTCGGAATAATTACTGTGLIP2 deletion:GACCamplification of promoter31LIP2LIP2-PRAAAGGCCATTTAGGCCACTTand terminator regions,GGGTATCAATTGAGGGCTTTSfi1 and Sfi1* andCverification of LIP232LI P2LIP2-TFTATGGCCTTGATGGCCTGTCdisruptionTCGGAGGAGCTGCAGCCC33LI P2LIP2-TRTTGCTTAACACCAGTATCAGAACACAGAC34LIP2LIP2-VACGGAAGCGAAGTACCTGTCAC35ACT1ACT-FGGCCAGCCATATCGAGTCGfor RT-qPCR of ACT1CAgene36ACT1ACT-RTCCAGGCCGTCCTCTCCC37EYK1EYKq-FCTTCTGCTGGACCCTCTGTCfor RT-qPCR of EYK138EYK1EYKq-RACTGCACGTAGGGGATCAACgene39EYD1EYD qper FoCATGACCTTTCGGAACCAGTfor RT-qPCR of EYD140EYD1EYD qperAACGCCTCTCTGGTCTTCAAgeneRev41P300pEYK300 FGACATCGATGCATCTACTTTPromoter ampli,TCTCTATACTGTClaI42P300pEYK RGACGGATCCAGTAGATGTGTPromoter ampli, BamHIAAGTGTGTAGAAG43P450MT-ACGATCGATTTTGTGCAAGTPromoter ampli, ClaITATAampli-GTGTGTGTGTGF44P450MT-ACGACTAGTCAGGTCATCGGPromoter ampli, SpeITATAampli-ATTATGCAAGGR45ST043pEYK-mut1CGATGCATCTACTTTTCTCTAWT native Box ATACTGTACGTTTCAATCTGGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCC46ST044pEYK-CGATGCATCTACTTTICTCTAMutated Box Amut1BISTACTGTACGTTTCAATCTGGGGAAGCGGAATCCCAAAAGGACGCGTCGCCGCATTAAGCTCCACAGCC47ST045pEYK-mut2TTGCATAATCCGATGACCTGWT native Box BA48ST046pEYK-TTGTACGCGTAGATGACCTGMutated Box Bmut2BISA49ST047pEYK-mutAGGCGTAATTCGAGGTGTCGWT native Box AGAACGTATTAGGCTACTGGAcomplementary strandCTGATC50ST048p EYK-GGCGTAATTCGAGGTGTCGMutated Box AmutABISGAACATGCGCATCTACTGGAcomplementary strandCTGATC51ST049pEYK-mutBTACGTAGATGAAAAGAGATAWT native Box BTGACATGCAAAGTTAGACCCcomplementary strandCTTCGCCTTAGGGTTTTCCCTTTCGCG52ST050pEYK-TACGTAGATGAAAAGAGATAMutated Box BmutBbisTGACATGCAAAGTTAGACCCcornplementary strandCTTCGCCTTAGGGTTTTCCTGCGCACG53MT-URA3-GCGTAGGTGAAGTCGTCAATFor promoter sequenceforverification (forward)54MT-YFP-revCAGATGAACTTCAGGGTCAGFor promoter sequenceCverification (reverse)55pTEF-startGGGTATAAAAGACCACCGTCFor gene verificationC(forward)5661stopGTAGATAGTTGAGGTAGAAGFor gene verificationTTG(reverse)57Sfi1GGCCTAAATGGCC58Sfi1*GGCCATCAAGGCC* Modified sequences are in bold, restriction sites introduced are underlined.
Construction of PEYK300

The promoter region of EYK1 gene (pEYK300) was amplified from genomic DNA ofY. lipolyticastrain W29 with primer pair pEYK300 F/pEYK R, designed to introduce a ClaI and BamHI restriction sites, respectively, in the amplified fragment. The resulting amplicon was purified and cloned into pJET1.2, to yield plasmid FCP007. The pEYK300 fragment was then released from FCP007 and cloned at the corresponding site of JMP1427, yielding the plasmid JMP3934.

Construction of PEYK450, PEYK300Ab and PEYK300aB Promoters

Plasmid containing pEYK450 was obtained by PCR amplification of the intergenic region between genes YALI0F01628g and YALI0F01606g with primer pair MT-TATAampli-F/MT-TATAampli-R. This resulted in a 252 bp fragment carrying T, A and B boxes within a ClaI-SpeI fragment at the 5′ and 3′ ends, respectively. This fragment was ligated into FCP013 digested by ClaI-SpeI, to yield the plasmid JMP3994 (pEYK450). Plasmids containing pEYK300Ab and pEYK300aB were obtained by exchange of the ClaI-SpeI fragment of JMP3934 (pEYK300) by two ClaI-SpeI DNA fragments carrying the A (aB) or B (Ab) mutated regions, respectively. They were obtained by annealing oligonucleotides ST044/ST045/ST050/ST047 (fragment aB) and ST043/ST046/ST049/ST048 (fragment Ab) (Table 2). The oligonucleotides ST044 and ST046 contains a MluI site for the verification of the insertion of the mutation. The resulting plasmids were designated as JMP3988 (pEYK300aB) and JMP3991 (pEYK300Ab), respectively.

Construction of Hybrid PHU4EYK300 Promoter

The fragment carrying four tandem repeats of the UAS1XPR2(HU4 deriving from pHp4d) was obtained by ClaI-BstBI digestion from the JMP2027 vector (Dulermo et al., 2017). After gel purification, it was then ligated at the ClaI site of JMP3934 (previously digested by ClaI and dephosphorylated). Correct orientation of the HU4 region was verified by ClaI-BamHI restriction and DNA sequencing. The resulting plasmid was named JMP3998 (pHU4EYK300).

Construction of Hybrid EYK Promoter

Synthetic promoters carrying three repeated of UAS1-eyk1 upstream of the wild-type Box B (A3B, JMP4123) and the mutated Box B (A3b, JMP4124) were synthesised by GenScript (www.genscript.com/) with ClaI and SpeI sites at the 5′ and 3′ ends, respectively. The ClaI-SpeI fragment from JMP4123 and JMP4124 were ligated into JMP918 digested by ClaI-SpeI, yielding the plasmid JMP4137 (pEYK300A3B) and JMP4139 (pEYK300A3b), respectively.

Deletion of the EYK1 Gene

The EYK1 disruption cassette was generated by PCR amplification. First, the upstream (Up) and downstream (Dn) regions of the EYK1 gene were amplified usingY. lipolyticaW29 genomic DNA as the template with the EYK-P-L/EYK-P-R-SfiI and EYK-T-L-SfiI/EYK-T-R as primer pairs. URA3ex marker was amplified from JMP113 with the primer pair LPR-L-SfiI/LPR-R-SfiI (Table 2). Amplicons were digested with SfiI before being purified and ligated, using T4 DNA ligase. The ligation product was amplified by PCR using the primer pair EYK-P-L/EYK-T-R. The eyk1::URA3ex disruption cassette was finally used to transformY. lipolyticastrain Po1d. The resulting strain was designated RIY147 (eyk1::URA3ex, Leu−). The auxotrophic derivative RIY176 was isolated after transformation with the replicative plasmid pRRQ2 according to Fickers et al. (2003) for marker rescue (Table 1). The primers EYK-V1 and EYK-V2 (Table 2) were used for gene disruption verification.

Deletion of the EYD1 Gene

The EYD1 disruption cassette was generated by PCR amplification. First, the upstream (Up) and downstream (Dn) regions of the EYD1 gene were amplified usingY. lipolyticaW29 genomic DNA as the template with the EYD-P-Fo/EYD-P-sfi-Rev and EYD-T-Sfi-Fo/EYD-T-Rev as primer pairs. URA3ex marker was amplified from JMP113 with the primer pair LPR-F/LPR-R (Table 2). Amplicons were digested with SfiI before being purified and ligated, using T4 DNA ligase. The ligation product was amplified by PCR using the primer pair P1-EYD/T2-EYD. The eyd1::URA3ex disruption cassette was finally used to transformY. lipolyticastrain Po1d. The resulting strain was designated RIY212 (eyd1::URA3ex). The auxotrophic derivative RIY225 was isolated. The primers EYD-V and URA3-P-R1 (Table 2) were used for gene disruption verification.

Construction of Plasmid JMP3739

Y. lipolyticaLip2prepro-CalB protein was codon optimized using in house codon optimization software (Biocatalysts LTD), synthesized by Geneart (15ACCYPP_1762989_LIP2-CalB-YI-Opt) and cloned into JMP1046 giving rise to JMP3739 (15ACCYRP_1762990_pJME1046-CalB). The Lip2prepro-CalB sequence is provided as set forth in SEQ ID NO: 59:

ATGAAGCTGCTGTCTCTGACCGGTGTGGCTGGTGTTCTGGCCACCTGTGTCGCTGCCACCCCTCTGGTGAAGCGACTGCCTTCTGGATCTGACCCTGCCTTCTCTCAGCCCAAGTCTGTTCTGGACGCTGGTCTGACCTGTCAGGGAGCTTCTCCTTCTTCTGTGTCTAAGCCCATTCTCCTGGTGCCTGGAACCGGAACCACCGGTCCTCAGTCTTTCGACTCGAACTGGATTCCTCTGTCTACCCAGCTGGGATACACCCCCTGTTGGATTTCTCCTCCTCCTTTCATGCTGAACGACACCCAGGTGAACACCGAGTACATGGTGAACGCCATTACCGCTCTGTACGCTGGCTCTGGAAACAACAAGCTGCCCGTTCTGACCTGGTCTCAGGGAGGTCTGGTGGCTCAGTGGGGTCTGACCTTCTTCCCTTCTATTCGATCTAAGGTGGACCGACTGATGGCCTTCGCTCCCGACTACAAGGGAACCGTTCTGGCTGGTCCTCTGGACGCTCTGGCTGTCTCTGCTCCTTCTGTGTGGCAGCAGACCACCGGCTCTGCTCTGACCACCGCTCTGCGAAACGCTGGAGGTCTGACCCAGATTGTCCCCACCACCAACCTGTACTCTGCCACCGACGAGATTGTCCAGCCTCAGGTGTCTAACTCTCCTCTGGACTCTTCGTACCTGTTCAACGGAAAGAACATTCAGGCTCAGGCTGTCTGTGGACCTCTGTTCGACATTGACCACGCTGGCTCTCTGACCTCTCAGTTCTCCTACGTGGTTGGACGATCTGCTCTGCGATCTACCACCGGTCAGGCTCGATCTGCTGACTACGGTATCACCGACTGTAACCCTCTGCCTGCCAACGACCTGACCCCTGAGCAGAAGGTGGCTGCTGCTGCTCTGCTGGCTCCCGAGGCTGCTGCCATTGTCGCTGGTCCCAAGCAGAACTGCGAGCCCGACCTGATGCCTTACGCTCGACCCTTCGCTGTTGGAAAGCGAACCTGTTCTGGTATTGTCACCCCTTAA(Lip2 prepro sequence is in bold and underlined).

Plasmid JMP1046 containing the pTEF promoter present the typical structure of the expression vector JMP62 (Nicaud et al., 2002) carrying an excisable marker (I-scel fragment flanked by LoxP/LoxR, a promoter as a ClaI-BamHI fragment, BamHI and AvrII sites for cloning a gene of interest and zeta region for random or zeta platform expression cassette integration, flanked by NotI site for the release of the expression cassette prior transformation.

Construction of JMP4266.

Promoter exchange was performed by digestion of JMP1046 plasmid by ClaI-BamHI for insertion of the inducible promoter pEYK3AB ClaI-BamHI fragment from JMP4123 resulting in plasmid JMP4266.

Construction of CalB Expression Vectors

First, the BamHI-AvrII fragment carrying preproLip2-CalBop was isolated from BamHI-AvrII digested JMP3739 and cloned at the corresponding site into JMP4266, giving rise to JMP4365. In a second step, the derivative plasmid JMP4384 was constructed by exchange of the IScel-URA3ex fragment with the corresponding I-SceI-LYS5ex fragment carrying the LYS5ex marker from JMP3265.

Construction of JMY7126.

The construction has been realized by successive gene deletion and marker rescue according to Fickers et al. (2003) as described inFIG.15using lys5::URA3ex (JME3265) and eyk1::URA3ex (JME4056) disruption cassettes. Marker rescue was obtained after transformation with the replicative plasmid carrying the hygromycin-B resistance hph marker (pUB4-cre-hph; JME547) and the catabolic marker EYK1 marker (Cre-ARS68-pTEF-EYK1; JME4265) described in Table 1. To cure cells of the cre expression plasmid, cells were grown on YPD. The loss of the replicative plasmids was checked by replica plating on YPD, and YPDhyg for pUB-cre-hph and on YNBglucose and YNBerythritol for pGEM6-easy-cre-EYK1. Correctness of marker excision was verified on YNBA glucose and YNBA erythritol supplemented with lysine.

Construction JMY7240

Strain JMY7126 was co-transformed with the two expression cassettes URA3ex-pEYK3AB-CalB (JMP4365) and LYS5ex-pEYK3AB-ClaB (JMP4384) (Table 1). Transformants were selected on YNBD agar plate.

Construction of JMY7126

Strain JMY7126, deriving from JMY1212, has been obtained after successive gene deletion (LYS5 and EYK1) and marker rescue (FIG.15). The PUT plasmids (Promoter-URA3ex marker-Terminator) were constructed for gene disruption according to Fickers et al 2003 for LYS5 and according to Vandermies et al., 2017 for EYK1. The disruption cassettes were prepared by digesting PUT plasmids followed by transformation intoY. lipolyticastrain. Transformants were selected on YNB Leucine, YNB Leucine Lysine medium depending on their genotype. The replicative plasmids (JME547, RIE132-JME4265) harboring Cre recombinase gene were used for excising URA3ex marker.

Construction of Strain for Promoter Studies

Strains for promoter studies are described in Table 1. Plasmids for promoter analysis, assembled by Golden Gate assembly, were digested by NotI to allow the release of the expression cassette priorY. lipolyticaJMY1212 and JMY7126 transformation. After transformation with 100 ng DNA via the lithium acetate method (Le Dall et al., 1994), transformants were selected utilizing YNB or YNB Lysine medium depending on their genotype. After florescence test of twelve transformants of each construct, representative clone was selected (Table 1, Example 12).

Analytical Methods

Erythritol, erythrulose, glucose and glycerol concentrations in the culture supernatant were measured by HPLC (Agilent Technologies 1200 series) using a Aminex HPX-87H ion exclusion column, (Biorad 300×7.8 mm). Elution was performed using 15 mM trifluoroacetic acid as the mobile phase at a flow rate of 0.5 mL/min and a temperature of 65° C. Erythritol, glucose and glycerol were detected using a refractive index detector (RID, Agilent Technologies), while erythrulose was measured at 210 nm with a UV detector (Agilent Technologies).

Protein Analysis.

Protein concentration was determined as described by Bradford (Bradford, M. M. 1976). The standard curve was obtained by serial dilutions of Pierce™ Bovine Serum Albumin Standard Ampules (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on a Novex™ 12% Tris-Glycine Mini Gel (Thermo Fisher Scientific) as described by Laemmli (Laemmli, U. K. 1970). Four μl of pre-stained Protein Marker IV (AppliChem GmbH, Darmstadt, Germany) were used as molecular mass standards. Cell culture protein amounts equivalent to 5 μl of cell supernatant were loaded per lane.

Lipase Activity.

Lipase activity on solid media was monitored on tributyrin plates (YNBDETo medium) after 120 h of incubation as previously described (Pignede et al., 2000). Lipase activity in culture supernatants was determined by monitoring the hydrolysis of para-nitrophenyl butyrate (p-NPB), according to Fickers et al. 2003. Briefly, p-NPB dissolved in acetonitrile (20% v/v) was added dropwise into vigorously stirred 100 mM phosphate buffer, pH 7.2, containing 100 mM NaCl to a final concentration of 1 mM. The resulting solution was sonicated for 2 min on ice. The reaction was initiated by addition of 20 μl of culture supernatant (pure or diluted) to 1 mL of p-NPB solution. The release of para-nitrophenol (εPNP=0.0148 μM−1 cm−1) was monitored for three min at 405 nm (A405). Supernatant samples were diluted to obtain initial velocities below A405 of 0.3 U/min. All lipase activity assays were performed at least in duplicate from two independent cultures. One unit of lipase activity was defined as the amount of enzyme releasing 1 μmol p-NPB per minute at 25° C. and pH 7.2 (U/ml). Specific lipase activity was defined as lipase activity per gram of CDW (U/gCDW). Lipase volumetric production rate was defined as lipase activity per hour of culture (U/mL·h). Lipase specific production rate was defined as lipase activity per gram of CDW per hour of culture (U/gCDW·h).

TABLE 3List of primers used for promoter analysis usingRedStarII reporter geneSEQIDNO:PrimersSequences*Use60P1 TEF FWGGTCTCTACGGGGGTTGGCGGCGAmplification61P1 TEF RVGGTCTCTCATTCTTCGGGTGTGAGTTACfor building62P1 EYK FWGGTCTCTACGGCCCATCGATGGAAACCTTAAblockTAGGAGACTACTTCCconstruction63P1 EYK RVGGTCTCTCATTGGATCCAGTAGATGTGTAAGTG64P1 EYD FWGGGGGGTCTCTACGGCCCATCGATGGAAACCTTAATAGGAGACTACTTCC65P1 EYD RVCCCGGTCTCTCATTTGTGTATGTGTGTGTGTGTGTGTG66EYD UAS1 MluICCTTAATAGGAGACTACTTCCGACGCGTAATAddition ofFwTAGGMluI site for67EYD UAS1 MluICCTAATTACGCGTCGGAAGTAGTCTCCTATTEYD1 UASRVAAGGmutation68EYD UAS2 MluIGAACTCGATACGCGTGCCGTACTCTGGAAAFw69EYD UAS2 MluITTTCCAGAGTACGGCACGCGTATCGAGTTCRV70ZetaUp-internal-TATCTTCTGACGCATTGACCACVerification ofFWGolden Gate71URA3-internal-FWCATCCAGAGAAGCACACAGGassembly72URA3-internal-RVCAACTAACTCGTAACTATTACC73Redstar-internal-AAGACGGTGGCGTTGTTACTFW74RedStar-internal-GACTTGCTTCTTGGCCTTGTRV75Tlip2-internal-FWTGCGTTCCTCTAAGACAAATC76Tlip2-internal-RVGATTTGTCTTAGAGGAACGCATA77ZetaDown-internal-GGTAACGCCGATTCTCTCTGRV*Bold-underlined correspond to the Bsal site with the overhang in italic.

Example 1. EYK1 Promoter is Induced by Erythritol and Erythrulose

To date, two different pathways have been reported for erythritol catabolism. In a first one, erythritol is phosphorylated into erythritol-phosphate and then oxidized in erythrulose-phosphate (Barbier et al 2014). In a second one, erythritol is first converted into erythrulose before being phosphorylated into erythrulose-phosphate (Paradowska and Nikta, 2009). We have recently identified and characterized EYK1 gene (YALI0F1606g) inY. lipolytica. Disruption of the latter abolished yeast growth on erythritol medium, showing that EYK1 gene is involved in erythritol catabolism. In addition, a Δeyk1 mutant was found to accumulate L-erythrulose. From this, it has been concluded that EYK1 encode an erythrulose kinase and that erythritol catabolism inY. lipolyticafollows the pathway depicted inFIG.1.

Example 2. Expression Levels of EYK1 and EYD1 in Erythritol or Glucose Medium

In order to evaluate the expression level of genes EYK1 and EYD1 in erythritol or glucose medium, qPCR experiments were performed in the W29 wild-type strain. Shake-flask cultures were grown for 12 hours in YNB medium supplemented with either glucose or erythritol. Cells were then collected at an OD600 of 1.0 and stored at −80° C. RNA extraction and cDNA synthesis were performed as previously described (Sassi et al., 2016). Amplification was performed using primer couples ACT-F/ACT-R, EYKq-F/EYKq-R and EYDq-F/EYDq-R for actin, EYK1 and EYD1 respectively (Table 2). Gene expression levels were standardized using the expression level of actin as the reference (ΔCT method). The fold difference in EYK1 or EYD1 expression between YNB+glucose and YNB+erythritol medium was calculated as 2−ΔΔCT(Livak and Schmittgen, 2001). Samples were analyzed in duplicate. Results are onFIG.2and showed a 41-fold increase of EYK1 expression level and a 46-fold increase of EYD1 expression level when cells were grown on erythritol, indicating that this gene is induced by the presence of erythritol. (A) EYK1 value 0.8 (glucose) and 40.9 (erythritol); B) EYD1 value 0.8 (glucose) and 45.9 (erythritol)).

Example 3. Identification and Study of EYK1 and EYD1 Regulatory Elements

Gene EYK1

In order to identify the regulatory element (i.e. UAS) the EYK1 promoter region, we analysed the nucleotide sequence using the intergenic region between YALI0F01628g and YALI0F01606g (FIG.3). Blast analysis of the EYK1 promoter did not evidenced any conserved motif withinYarrowia lipolyticagenome (data not shown). Therefore, we compared the promoter region of the EYK1 gene to those present in species of theYarrowiaclade other thanYarrowia lipolytica(SEQ ID NO: 78) (namely,Yarrowia phangngensis(SEQ ID NO: 79),Yarrowia yakushimensis(SEQ ID NO: 80),Yarrowia alimentaria(SEQ ID NO: 81) andYarrowia galli(SEQ ID NO: 82) that have been recently sequenced and annotated in our laboratory. Alignment of the EYK1 promoter sequences (FIG.3) highlighted three putative conserved elements; a putative TATA box (Box TATA) and a conserved motif A (Box A) and a conserved motif B (Box B).

Gene EYD1

In order to identify the regulatory element (i.e. UAS) within the EYD1 promoter region, we analysed the intergenic region between YALI0F01650g and YALI0F01672g. Since that this intergenic region was greater than 5500 bp (5591 bp), we analysed the upstream region using the 800 bp nucleic acid sequence upstream of YALI0F01650g. Blast analysis of the EYD1 promoter did not evidenced any conserved hit withinYarrowia lipolyticagenome (data not shown). Therefore, we compared the promoter region of the EYD1 gene to those present in other species of theYarrowiaclade (namely,Yarrowia phangngensis, Yarrowia yakushimensis, Yarrowia alimentariaandYarrowia galli) that have been recently sequenced and annotated in our laboratory. Boxed ATG correspond to the start codon of the YALI0F01650g. Sequence are fromY. lipolyticaE150 (YALI; YALI0F01606g (SEQ ID NO: 83)),Yarrowia galli(YAGA, gene YAGA0A02014g (SEQ ID NO: 84)),Yarrowia phangngensis(YAPH-pEYD1, (SEQ ID NO: 85)),Yarrowia yakushimensis(YAYA-pEYD1 (SEQ ID NO: 86) andYarrowia alimentaria(YAAL-pEYD1 (SEQ ID NO: 87)). Alignment of the EYD1 promoter sequences highlighted conserved motifs observed only within the 300 bp upstream region (FIG.4) three putative conserved elements; a putative TATA box (Box TATA; GATATAWA) and a conserved motif (Box A) and a conserved motif (Box B) and a variable number of CA repeats just before the ATG.

In order to assess the regulation of the EYK1 promoter, two fragments of 450 bp and 300 bp, (EYK450 and EYK300, respectively), corresponding to the intergenic region of genes YALI0F01606g and YALI0F01628g were used to construct a reporter gene system based on a yellow fluorescent reporter protein (YFP) and the YFP fluorescence was used to quantify the promoter induction level (FIG.5).

Fragments EYK450 and EYK300 that span over 438 bp and 291 bp upstream of the EYK1 start codon (FIG.5a) were cloned JMP1427 as described in material and methods to yield plasmid JMP3934 (pEYK300) and JMP3994 (pEYK450), respectively (FIG.6). They were then used to transformY. lipolyticastrain JMY2101. Several independent transformants (3 to 6) were randomly selected for each construct and the corresponding YFP fluorescence measured during cell growth on erythritol medium (YNBE). Since no differences in YFP fluorescence level, and thus promoter induction, could be observed (data not shown), one transformant of each construct was used for further studies, namely strains JMY6245 (pEYK300-YPF) and JMY6375 (pEYK450-YFP), respectively (Table 1).

Cell growth and YFP fluorescence were quantified over time during culture of strain JMY6245 in YNB minimal media supplemented with glucose (YNBD), glycerol (YNBG), erythritol (YNBOL) and erythrulose (YNBOSE).

In medium containing erythritol (YNBOL) and erythrulose (YNBOSE), YFP fluorescence, and therefore pEYK300 induction levels were significantly higher than in the presence of glucose (YNBD) and glycerol (YNBG) (3157 and 4844 mSFU as compared to 344 and 357 mSFU, respectively) (FIG.7a). This clearly highlights that erythrulose and erythritol positively regulate pEYK300 induction by contrast to glucose and glycerol. However, the low fluorescence levels observed in YNBD and YNBG medium, suggest that pEYK300 is slightly induced by glucose and glycerol. After 60 h of culture, the fluorescence level in medium supplemented with erythrulose was 1.5-fold higher than in the presence of erythritol (3536 SFU and 5904 SFU, respectively). This suggests that erythrulose could be a better inducer than erythritol. Experiments performed with strain JMY6375 (pEYK450-YFP) in the same experimental conditions yielded to similar results (data not shown). Therefore, the pEYK300 promoter seems to encompass the different regulatory elements requested for gene expression (UAS and URS).

In order to assess the strength of pEYK300 induction by erythritol and erythrulose, it was compared to that of the strong constitutive pTEF promoter. YFP fluorescence of strain JMY2876 (pTEF-YFP) was measured in the same experimental conditions and compared to that of strain JMY6245. As shown in Figure. 7b, pTEF expression was similar in the four media tested, with fluorescence values being 1192, 1369, 1485 and 1016 mSFU in YNBOL, YNBOSE, YNBDD and YNBG, respectively. Expression level for pEYK300 in YNBOL and YNBOSE were in average 2.6- and 3.5-fold higher than the expression level of pTEF, respectively.

The comparison of YFP fluorescence under pEYK450 and pEYK300 indicates that the TATA box may be involved in the expression of gene YALIPF01628g rather than gene YALI0F01606g. Thus, in order to determine the role of Box A and Box B in pEYK regulation, two mutated promoters, namely pEYK300aB and pEYL300Ab, were constructed as described in material and method by exchange of the ClaI-SpeI fragment. Mutation of the conserved Box A and Box B were performed by introducing a MulI site. The sequence [GGAAAGCCGCC] was replaced by [GGAACGCGTCC] and named a. The sequence [CTTGCATAATCCGATGAC] was replaced by [CTTGTACGCGTAGATGAC] and named b. This yielded to pEYK300aB and pEYK300Ab, respectively (FIG.5b). The mutated pEYK300aB and pEYK300Ab were introduced into strain JMY2101 (Po1d Leu+) to give rise to representative strains JMY6369 and JMY6372, respectively (Table 1). For strain JMY6369 carrying the pEYK300aB mutant promoter, YFP fluorescence was remarkably reduced in the presence of erythritol (YNBOL) and erythrulose (YNBOSE) (683 and 1481 mSFU, respectively) (FIG.8a). This observation suggested that the Box A is part of the upstream activating sequence (UAS1EYK1) required for the promoter induction by both erythritol and erythrulose.

On the opposite, the mean relative YFP fluorescence measured for strain JMY6372 carrying the pEYK300Ab mutated promoter (FIG.8b), was 2.4-fold higher in the presence of erythritol (YNBOL medium) than for the non-mutated pEYK300 promoter in the same conditions (8389 and 3536 SFU after 60 h, respectively). In contrast, YFP fluorescence was in the same range in the presence of erythrulose (YNBOSE medium) than that of the non-mutated promoter. Furthermore, pEYK300Ab was less repressed on glucose media as compared to pEYK300 (with a mean specific fluorescence of 718 versus 279 mSFU) suggesting that the Box B may be involved in glucose repression. This clearly demonstrate that Box A is involved in erythritol and erythrulose induction and that Box B may be involved in glucose repression since expression of the pEYK300Ab increased at the end of the culture in glucose media, which is not the case in glycerol media.

Example 4. Tandem Repeats of UAS1EYK1Increase Promoter Strength

Multicopy repeats of UAS elements upstream of a promoter have been shown to increase promoter strength (Madzak et al. 2000; Blazeck et al. 2011; Blazeck et al. 2013; three repeats of the 48 bp UAS1EYK1fragment

(GGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGC; SEQ ID NO: 13)
encompassing the Box A (in bold and underlined), upstream of the wild-type pEYK300 promoter (FIG.5c). The resulting construct was introduced into strain JMY2101 to give rise to strains JMY6681.

Promoter strength was monitored in the presence of glucose (YNBD), glycerol (YNBG), erythritol (YNBOL) and erythrulose (YNBOSE) and compared to that of pEYK300 (strain JMY6245). As shown inFIG.9a, YFP fluorescence measured for pEYK300A3B was 3.4-fold higher in average in the presence of erythritol as compared to pEYK300 (10538 mSFU and 3157 mSFU, respectively). In contrast, induction of pEYK300A3B was found similar in average in the presence of erythrulose as compared to pEYK300 (5034 mSFU and 4844 mSFU, respectively). By contrast to previous observation with pEYK300 (FIG.7a) pEYK300A3B induction level was 2.1 fold higher in average in the presence of erythritol than for erythrulose (10538 mSFU and 5034 mSFU, respectively). Similar experiments performed with strain JMY6684 (pEYK300A3b), showed that the induction profile on YNBOL were not significantly different from that of JMY6681 (pEYK300A3B) except that induction was significantly less repressed by glucose and glycerol, confirming thus previous observations (data not shown).

Since the insertion of several copies of the 48 bp UAS1EYK1encompassing the Box A resulted in a stronger promoter induction level, it could be assumed that increasing the copy number of UAS1EYK1would allow to fine tune the strength of promoter induction. Indeed, several strong synthetic hybrid promoters have been created by fusing tandem repeats of upstream activation sequence (UAS) upstream to a core promoter region. The first one (hp4d) was based on four tandem repeats of the 108 bp UAS1XPR2of the XPR2 gene upstream on the minimal LEU2 core promoter (Madzak et al, 2000). Later Blazek and coworker's constructed hybrid promoter containing up to 32 copies of UAS1XPR2of the XPR2 gene upstream on the minimal LEU2 core promoter and 16 copies of UAS1XPR2of the XPR2 gene upstream of TEF core promoters of different length (Blazek et al, 2011). Promoter strength increase with copy number of the UAS, and the best one showed a 10-fold increase expression compared to the pTEF promoter. Similar expression levels were obtained by inserting three tandem copies of the 230 bp UAS1TEFupstream of the pTEF promoter (Blazek et al, 2013) and its expression did not vary significantly with carbon source (glucose, sucrose, glycerol and oleic acid). The only strong inducible promoter is the POX2 one (Juretzek et al. 2000). Oleic acid inducible hybrid synthetic promoters were obtained comprising eight copies of UAS1xpr2upstream of the 100 bp proximal core POX2 promoter. This UAS-core promoter chimera showed a 4.2-fold higher expression level in oleic acid media than in glucose in contrast to a 2-fold higher expression level for the 8 copies of UAS1xpr2upstream of the 136 bp proximal core TEF promoter (Hussain et al. 2016). Here we showed that an hybrid promoter containing two additional tandem copies of the short 48 bp UAS1EYK1upstream of the EYK1 promoter results in a 3.3-fold stronger promoter, thus stronger erythritol/erythrulose inducible promoter may be constructed by introducing additional tandem repeats of the UAS1EYK1.

Example 5. UAS1B from XPR2 Enhanced Promoter Strength without Affecting Erythritol and Erythrulose Induction

Madzak and colleagues reported that the fusion of four tandems repeats of UAS1B of XPR2 gene upstream of a minimal promoter of the LEU2 gene (yielding the so-called hp4d hybrid promoter) allowed a significant transcriptional activity (Madzak et al. 2000). In the same line, we combined four copies of UAS1XPR2(UAS1B) with the pEYK300 promoter leading to promoters HU4EYK300 (JME3998) (FIG.5c). The latter was introduced into JMY2101 giving rise to strain JMY6380. The regulation of the pHU4EYK300 was investigated by monitoring cell growth and YFP fluorescence levels during culture of strain JMY6380 in YNB medium supplemented with erythritol (YNBOL), erythrulose (YNBOSE), glucose (YNBD) and glycerol (YNBG). As shown inFIG.9b, YFP fluorescence, and therefore promoter induction were 17.1- and 9.8-fold higher in the presence of erythritol (YNOL medium) and erythrulose (YNBOSE) than for pEYK300 promoter (54063 mSFU and 47487 mSFU as compared to 3157 mSFU and 4844 mSFU, respectively). pHU4EYK was induced in stationary phase (i.e. after 60 h of culture, 63380 SFU) on glucose media (YNBD) in contrast to pEYK300 (344 SFU). Nevertheless, pHU4EYK was not found highly expressed on glycerol media.

Example 6. Hybrid Promoter HU4EYK300 is Inducible by Erythritol and Erythrulose

The regulation of the hybrid promoter pHU4EYK300 was characterized in the presence of a mixture of glycerol/erythritol or glycerol/erythrulose. These experiments were performed at steady state in chemostat culture in YNBG medium with CSP of erythritol or erythrulose. The regulation of pHU4EYK300 was investigated in regards to the growth rate of strain JMY6380 and the composition of the culture medium, more specifically in the presence of a mixture of glycerol/erythritol or glycerol/erythrulose.

Erythritol and Erythrulose Concentration Modulate the Strength pHU4EYK300 Induction in the Presence of Glycerol

To assess the influence of inducer concentration on the regulation of pHU4EYK300, chemostat cultures of JMY6380 were performed on YNBG medium at a dilution rate of 0.2 h−1. At steady state, different amount of erythritol or erythrulose were injected in the bioreactor to reach a final concentration of 0.2 and 0.6% (hereafter 0.2 CSP and 0.6 CSP, respectively) Glycerol, erythritol, erythrulose and YFP fluorescence were monitored for 8 h after inducer addition. In all experimental conditions tested, glycerol concentration remained almost constant (i.e. 3 g/I) in the bioreactor, confirming that a steady state was maintained in those experimental conditions.

As shown inFIG.10, pHU4EYK300 induction level seems to be modulated by the inducer concentration in those experimental conditions (i.e. in the presence of glycerol). For 0.2 CSP, induction increased during the three first hours after inducer (erythritol and erythrulose) addition (FIG.10a,10c). Then after, when inducer concentration was below 1 g/L, it remained almost constant for the next six hours. By contrast, for 0.6 CSP, induction increased almost linearly during 8 h after inducer addition (FIG.10b,10d). It is worth mentioning that the amplitude of induction also seems to be correlated to the inducer concentration. The maximal YFP fluorescence and thus pHU4EYK300 induction, obtained after 8 hours of erythritol addition was higher for the 0.6 CSP than for the 0.2 CSP (1.4×103and 1.1×103RFU, respectively). Similar observations were made for erythrulose. The maximal YFP fluorescence obtained 8 hours after erythrulose addition was higher for the 0.6 CSP than for the 0.2 CSP (2.5×103and 1.1×103RFU, respectively). It could also be deduced fromFIG.10, that erythrulose yield to higher induction level than erythritol, even in the presence of 3 g/l of glycerol. These results obtained from a chemostat experiment confirm the observation made inFIG.9b, i.e. pHU4EYK300 is a strong inducible promoter, responding to erythritol and even more to erythrulose as an inducer.

Example 7. Deletion of EYK1 Enhanced pEYK Expression

A disruption cassette was constructed as described in material and methods. The disruption cassette, carrying a URA3 marker, was introduced into Po1d, yielding strain RIY147 (eyk1::URA3). The marker was then excised with pRRQ2 (Fickers et al 2003), yielding an Δeyk1 strain (RIY176, Table 1). The expression cassette carrying pEYK300-YFP-LEU2ex was then introduced into RIY176, giving rise to strain RIY180 (JMY6637). Since Δeyk1 could not grow on erythritol and erythrulose as sole carbon source, strain JMY6637 was grown in the presence of glucose or glycerol, used as energy source. Therefore, JMY6245 (pEYK300-WT) and JMY6637 (pEYK300-eyk1Δ) grown in YNBDOL (glucose, erythritol), YNBGOL (glycerol, erythritol), YNBDOSE (glucose, erythrulose), YNBGOSE (glycerol, erythrulose). Induction of the promoters was followed over time in microplates with glucose or glycerol for growth (0.25%) and with erythritol or erythrulose for induction (0.25%).

As shown inFIG.12, YFP expression in wild-type and eykΔ in the presence of erythritol occurred during the growth phase in media containing 0.25% of glucose or 0.25% of glycerol (FIG.12a,12b, respectively). YFP fluorescence at 34 h of growth being 8.3-fold and 7.8-fold higher in the EYK1 deleted strain compared to the wild-type in glucose and glycerol, respectively (25672 SFU versus 3078 SFU with glucose and 19478 SFU versus 2500 SFU with glycerol). In contrast, in presence of erythrulose, YFP expression in wild-type and Δeyk was somewhat delayed from the growth phase in media containing 0.25% of glucose or 0.25% of glycerol (FIG.12c,12d, respectively). However, YFP fluorescence being 4.9-fold and 2.6-fold higher in the deleted strain compared to the wild-type in glucose and glycerol, respectively (9106 SFU versus 2993 SFU with glucose and 7934 SFU versus 3564 SFU with glycerol).

For strain JMY6245 (pEYK300-WT), the rates of the increase of the YFP fluorescence in presence of erythritol were 97 FU/h and 83 FU/h in glucose and glycerol, respectively. While, in the mutant Δeyk, the rates of the increase of the YFP fluorescence were 10.5-fold higher (1034 FU/h and 875 FU/h in glucose and glycerol, respectively).

Similarly, in the presence of erythrulose higher induction levels were obtained for the Δeyk mutant (pEYK300-Δeyk1) as compared to the non-disrupted mutant (pEYK300-EYK1) The rate of YFP production in the mutant strain was 6.1-fold higher in glucose as compared to the wild-type strain (4000 FU/h and 347 FU/h, respectively). In the presence of glycerol, this increase was equal to and was 7.3-fold (2527 FU/h and 875 FU/h, respectively).

These results demonstrate that expression level could be further improved by using a strain deleted for the EYK1 gene. In such strain erythritol or erythrulose could be used as inducer and could be used independently having induction either during the growth phase or delayed from the growth phase.

Similarly, a disruption cassette was constructed as described in material and methods for the deletion of EYD1 gene. The disruption cassette, carrying a URA3 marker, was introduced into JMY2101, yielding strain RIY212 (eyd1::URA3). The marker was then excised with pRRQ2 (Fickers et al 2003), yielding an Δeyk1 strain (RIY225, Table 1). Such strain could also be used as recipient for gene expression or metabolic engineering using either pEYK1 or pEYK1 derivative promoters and/or pEYD1 or pEYD1 derivatives promoters.

Growth Rate has No Effect on PHU4EYK300 Induction.

Yeast cell physiology is directly influenced by the growth rate. With the aim to evaluate the influence of cell growth rate on pHU4EYK300 induction by erythritol, chemostat cultures were performed in YNBOL medium at two distinct dilution rates (i.e. 0.16 h−1and 0.08 h−1). The fluorescence levels of YFP were monitored by flow cytometry in order to assess the induction level at the single cell level. No significant difference in the promoter induction levels could be observed for the two dilution rates tested (data not shown). Indeed, the mean relative fluorescence of the cell population was equal to 8.86±0.62 0.104 RFU at D=0.16 h−1, and to 9.47±0.31 0.104 RFU at D=0.08 h−1. Moreover, cytograms showed that the cell population is homogenously induced in presence of erythritol (FIG.11).

Example 8. EYK1 Encoding Erythrulose Kinase as a Catabolic Selectable Marker for Genome Editing in the Non-Conventional YeastYarrowia lipolytica

Selectable markers are a central component of genome edition technologies. In the yeastYarrowia lipolytica, these markers are based on auxotrophy (leucine, uracil), antibiotic resistance (hygromycin B) or carbon source utilisation (SUC2) (Barth and Gaillardin, 1996). Multi-step genome editions imply the use of multi-auxotrophic strains, and as a drawback their final utilization requires to complement, at least partly, the culture medium accordingly or to render strains prototroph. Dominant markers, such as theE. colihph gene, conferring resistance to hygromycin B, could also be used. However, they remain difficult to handle in practice due to a high level of spontaneous resistance in transformed cells. Genes related to the catabolism of carbon sources, hereafter “catabolic selectable markers” (CSM), present the advantage of not being involved in essential metabolic pathways. For instance, SUC2 fromSaccharomyces cerevisiaeencoding invertase and conferring the ability of the recombinant strains to grow on sucrose has been developed as a CSM (Nicaud et al., 1989). However, its utilization is impaired by residual growth on sucrose impurities.

Here, EYK1 (YALI0F01606g) encoding an erythrulose kinase is reported as a novel CSM. Therefore, a Δeyk1 strain transformed with a DNA fragment carrying EYK1 under the control of the strong constitutive pTEF promoter could be screened for Ery+(erythritol positive) phenotype on YNB medium supplemented with erythritol (10 g/L, YNBE medium). To assess the fidelity of this novel CSM, LIP2 encoding the extracellular lipase lip2 (Pignede et al., 2000) was disrupted using EYK1 as CSM and compared to URA3 and LEU2 selectable markers for its efficiency. The disruption cassettes (DC) were constructed using a cloning-free strategy derived from the previously reported Cre-lox method (Fickers et al., 2003). This update combines directed fragment assembly based on SfiI recognition sequence (SRS) and PCR amplification. Indeed, an appropriate design of the five inner-degenerated nucleotides of SRS (i.e. GGCCNNNN↓NGGCC) allows a directed assembly of the DC constitutive elements prior its final release by PCR amplification. In a first step, the 5′ and 3′ flanking regions of the gene to be disrupted (i.e. LIP2, PLIP2and TLIP2fragments, respectively) and a selectable marker (i.e. URA3, LEU2, EYK1; rescued from JMP113, JMP114, RIP131, respectively) were amplified by PCR using primers LPR-F/LPR-R in order to introduce compatible SRS as illustrated inFIG.13A. In a second step, amplicons were purified, digested with SfiI and ligated for 10 min at 25° C. after endonuclease elimination in an equimolar ratio (0.4 μM) with T4 DNA ligase (1 U, 20 μl final volume). Then, one μl of ligation product was used as a template for PCR amplification using primer pair LIP2-PF/LIP2-TR. The final DCs (MUT, MLT, MET;FIG.13B) were purified, and used to transformY. lipolyticastrain Po1d (ura3-302, leu2-270, xpr2-322) or RIY146 (ura3-302, leu2-270, xpr2-322, eyk1::LEU2) using the lithium acetate method (Le Dall et al., 1994). Transformants were plated on YNBG (10 g/L glucose, MUT, MLT transformants) or YNBE (MET transformants) supplemented to meet the requirements of auxothrophs (Barth and Gaillardin, 1996) and grown at 30° C. Transformants carrying MET DC appeared on plates after 16 h by contrast to those carrying the auxotrophic marker that appeared between 48 and 72 h. Correctness of the disruption in strains RIY148, RIY149 and RIY201 (Table 1) was verified by analytical PCR using primer pair LPR-F/LIP2-V (FIG.13A, data not shown). Correct disruption of LIP2 was found in 50% of the transformants with Ery+phenotype while a significantly lower yield (17% and 20%, respectively) was obtained for transformants with Leu+and Ura+phenotype, respectively.

To extend the utilisation of EYK1 as a CSM, a replicative vector, allowing transient expression of the Cre recombinase for marker excision (Fickers et al., 2003), was constructed based on that selectable marker. Briefly, pTEF-EYK1 fragment was amplified from RIP131 with primer pair EYK1-AF/EYK1-KR, digested by Apa1 and Kpn1, before being cloned at the corresponding site of pRRQ2 to yield RIP132 (hosted in strain RIE132). In strain RIY147 (Po1d eyk1::URA3), URA3 marker was excised with an efficiency of 80% and 50%, respectively, by using RIP132 (Cre-EYK1) and pRRQ2 (Cre-LEU2). In strain RIY203, correctness of URA3 excision was verified by analytical PCR (FIG.14).

Through these results, EYK1 has been demonstrated as a suitable catabolic selectable marker for both targeted gene disruption and vector transformation. Compared to URA3 and LEU2 auxothrophic markers, transformants harboring EYK1 marker grow faster and marker excision was found to occur at a higher rate. Moreover, the cloning free method reported here for the construction of disruption cassettes renders genome edition inY. lipolyticamore straightforward.

Similarly, EYD1 could be used as a catabolic marker in strain RIY225 (Δeyd1) or any strain bearing a deletion in EYD1 gene.

Example 9. Construction of the New Host Strain JMY7126 for Protein Expression and Secretion Carrying Deletion of EYK1 Gene

The host strain JMY1212 is deleted for the main protease, the alkaline extracellular protease Aep encoded by the XPR2 gene and the main lipases encoded by the LIP2, LIP7 and LIP8 genes. It contains a single auxotrophy for uracil (deletion of URA3 gene) (see Table 1). Therefore, only a single expression cassette could be introduced using URA3 marker. To perform further modifications such as insertion of an additional expression cassette or to introduce a gene deletion to improve secretion, the URA3 marker must be rescued using a replicative cre-hph vector by select on YPD hygromycin plate (Fickers et al. 2003).

The new host strain JMY7126 was constructed as described inFIG.15in order to introduce two additional gene deletions.

First, in order to introduce additional auxotrophy, we deleted the LYS5 gene coding for the saccharopine dehydrogenase (Xuan et al., 1990) resulting in lysine auxotrophy. The resulting strain JMY5207 was then transformed with the pUB-cre-hph to rescue uracyl auxotrophy. Secondly, deletion of the EYK1 gene involved in the catabolism of erythritol was introduced in order to, on one hand be able to use the newly developed method of marker rescues using cre-EYK1 replicative vector (FIG.14B) recently described by Vandermies et al, (2017) and on the other hand, to use the inducible pEYK1 hybrid promoters according to Trassaert et al 2017.

The host strain JMY7126 could be therefore used for enzyme engineering as JMY1212 taking advantage of the zeta platform or used for the construction of overproducing enzymes by construction of multiple copy strains using the two auxotrophies available uracil and lysine. This new strain contains the deletion of the EYK1 gene which allows better expression and induction upon erythritol induction.

Example 10. Expression of CalB Lipase Using Erythritol Inducible Promoter pEYK3AB (Also Named pEYK300A3B in FIG.9and Equivalent to pEYK1-3AB in FIG.18)

For the construction of CalB overexpressing strains, expression cassettes pEYK3AB-CalB-URA3ex and pEYK3AB-CalB-LYS5ex were co-transformed into JMY7126 and selected on minimal YNB glucose medium. The transformants were first screened for their growth and lipase production. The transformant JMY7240, having the highest specific lipase activity, was selected for fermentation studies.

In a previous study, Trassaert and colleagues (2017) showed that the induction levels of pEYK1-derived promoters were dependent of the erythritol concentration in the culture medium, hence of the erythritol uptake by the cells. Recently, it was demonstrated that a high glycerol concentration negatively affects erythritol uptake by the cells (Carly et al., 2018). Fed-batch culture was tested to minimize glycerol concentration in the culture medium (in order to increase pEYK300A3B induction level), while providing sufficient energy to the cells. Strain JMY7240 was cultivated for 48 h in a 2 L fed-batch bioreactor, in YNBE liquid medium initially supplemented with 1 g/L glycerol. Based on anterior results (Carly et al., 2018), additional glycerol was added to the reactor at a feeding rate of 0.41 g/L·h for 24 h of culture, and then of 0.82 g/L·h for the next 24 h. These feeding values are both lower than the glycerol uptake capacity ofY. lipolyticaat the considered biomass concentration. As shown inFIG.16A, the first feeding phase (0.41 g/L·h) allowed to reach a biomass higher than 3.3 g CDW/L (OD600=11.46±0.24), while the second feeding phase (0.82 g/L·h) provided a supplement of energy to the cells in order to stimulate the production of lipase CalB. Thus, lipase CalB was efficiently accumulated in the culture medium over time (FIG.16B), and a final lipase activity of 50,012±5,123 U/mL was reached (FIG.16A). Due to the higher increase of culture medium volume through glycerol feeding during the second phase, a lower volumetric production rate than in the first phase was observed (944±448 U/mL·h versus 1,140±234 U/mL·h). Nevertheless, the specific production rate was improved from the first phase to the second phase (342.34±63.45 U/gCDW·h versus 456.63±34.23 U/gCDW·h), which proves the efficiency of the glycerol feeding strategy.

Example 11. Tandem Repeats of UAS1 EYK1 Increase Promoter Strength Both in EYK1 Wild-Type (JMY1212) and in Mutant eyk1A Strain (JMY7126) Background

Promoter Bricks Construction

The first step was to construct different bio-brick for promoter analysis that will be compatible with our Golden Gate Assembly method (Celińska et al. 2017). We used different strategies to construct promoters bricks compatible with theY. lipolyticaGGAS. Firstly, the presence of internal BsaI sites within the promoter sequence was analyzed. Depending on the number of BsaI site, either they were eliminated by PCR mutagenesis, either a synthetic DNA fragment was purchased at GeneScript Biotech. Secondly, we added BsaI sites at both end of the promoter by PCR with the overhang required for a specific position of the GGAS. We designed P1 Promoters with the upstream overhang C (ACGG) and the downstream overhang D (AATG). Third, we purified the PCR product by gel extraction, cloned them into a TOPO vector (Table 1), and selected the recombinant plasmids inE. coli. Promoter cloning in TOPO was first verified by PCR onE. colicolonies followed by a migration of the PCR product on agarose gel. Finally, DNA was extracted from positive clones and verified by sequencing. Alternatively, promoters were purchased from GeneScript Biotech as DNA fragment or cloned into GeneScript Biotech vector (See Table 1).

Creation of Expression Cassettes by Golden Gate Assemblies for Promoter Analysis.

We decided to create assemblies with the GGAS between promoters, the fluorescent protein RedStarII and the Lip2 terminator as described inFIG.17using the BsaI sites C, D and L overhang according to the protocol described previously (Celińska et al. 2017). First, an intermediate GGAS was performed using GG bio-bricks containing the promoter with the overhang C (ACGG) and D (AATG) together with the fragment carrying the RedStarII with the Lip2 terminator as a RedStarII-Tlip2 (G1-T3) fragment with the overhangs D (AATG) and L (GAGT). The destination vector GGE114 (Table 1) was used for the GGAS, it contains the chromophore RFP (red fluorescent protein) giving redE. colicolony. The three corresponding fragments were assembled by adding equimolar concentration of each fragment followed by a digestion/ligation PCR as described in Material and methods.

The description of promoter construction with the promoter name, the forward and reverse primer pair used for amplification, the template used for PCR amplification, theE. colistrain containing the corresponding Golden Gate assembly and the representativeY. lipolyticatransformant used for promoter analysis are summarized in Table 4.

TABLE 4PrimerTemplateE.coliY.lipolyticastrainPromoterForwardReverseusedstrainJMY1212JMY7126TEF1P1 TEF FWP1 TEF RVJME2928GGE085GGY037GGY109EYK1P1 EYK FWP1 EYK RVJME3934GGE238JMY7382JMY7384EYK1-2ABP1 EYK FWP1 EYK RVsynthesizedGGE0130GGY027GGY056EYK1-3ABP1 EYK FWP1 EYK RVsynthesizedGGE0104JMY7345JMY7394EYK1-4ABP1 EYK FWP1 EYK RVsynthesizedGGE0132GGY033GGY068EYK1-5ABP1 EYK FWP1 EYK RVSynthesizedGGE250JMY7390JMY7392EYK1-4AB-——SynthesizedJME4417JMY7325JMY7400Core TEFEYD1ABP1 EYD FWP1 EYD RVY.lipolyticaGGE140JMY7386JMY7388genomic DNAEYD1A*BP1 EYD FWEYD UAS1GGE140GGE172JMY7398JMY7349EYD UAS1Mlul RVMlul FWP1 EYD RVEYD1AB*P1 EYD FWEYD UAS2GGE140GGE174JMY7396JMY7351EYD UAS2Mlul RVMlul FWP1 EYD RV

The resulting sequences of promoters are summarized in Table 5.

TABLE 5Promoter sequenceSEQIDNO:PromoterSequence *88TEFGGTCTCTACGGGGGTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAGAATGAGAGACC89Core-TEFGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAGgatcccacaAATGAGAGACC90EYK1GGTCTCTACGGatcgatTGCATCTACTTTTCTCTATACTGTACGTTTCAATCTGGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCCTTGCATAATCCGATGACCTGACTAGTGCGGACAAAGACTATTATTTCGAGGCAAGGCCACCACGTACCGCGGTCCCAAACTTTTGCAAAGCTGAAAACAGCGTGGGGGTCAACGTGGATCAGAAAGAGGGGCAGATCAGCTTCTATAAGAAGCTCCTTTCCCCACAATTGGCCCACACGACACTTCTACACACTTACACATCTACTggatccATGAGAGACC91EYK1-2ABGGTCTCTACGGCGATACGCGTATCGATGCATCTACTTTTCTCTATACTGTACGTTTCAATCTGGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCCTTGCATAATCCGATGACCTGACTAGTGCGGACAAAGACTATTATTTCGAGGCAAGGCCACCACGTACCGCGGTCCCAAACTTTTGCAAAGCTGAAAACAGCGTGGGGGTCAACGTGGATCAGAAAGAGGGGCAGATCAGCTTCTATAAGAAGCTCCTTTCCCCACAATTGGCCCACACGACACTTCTACACACTTACACATCTACTggatccATGAGAGACC92EYK1-3ABGGTCTCTACGGCGATACGCGTatcgatGCATCTACTTTTCTCTATACTGTACGTTTCAATCTGGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCCTTGCATAATCCGATGACCTGACTAGTGCGGACAAAGACTATTATTTCGAGGCAAGGCCACCACGTACCGCGGTCCCAAACTTTTGCAAAGCTGAAAACAGCGTGGGGGTCAACGTGGATCAGAAAGAGGGGCAGATCAGCTTCTATAAGAAGCTCCTTTCCCCACAATTGGCCCACACGACACTTCTACACACTTACACATCTACTggatccATGAGAGACC93EYK1-4ABGGTCTCTACGGCGATACGCGTatcgatGCATCTACTTTTCTCTATACTGTACGTTTCAATCTGGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCCTTGCATAATCCGATGACCTGACTAGTGCGGACAAAGACTATTATTTCGAGGCAAGGCCACCACGTACCGCGGTCCCAAACTTTTGCAAAGCTGAAAACAGCGTGGGGGTCAACGTGGATCAGAAAGAGGGGCAGATCAGCTTCTATAAGAAGCTCCTTTCCCCACAATTGGCCCACACGACACTTCTACACACTTACACATCTACTggatccATGAGAGACC94EYK1-5ABGGTCTCTACGGCGATACGCGTatcgatGCATCTACTTTTCTCTATACTGTACGTTTCAATCTGGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCCTTGCATAATCCGATGACCTGACTAGTGCGGACAAAGACTATTATTTCGAGGCAAGGCCACCACGTACCGCGGTCCCAAACTTTTGCAAAGCTGAAAACAGCGTGGGGGTCAACGTGGATCAGAAAGAGGGGCAGATCAGCTTCTATAAGAAGCTCCTTTCCCCACAATTGGCCCACACGACACTTCTACACACTTACACATCTACTggatccATGAGAGACC95Core-EYK1CTGACTAGTGCGGACAAAGACTATTATTTCGAGGCAAGGCCACCACGTACCGCGGTCCCAAACTTTTGCAAAGCTGAAAACAGCGTGGGGGTCAACGTGGATCAGAAAGAGGGGCAGATCAGCTTCTATAAGAAGCTCCTTTCCCCACAATTGGCCCACACGACACTTCTACACACTTACACATCTACTggatccATGAGAGACC96EYK1-4AB-GGTCTCTACGGCGATACGCGTatcgatGCATCTACTTTTCTCTATACTGTACGTCore TEFTTCAATCTGGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCGGGAAGCGGAATCCCAAAAGGGAAAGCCGCCGCATTAAGCTCCACAGCCTTGCATAATCCGATGACCTGACTAGTGCGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAggatccCACAATGAGAGACC97EYD1ABGGTCTCTACGGCCCatcgatGGAAACCTTAATAGGAGACTACTTCCGTTTCCTAATTAGGACTTCCGCGACCCCAGACAAAGCGGCTTGGAGTAGGCCTCGTGTCCGGCCTAGGGCAGAAACAGCTCCGGAACTCGATTGAGAAGCCGTACTCTGGAAAGTCTAGAGGAAGTTCCAAGGTCGAGTCTCTTCGATATAAAAGGACGCCATGGAAG CTCTGTAGTTCGATATCAAATACTGACAACAGTTTCCAAACACACAAACACACACACACACACACACACACATACACACATGAGAGACC98EYD1A*BGGTCTCTACGGCCCatcgatGGAAACCTTAATAGGAGACTACTTCCGACGCGTTAATTAGGACTTCCGCGACCCCAGACAAAGCGGCTTGGAGTAGGCCTCGTGTCCGGCCTAGGGCAGAAACAGCTCCGGAACTCGATTGAGAAGCCGTACTCTGGAAAGTCTAGAGGAAGTTCCAAGGTCGAGTCTCTTCGATATAAAAGGACGCCATGGAAGCTCTGTAGTTCGATATCAAATACTGACAACAGTTTCCAAACACACAAACACACACACACACACACACACACATACACACATGAGAGACC99EYD1AB*GGTCTCTACGGCCCatcgatGGAAACCTTAATAGGAGACTACTTCCGTTTCCTAATTAGGACTTCCGCGACCCCAGACAAAGCGGCTTGGAGTAGGCCTCGTGTCCGGCCTAGGGCAGAAACAGCTCCGGAACTCGATACGCGTGCCGTACTCTGGAAAGTCTAGAGGAAGTTCCAAGGTCGAGTCTCTTCGATATAAAAGGACGCCATGGAAG CTCTGTAGTTCGATATCAAATACTGACAACAGTTTCCAAACACACAAACACACACACACACACACACACACATACACACATGAGAGACC100Core-EYD1TAGAGGAAGTTCCAAGGTCGAGTCTCTTCGATATAAAAGGACGCCATGGAAGCTCTGTAGTTCGATATCAAATACTGACAACAGTTTCCAAACACACAAACACACACACACACACACACACACATACACACATGAGAGACC* BsaI site are underlined with the 4 bp overhang bolded, MluI site is underlined for EYD1A*B et EYD1 AB*. The ClaI and BamHI are in lower case, introduced to be compatible for promoter exchange into JMP62 type vector.
Tandem Repeats of UAS1EYK1Increase Promoter Strength in Both JMY1212 and JMY7126.

We showed that promoter strength was increased with the hybrid promoter pEYK300A3B composed of three repeats of the 48 bp UAS1eyk1. Four new hybrid promoters were generated by fusing two, three, four, or five UAS1EYK1tandem elements taken from EYK1 promoter, named EYK1-2AB, EYK1-3AB, EYK1-4AB, and EYK1-5AB, respectively (FIG.18b). All these promoters were synthesized with BsaI recognition site and 4 nt overhang for Golden Gate assembly from Genescript and cloned to Genescript plasmid. Each plasmid with synthetic promoter was assembled with RedStarII and Terminator LIP2 as described above. Then, the expression cassettes with several synthetic promoters were transformed intoY. lipolyticastrain with two different genetic backgrounds, JMY1212 (EYK1) and JMY7126 (Δeyk1), respectively. Hybrid EYK1 promoter expression and strength were determined by following RedStarII expression and measuring the mean specific fluorescence rate (SFU/h) using erythritol for JMY1212 or Glucose+erythritol for JMY7126 as carbon source compared to glucose as carbon source, the results of which are in Table 6.

In strain JMY1212, activity increased slightly concomitantly to UAS1eyk1 copy number ranging from 0.54 to 4.42 SFU/h (Table 6). While SFU rate increased more significantly on erythritol medium, increasing from 2.28 SFU/h for EYK1 (one copy) up to 48.12 SFU/h for EYK1-5AB (5 copies). The fold induction also increased from 4.3-fold up to 19.0-fold. The optimum been observed for EYK1-4AB. In this growth condition, on glucose media EYK1 present a low expression, 0.54 SFU/h compared to TEF promoter which as an activity of 67.16 SFU/h. While in erythritol medium TEF promoter as a similar strength than on glucose, 65.42 SFU/h and EYK1-4AB is 48.12 SFU/h. Thus, the EYK hybrid promoter has a similar activity in inducible condition than the TEF promoter and has the strong advantage to be inducible.

In strain JMY7126, activity also increased concomitantly to UAS1eyk1 copy number ranging from 0.76 up to 13.15 SFU/h (Table 6). While SFU rate increased more significantly on erythritol media increasing from 7.13 for EYK1 (one copy) up to 90.15 for EYK1-5AB (5 copies).

The fold induction also increased from 9.4-fold up to 45.8-fold. The optimum been observed for EYK1-2AB. In this growth condition, on glucose media EYK1 present a low expression, 0.76 SFU/h compared to TEF promoter which as an activity of 24.11 SFU/h. While in erythritol medium TEF promoter as a slightly reduced strength, 17.45 SFU/h and EYK1-5AB is 90.15 SFU/h. In this condition and this strain background (strain deleted for EYK1 gene), the EYK1 hybrid promoter surpasses the TEF promoter being 5.16-fold stronger.

Example 12. Both UAS1EYD1and UAS2EYD1Give Rise to Inducible Promoter in Both EYK1 Wild-Type (JMY1212) and in Mutant eyk1A Strain (JMY7126) Background

Two putative regulatory elements for the expression and regulation of the EYD1 gene (YALI0F01650g) by erythritol have been found by comparing the upstream DNA sequences of EYD1 homologs of theYarrowiaclade. We thus identified two-conserved consensus sequences of [ANTTNNNTTTCCNNATNNGG] (within UAS1EYD1of sequence SEQ ID NO: 101 AAACCTTAATAGGAGACTACTTCCGTTTCCTAATTAGGACTTCCGCGACCCC) and [CGGNNCTNNATTGAGAANNC] (within UAS2EYD1of sequence SEQ ID NO: 102=GGGCAGAAACAGCTCCGGAACTCGATTGAGAAGCCGTACTCTGGAAAGTC) within a 0.3 kb promoter region (FIG.4). We analyzed both the expression and induction fold of the WT and mutated promoters (FIG.18d). Mutation of the conserved (UAS1EYD1) and (UAS2EYD1) were performed by introducing a MluI site. The Box A, [ACTTCCGTTTCCTAATTAGG] was replaced by [ACTTCCGACGCGTAATTAGG] and named A*. The Box B, [CGGAACTCGATTGAGAAGCC] was replaced by [CGGAACTCGATACGCGTGCC] and named motif B*. This yielded to EYD1A*B and EYD1AB* promoters, respectively. Promoter strength and induction level was compared with the EYK1 and EYD1 promoter in the EYK1 Wild-type (JMY1212) and in mutant Δeyk1 strain (JMY7126) background (Table 6).

In strain JMY1212, pEYD1 allowed similar level of expression of RedStarII on glucose medium, 0.85 SFU/h, compared with the 0.54 SFU/h for pEYK1 in the same media. This promoter is also induced by erythritol giving 11.5 SFU/h compared with the 2.28 SFU/h for EYK1 (Table 6). Mutation of Box A completely abolish the expression of RedStarII on glucose medium, while it remains slightly expressed on erythritol giving 0.16 SFU/h, thus indicating that UAS1EYD1was important for expression and induction. On the opposite, mutation of Box B resulted only in a 2-fold reduction of RedStarII expression on glucose medium (0.43 SFU/h), while it remains more expressed on erythritol giving 2.57 SFU/h, thus indicating that UAS2EYD1was less important for expression and induction (Table 6). In contrast, unexpected expression level and fold induction were observed in JMY7126 which contains a deletion of the EYK1 gene on glucose+erythritol medium (Table 6). While expression on glucose medium, expression level remains low, at about 0.54 SFU/h, EYD1 promoter displayed greater expression level ranging from 245.27 to 457.51 SFU/h on glucose+erythritol media showing a tremendous fold induction ranging from 357.6 to 896.1-fold. Thus, indicating that both UAS1EYD1and UAS2EYD1were important for expression and induction in this genetic background and growth condition.

TABLE 6Overall summary of promoter expression and induction level in the two strains.JMY1212JMY7126FoldGlucose +FoldPromoterGlucoseErythritolchangeGlucoseErythritolchangeTEF67.16 ± 3.8765.42 + 0.171.024.11 ± 1.8817.45 ± 0.390.7EYK10.54 ± 0.232.28 ± 0.044.30.76 ± 0.137.13 ± 0.519.4EYK1-2AB2.63 ± 0.3815.55 ± 0.555.91.41 ± 0.5764.48 ± 0.4945.8EYK1-3AB1.68 ± 1.4426.76 ± 0.3815.93.23 ± 1.3984.41 ± 4.5526.1EYK1-4AB2.39 ± 0.8845.50 ± 2.7019.08.18 ± 0.0784.29 ± 5.2110.3EYK1-5AB4.42 ± 0.0948.12 ± 3.4310.913.15 ± 0.8190.15 ± 0.306.9EYK1-4AB-23.57 ± 1.3780.14 ± 7.063.435.53 ± 3.73340.52 ± 16.459.6coreTEFEYD1AB0.85 ± 0.5411.50 ± 0.2513.40.67 ± 1.52457.51 ± 11.37682.5EYD1A*B—0.16 ± 0.32—0.54 ± 0.88194.50 ± 11.50357.6EYD1AB*0.43 ± 1.092.57 ± 0.665.90.27 ± 0.15245.27 ± 14.56896.1

CONCLUSIONS

Several groups have constructed hybrid promoters based on combination of tandem repeats of upstream activating sequence (UAS), TATA box and core promoter for gene expression inYarrowia lipolytica(Madzak et al. 2000; Blazeck et al. 2011; Blazeck et al. 2013; Hussain et al. 2016). This gave rise to hybrid promoters with various strengths, up to 10-fold higher expression than the constitutive pTEF promoter (Muller et al, 1998). This later one being a constitutive strong promoter commonly used for gene expression and for promoter strength comparison. Among them they are few strong inducible promoters such as ICL1, LIP2, POX2 (Juretzek et al 2000, Pignede et al 2000, Sassi et al 2016). The LIP2 and POX2 promoters are inducible by oleic acid which has the drawback to require oil emulsion for induction. The inventors have identified a short nucleotide sequence acting as an upstream activating sequence may be conferring inducibility by erythritol or by erythrulose. The present invention provides new promoters allowing at least a 10-fold higher expression than the pTEF promoter. This open the path to the design of new synthetic promoters containing UASEYKand/or URSEYKwith higher tandem repeats number or with various core promoters to further wide the expression range and the induction profiles.

The promoters of the invention are poorly induced by glucose or glycerol. They could be induced by erythritol or by erythrulose with a tremendous advantage of being dose dependent thus allowing fine tuning of induction which will permit to modulate the degrees of expression that could be obtained. The inducible promoters and the nucleotide sequence according to the invention contained therein, expand the parts available for protein synthesis and for the development of tools for genetic engineering such as additional marker for gene deletion or marker rescue and for inducible expression of genes, in particular for genome editing. The present invention could be also a powerful tool for fundamental research.

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