Methods for producing fatty alcohols and applications thereof

Abstract

We have shown that cprA expression leads to the production of outer membrane vesicles (OMVs) that block autophagic flux and have a greater capacity to activate the non-canonical inflammasome pathway. In a murine model of sepsis, a P. aeruginosa strain deleted for cprA was less virulent than the wild-type strain. These results demonstrate the important role of CprA in the pathogenicity of P. aeruginosa. It is worth noting that CprA is also a functional ortholog of HlyF, which is encoded by virulence plasmids of Escherichia coli. We have shown that other cryptic SDRs encoded by mammalian and plant pathogens, such as Yersinia pestis and Ralstonia solanacearum are functional orthologs of CprA and HlyF. These SDRs also induce the production of OMVs which block autophagic flux. This study uncovers a new family of virulence determinants in Gram-negative bacteria, offering potential for innovative therapeutic interventions and deeper insights into bacterial pathogenesis. The present invention relates to a method for producing fatty alcohols in a medium comprising HlyF protein, or an ortholog of HlyF protein, and fatty acyl-CoA.

Description

[0001] METHODS FOR PRODUCING FATTY ALCOHOLS AND APPLICATIONS THEREOF

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a method for producing fatty alcohols in a medium, said medium comprising HlyF protein, or an ortholog of HlyF protein, and fatty acyl-CoA.

[0004] BACKGROUND OF THE INVENTION:

[0005] Pseudomonas aeruginosa is a leading cause of chronic and healthcare-associated infections, exhibiting alarming levels of antimicrobial resistance, morbidity and mortality1. Contributing significantly to global bacterial fatalities, P. aeruginosa ranks among the top five pathogens, accounting for half of these deaths2. The escalation of this threat is propelled by the pathogen’s remarkable ability to develop resistance through chromosomal mutations and the rising prevalence of transferable resistance determinants3. Annually, over 300,000 deaths are attributed to antibiotic-resistant P. aeruginosa infections, warranting urgent attention from the WHO for novel research and development4. Polymyxins (i.e. polymyxin B and colistin), represent a crucial class of cyclic lipopeptides antibiotics, that maintain efficacy against extensively-drug resistant P. aeruginosa5. Among the proteins involved in polymyxin resistance, PA14 44311 (accession number uniport KB A0A0H2ZLT3), also known as CprA (for cationic peptide resistance), emerges as a hypothetical protein playing a role in P. aeruginosa resistance to polymyxins and antimicrobial peptides6. The expression of cprA is positively regulated by the two-component system PmrAB, a key regulator of LPS modifications involved in polymyxin resistance in / < aeruginosa1, but also indirectly by others two-component systems such as ParRS, CprRS and PhoPQ8–10. Activation of PmrB is triggered by various signals including polymyxins, antimicrobial peptides and low Mg2+7,11. The predicted CprA structure is typical of an extended short-chain dehydrogenase / reductase (SDR) family member12. Despite this insight, the function and substrate of CprA remain unknown. However, CprA exhibits homology with E. coli hemolysin F (HlyF), a protein which is encoded by a virulence plasmid found in various E. coli pathotypes and in Salmonella enterica serovar Kentucky13,14. It is important to note that HlyF itself lacks hemolytic activity but instead acts as a cytoplasmic enzyme triggering the formation of outer membrane vesicles (OMVs), capable of delivering ClyA, a bona fide hemolysin13. The initial assumption that HlyF was a hemolysinwas based on this phenotype15. Recently, we demonstrated that HlyF induces the formation of OMVs that not only transport various toxins but also have the intrinsic ability to block autophagic flux and exacerbate inflammasome activation in host cells16–19.

[0006] SUMMARY OF THE INVENTION:

[0007] The invention is defined by the claims. In particular, the present invention relates to a method for producing fatty alcohols in a medium, said medium comprising HlyF protein, or an ortholog of HlyF protein, and fatty acyl-CoA.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] Here, the Inventors demonstrate that CprA from P. aeruginosa exhibits similar properties to HlyF. Furthermore, their study reveals that functional HlyF / CprA orthologs are also encoded by various Gram-negative bacteria. This constitutes a newly identified family of virulence factors and of bacterial enzymes with particular properties.

[0010] In a first aspect, the present invention relates to the use of hemolysin F (hlyF) gene, of an ortholog of hlyF gene, of HlyF protein or of an ortholog of HlyF protein to produce fatty alcohols. In particular, the present invention relates to a method for producing fatty alcohols in a medium, said medium comprising HlyF protein, or an ortholog of HlyF protein, and fatty acyl-CoA.

[0011] In some embodiments, the present invention relates to a method for producing fatty alcohols, the method comprising the step of expressing or overexpressing hemolysin F (hlyF) gene or an ortholog of hlyF gene in an expression system, said hlyF gene or ortholog of hlyF gene encoding respectively a HlyF protein or an ortholog of HlyF protein.

[0012] As used herein, the term “expression system” refers to a system designed to produce a biological molecule (e.g. HlyF protein or an ortholog of HlyF protein) encoded by a gene. The expression system typically includes genetic material encoding the protein (e.g. hlyF gene or an ortholog of hlyF gene), regulatory elements for controlling gene expression and a molecular machinery.In some embodiments, the expression system is a heterologous production system. As used herein, the term “heterologous production system” refers to a system wherein a gene of interest is introduced into a living cell (e.g. microorganism, prokaryotic organism, bacteria, yeast, fungi, algae, artificially engineered microorganisms, eucaryote cells, plant cells, mammalian cells, insect cells) that does not naturally contains or expresses the gene of interest (e.g. hlyF gene or an ortholog of hlyF gene) in order to produce the corresponding protein (e.g. HlyF protein or an ortholog of HlyF protein). In some embodiments, the heterologous production system is a host organism selected from the list comprising Escherichia coli, Vibrio natriegens, Saccharomyces cerevisiae, Acinetobacter baylii, Yarrowia lipolytica, Pichia pastoris, Kluyveromyces marxianus, Lipomyces starkeyi, Candida tropicalis, Schizosaccharomyces pombe, Rhodotorula toruloides, Zygosaccharomyces bailli, Debaryomyces hansenii, Chlamydomonas reinhardtii, Phaeodactylum tricornutum, Nannochloropsis spp, Botryococcus braunii, Dunaliella salina, Chlorella spp, Isochrysis galbana, Scenedesmus obliquus, Tetraselmis spp, Auxenochlorella protothecoides, Pseudomonas putida, Clostridium acetobutylicum, Synechococcus elongatus, Ralstonia eutropha, Bacillus subtilis, Thermoanaerobacterium thermosaccharolyticum, Zymomonas mobilis, Corynebacterium glutamicum, Methanosarcina barker or Halomonas sp.

[0013] In some embodiments, the expression system is a yeast. As example, the method can comprise a step of expressing a gene encoding HlyF or an ortholog of HlyF with a codon bias optimized if necessary for the yeast strain through an expression system suitable for production. The yeast can be chosen from the commonly used hosts for production, e.g. Saccharomyces cerevisiae, Pichia pastoris or Yarrowia lipolytica. In particular, the yeast can be Y. lipolytica for its capacity to accumulate large amounts of lipids. Expression of HlyF and orthologs in Y. lipolytica can be carried out by use of a replicative plasmid for episomal expression (e.g. CEN plasmid), or integration of the coding gene and promoter directly in the chromosome (e.g. via JMP vector). The promoter inducing expression of the gene can either be constitutive (e.g. TEF and derivatives with UAS activating sequences for modulating expression) or inducible (e.g. POX). Additionally, the sequence coding for HlyF or orthologs could possess a N-terminal sequence (e.g. PTS2) or a C-terminal (e.g. PTS1) for peroxisome addressing. The strain of Y. lipolytica used could be further engineered for maximal production of lipids. For example, the chassis could be engineered by i) deletion of one or more lipid storage enzymes (e.g. diacylglycerol acyl transferase DGAT1 / 2, sterol acyl-CoA transferase ARE1,phospholipid:diacylglycerol acyltransferase LRO1), ii) deletion of fatty acid and derivatives degradation pathways (e.g. acyl-coenzyme A oxidases P0X1-6, fatty alcohol oxydase, alcohol dehydrogenases, Fatty aldehyde dehydrogenases), iii) overexpression of enzymes regulating the acyl-CoA / malonyl-CoA pool in the yeast (e.g. ATP-citrate lyase, acetyl-CoA synthase, acetylCoA carboxylase). Cultures for lipid production can be done in conditions favouring lipid accumulation (e.g. high C / N ratio, carbon source either glucose or glycerol). Solvent-mediated extraction fermentation could be used to favour excretion of the active lipid derivative in the culture medium. Dodecane or decane can be used for such purpose. If intracellular, lipids can be extracted from pelleted cells. For example, extraction could be done by addition of CH₂Cl₂ and anhydrous methanolic HC1 solution, and further incubated at 50°C for 3 h. After the incubation, water / hexane (1: 1) should be added and the whole suspension vortexed vigorously before a centrifugation step to separate organic and water layers. In another method, lipids could be extracted by incubation at room temperature of the pelleted cells with MeOH :CHCl₃ with a ratio 1:2 for Ih and further addition of H₂O and incubation 10 minutes. The mixture corresponding to MeOH :CHCl₃: H₂O with a ratio 1:2: 1 is then centrifuged to separate organic and water layers. Lipids composition of the organic layer, containing fatty alcohols and fatty acids or fatty acid methyl esters (FAME) depending on the protocol used for extraction and further trans-methylation, can be controlled by gas-chromatography coupled with mass spectrometry with suitable methods. As example Munkajohnpong, Pobthum et al. also describe methods to produce fatty alcohols (Munkajohnpong, Pobthum et al. “Fatty alcohol production: an opportunity of bioprocess.” Biofuels 14 (2020)).

[0014] In some embodiments, the expression system is a bacterium. As used herein, the term “bacteria” encompasses both Gram-positive and Gram-negative bacteria. In some embodiments, the bacterium is a non-pathogenic bacterium. In some embodiments, the bacterium is from the genera Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia and Lactobacillus. In some embodiments, the bacterium is selected from the list comprising or consisting in: Lactococcus lactis, Micrococcus luteus, Bacillus subtilis, Bacillus coagulens, Lactobacillus acidophilus, Lactobacillus plantarum, Streptomyces griseus, Streptomyces coelicolor, Staphylococcus epidermitis, Pediococcus acidilactici, Leuconostoc mesenteroides, Bacillus thuringiensis, Bacillus cereus, Propionibacterium freudenreichii, Lactobacillus rhamnosus, Carnobacterium piscicola, Corynebacterium glutamicum, Streptomycesavermitilis, Kocuria rhizophila, Lactococcus cremoris, Brevibacillus brevis, Bacillus megaterium, Lactobacillus casei, Lactobacillus delbrueckii, Geobacillus stearothermophilus, Saccharopolyspora erythrea, Nocardia brasiliensis, Clostridium acetobutylcum, Escherichia coli, Vibrio natriegens, Salmonella enterica Kentucky, Acinetobacter baumannii, Bordetella pertussis, Vibrio cholerae, Neisseria meningitis, Shigella flexneri, Cedecea neteri, Escherichia albertii, Enterobacter clocae, Klebsiella michiganensis, Klebsiella pneumoniae, Raoultella ornithinolytica, Klebsiella aerogenes, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter sakazakii, Serratia marcescens, Serratia odorifera, Yersinia enter ocolitica, Yersinia pseudotuberculosis, Yersinia pestis, Dickeya zeae, Dickeya dadantii, Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, Pseudomonas oryziphila, Pseudomonas mosselli, Pseudomonas aeruginosa, Pandoraea pnomenusa, Pandoraea apista, Pandoraea sputorum, Pandoraea commovens, Ralstonia pickettii, Ralstonia solanacearum, Collimonas fungivorans or Cupriavidus brasilensis.

[0015] In some embodiments, the expression system is a Gram-negative bacterium. As used herein, the term “Gram-negative bacterium” refers to a bacterium that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. Gram-negative bacteria, having two membranes, are also known as “diderm bacteria”. Their defining characteristic is their cell envelope, which consists of a thin peptidoglycan cell wall sandwiched between an inner membrane and an outer membrane. In some embodiments, the Gram-negative bacterium is a Gammaproteobacteria, in particular Pseudomonadales or Enterobacterales; a Betaproteobacteria, in particular Burkholderiales; or a Bacteroidia. In some embodiments, the Gram-negative bacterium is selected from the list comprising or consisting in Escherichia coli, Salmonella enterica Kentucky, Acinetobacter baumannii, Bordetella pertussis, Vibrio cholerae, Neisseria meningitis, Shigella flexneri, Cedecea neteri, Escherichia albertii, Enterobacter clocae, Klebsiella michiganensis, Klebsiella pneumoniae, Raoultella ornithinolytica, Klebsiella aerogenes, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter sakazakii, Serratia marcescens, Serratia odorifera, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia pestis, Dickeya zeae, Dickeya dadantii, Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, Pseudomonas oryziphila, Pseudomonas mosselli, Pseudomonas aeruginosa, Pandoraea pnomenusa, Pandoraea apista, Pandoraea sputorum, Pandoraea commovens, Ralstonia pickettii, Ralstonia solanacearum, Collimonas fungivoransor Cupriavidus brasilensis. In some embodiments, the Gram-negative bacterium is not Escherichia coli.

[0016] In some embodiments, the Gram-negative bacterium is Escherichia sp. As used herein, the term “Escherichia” denotes a family of Gram-negative bacteria that includes E. coli, E. albertii, E. fergusonii, E.hermannii, E. marmotae, E. ruysiae, E. vulneris, E. shigae or E. hibernae.

[0017] In some embodiments, the Gram-negative bacterium is Pseudomonas sp. As used herein, the term “Pseudomonas” denotes a large family of Gram-negative bacteria that includes, P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. agarici, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P.fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridian, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdale, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthi, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. Uni, P. lutea, P. moraviensis, P. otitidis, P. pachastr ellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophila, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septic, P. simiae, P. suis, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis or P. xanthomarina. In some embodiments, the Gram-negative bacterium is Pseudomonas aeruginosa.

[0018] In some embodiments, the expression system is a cell-free expression system. As used herein, the term “cell-free expression system” refers to a system wherein a gene of interest isexpressed in vitro without the use of living cells. These systems typically comprise a cell extract containing the molecular machinery necessary for transcription and translation, along with a nucleic acid template encoding the gene of interest (e.g. hlyF gene or hlyF gene ortholog). As example, a cell extract can be prepared by cultivating a suitable organism (e.g. Escherichia coli) in an appropriate culture medium until the cells reach an exponential growth phase; lysing the cells using mechanical or chemical methods (e.g. grinding, detergent); centrifugating the lysate to collect the soluble fraction containing ribosomes, RNA, translation enzymes and other required protein synthesis components; filtering the extract to remove cell debris and retain only the soluble fraction; store the cell extract until ready to use; using a plasmid that contains the gene of interest (e.g. hlyF or the ortholog of hlyF gene) under the control of a suitable promoter (e.g. T7 promoter); forming a reaction mixture comprising the cell extract and the plasmid, nutrients (e.g. amino acids, nucleotides), transcription and translation machinery (e.g. T7 RNA polymerase), energy reagents (e.g. ATP), water and buffer (e.g. Tris-HCL). The system cell-free system allows for a controlled and scalable production under defined conditions.

[0019] As used herein, the term “Hemolysin F gene” or ‘HlyF gene” refers to a gene which encodes a protein that was attributed a putative hemolysin function and which is expressed by certain strains of the Enterobacteriaceae family. An exemplary sequence for the gene hlyF is deposited in GenBank: AF 155222.1. For example, HlyF gene is located on the pS88 plasmid from strain S88 (Accession number CAQ87216).

[0020] As used herein, the term “gene orthologs” refers to a gene common to different species. These ortholog genes encode ortholog proteins, proteins having the same function in said different species. The term encompasses both natural (e.g. naturally expressed by a microorganism) or synthetic (e.g. designed, artificially synthetised) orthologs. In some embodiments, the ortholog of hlyF gene is selected in Table 1. In some embodiments, the ortholog of hlyF gene is cprA gene, encoding CprA protein. An exemplary CprA amino acid sequence is depicted in SEQ ID NO:1

[0021] SEQ ID NO: 1 > CprA MNMHADGEQITAIDTRERILLTGATGFLGGSVSAQLIAAGHGANLSFLVRAESRQQGLERLRGNLLMHGVDETDC LALRAEQILCGDFLDTSWLARETPRLMQVERVINCAAVASFSKNPTIWPVNVDGTFAFADVLSRSKRLKRFLHVG TAMCCGPQRESPISESWEFPAAEQQLVDYTASKAEIERRMREELPGLPLWARPSIWGHRTLGCQASGSIFWVF RMGFALESFTCGLDEQIDVI PVDYCAEALIGLALKPCLGHSLYHISAGHRAACTFGEIDEAFARANGAAPVGERYRKVEVDDLKELAKSFESRIGPANPRLVLRALRLYSGFADLNYLFDNSRLLEEGISAPPRFTDYLDVCVQSSSAVS IPAQMQWDFK

[0022] In some embodiments, the ortholog of HlyF protein is CprA. In some embodiments, the ortholog of HlyF is selected in Table 1. In some embodiments, the ortholog of HlyF protein is a short-chain dehydrogenase / reductase (SDR) comprising a NAD(P)H binding site and a catalytic site. As used herein, the term “short-chain dehydrogenase / reductase” or “SDR” refers to a family of enzymes known to be NADH- or NADPH-dependant oxidoreductases. Typically, the SDR have two main specific sequences, one responsible for the specific binding of the NAD(P)H coenzyme (“NAD(P)H binding site”) and the other including amino acids directly involved in the catalysis of various SDR substrates (“catalytic site”).

[0023] In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500 residues to 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500 residues, preferentially from 360 to 385 residues. In some embodiments, the ortholog of HlyF protein exhibits at least 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid identity with HlyF protein, preferentially, 40% amino acid identity with HlyF protein, even more preferentially 35% amino acid identity with HlyF protein. In some embodiments, the ortholog of HlyF protein includes a residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site. In some embodiments, the ortholog of HlyF protein includes a residue YTXSK (SEQ ID NO:3) in said catalytic site. In some embodiments, the ortholog of HlyF protein includes a residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues and includes a residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and a residue YTXSK (SEQ ID NO:3) in said catalytic site. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibit at least 35% amino acid identity with HlyF protein and includes a residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and a residue YTXSK (SEQ ID NO:3) in said catalyticsite. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibit at least 35% amino acid identity with HlyF protein, includes a residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and a residue YTXSK (SEQ ID NO:3) in said catalytic site and includes a residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibit at least 40% amino acid identity with HlyF protein and includes a residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and a residue YTXSK (SEQ ID NO:3) in said catalytic site. In some embodiments, the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibit at least 40% amino acid identity with HlyF protein, includes a residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and a residue YTXSK (SEQ ID NO:3) in said catalytic site and includes a residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region. In some embodiments, the ortholog of HlyF protein includes a residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region and said C-ter region of said ortholog of HlyF protein exhibits at least 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid identity with said C-ter region of said HlyF protein, preferentially, 31% amino acid identity with the C-ter region of said HlyF protein. In some embodiments, the ortholog of HlyF protein possesses a fatty acyl CoA reductase (FARs) domain. In some embodiments, the ortholog of HlyF protein lacks the FAR-C superfamily domain (i.e. C-terminal domain of FAR). In some embodiments, the ortholog of HlyF protein possesses a fatty acyl CoA reductase (FARs) domain and lacks the FAR-C superfamily domain. In some embodiments, the ortholog of HlyF protein has a thioester-reductase domain.

[0024] The term “expressing” refers to the most fundamental level at which the genotype gives rise to a phenotype or to the production of the desired protein. The term “overexpressing” refers to an increased expression of a gene (e.g. increased frequency of transcription). The terms “expressing” or “overexpressing” encompass both natural or artificial expression. According to the invention, hlyF gene or hlyF gene orthologs may be expressed or overexpressed by different means. In some embodiments, endogenous hlyF gene or hlyF gene orthologs expressed by the expression system may be overexpressed. In this case, expression system genome may be modified to obtain an overexpression of the hlyF gene or hlyF gene ortholog. For example, natural promoter of the hlyF gene or hlyF gene ortholog may be modified to obtain anoverexpression of this gene. In another embodiment, the medium used to cultivate the expression system may be modified to induce an expression or an overexpression of the hlyF gene or hlyF gene ortholog. For example, a depletion of Mg2+should be done to induce this overexpression. In still another embodiment, the expression system may be transformed with hlyF gene or an ortholog of hlyF gene. For example, the expression system may be transformed with a plasmid which expresses hlyF gene or an ortholog of hlyF gene. In some embodiments, the hlyF gene or ortholog of hlyF gene can be also expressed directly into the expression system chromosome or into a plasmid in the cytoplasm of the expression system. In a particular embodiment, the transformation of the expression system with the hlyF gene or ortholog of hlyF gene can be made using a plasmid. The term “using a plasmid” denotes the fact that the hlyF gene or ortholog of hlyF gene is expressed by a plasmid which is inserted into the expression system. Alternatively, the hlyF gene or ortholog of hlyF gene can be integrated into the expression system chromosome, in a neutral or silent location, under the control of an inducible or constitutive promoter. In a particular embodiment, the plasmid is a bacteria plasmid selected from the list comprising pGEX-6P-1 or pSA10 or pK184 (see for example Schlosser-Silverman E et al., 2000). In some embodiments, the hlyF gene or ortholog of hlyF gene expression is controlled by a promoter which induced a strong expression of the hlyF gene or ortholog of hlyF gene. In some embodiments, the promoter used is the IPTG inducible promoter pTAC or the arabinose inducible promoter pBAD, or native promoter from a well-produced ortholog.

[0025] In some embodiments, the fatty alcohols are saturated fatty alcohols, unsaturated fatty alcohols, branched fatty alcohols and / or cyclic fatty alcohols. In some embodiments, the fatty alcohols are CₙH₂ₙ₊₁OH, wherein n is the number of carbon atoms in the chain. In some embodiments, the fatty alcohols are CₙH₂ₙ₋₁OH, wherein n is the number of carbon atoms in the chain. In some embodiments, n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0026] In some embodiments, the fatty alcohols are methanol, ethanol, propanol (1-propanol, 2-propanol), butanol (1 -butanol, 2-butanol), pentanol (1 -pentanol), hexanol (1 -hexanol), heptanol (1 -heptanol), octanol (1 -octanol), nonanol (1 -nonanol), decanol (1 -decanol), undecanol (1-undecanol), lauryl alcohol (1 -dodecanol), myristyl alcohol (1 -tetradecanol), cetyl alcohol (1-hexadecanol), stearyl alcohol (1 -octadecanol), arachidyl alcohol (1-eicosanol), benhenylalcohol (1 -docosanol), lignoceryl alcohol (1-tetracosanol), ceryl alcohol (1-hexacosanol), montanyl alcohol (1-octacosanol), melissyl alcohol (1-triacontanol), isooctyl alcohol (2-ethylhexanol), isostearyl alcohol, oleyl alcohol (cis-9-octadecen-l-ol), linoleyl alcohol, linolenyl alcohol, elaidyl alcohol, octadecadienol, octadecatrienol, eicosapentaenol, docosahexaenol, isooctyl alcohol (2-ethylhexanol), isostearyl alcohol, 2-butyloctanol, 2-hexyldecanol, cyclohexanol, erythritol or sorbitol. In some embodiments, the fatty alcohols are 12-hydroxy-9-octadecenoic acid, 9-hydroxy-octadec- 12-enoic acid, 1 l-hydroxy-9-dodecenoic acid, 15-hydroxy -palmitoleic acid, 9-hydroxy-octadec- 10-enoic acid, 5,9 dihydroxy-11-octadene, 9, 10-dihydroxy-octadec- 12-enoic acid, 9-hydroxy-octadec- 13 -enoic acid, 10-hydroxy-8-octadecenoic acid, 7-hydroxy-9-dodecenoic acid, 9-hydroxy-octadec- 13 -enoic acid, 10-hydroxy-8-octadecenoic acid, 7-hydroxy-9-dodecenoic acid, 9-hydroxy-l 1 -octadecenoic acid, ll-hydroxy-octadec-9-enoic acid, 13-hydroxy-octadec-9-enoic acid, 3-hydroxy-octadec-9-enoic acid, 16-hydroxy-9-octadecenoic acid, 7-hydroxy-octadec-9-enoic acid, 5-hydroxy-octadec-9 -enoic acid and / or 9-hydroxy-8-octadenoic acid.

[0027] In some embodiments, the present invention also relates to a mixture of fatty alcohols obtained with any of the methods of the invention.

[0028] As used herein, the term “mixture of fatty alcohols” refers to a mixture of alcohols derived from fatty acids. A mixture typically comprises various fatty alcohols with different chain lengths and degrees of saturation or unsaturation.

[0029] Fatty alcohols are useful in various fields, e.g. pharmaceuticals, biotechnologies, personal care and cosmetic, detergent and cleaning products, food industry, agriculture, environment, biofuels, industrial applications or material science.

[0030] Accordingly, in some embodiments, the present invention also relates to a product comprising the mixture of fatty alcohols or at least one fatty alcohol obtained by or produced with any of the methods of the invention. In some embodiments, the product is a detergent, a surfactant, an emulsifier, a lubricant, a cream, a lotion, a conditioner, a thickening agent, a stabilizer, a plasticizer, an emollient, a biofuel, a cosmetic, a fragrance fixative, a foaming agent, a wetting agent, a pharmaceutical, an antiviral agent, a drug delivery, an excipient, a preservative, a flavorcarrier, a cleaner, a paint, a coating, a printing ink, a pesticide, a pesticide adjuvant, a plant growth regulator, a fertilizer coating, a substrate for fermentation or a leather treatment.

[0031] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0032] FIGURES:

[0033] Figure 1. Amino acid homology and structural prediction of CprA and HlyF. a, Multiple sequence alignment of HlyF and CprA orthologous proteins from E. coli strain SP15 and P. aeruginosa strains PAK and PAO1 was performed using Clustal Omega. The mutant of HlyF was constructed by site-directed mutagenesis of the predicted catalytic site leading to the two substitutions Y163F and K167A. The CprA protein from P. aeruginosa strain PAO1 is truncated due to a deletion of the cytosine at the position 670 of the gene. This results in a frame shift and a premature stop codon at position 245. The residues' identity and similarity are highlighted in dark gray and light gray, respectively, b, Alphafold 2 protein structure prediction was used to show the three-dimensional structure of the HlyF and CprA proteins from E. coli and P. aeruginosa. In both panels, the NAD(P)H binding sites and catalytic sites are depicted in green and blue, respectively, c, the iPBA web server was used to compare the secondary structure of CprA (in blue) and HlyF (in pink). (https: / / www.dsimb.inserm.fr / dsimb_tools / ipba / index.php).

[0034] Figure 2. Amino acid homology between HlyF, CprA and their orthologous proteins: focus on two most distant functional orthologs in E. coli SP15 and R. solanacearum GMI1000. a, Plot of HlyF orthologs GC% on corresponding genomes GC%. b, Structural comparison of the secondary structure of the R. solanacearum GMI100 ortholog (in orange) with CprA (in blue) or HlyF (in pink), using the iPBA web server (https: / / www.dsimb.inserm.fr / dsimb_tools / ipba / index.php). c, Three-dimensional structure of the HlyF and its ortholog protein from R. solanacaerum using Alphafold 2 protein structure prediction, d, Multiple sequence alignment of HlyF and its ortholog proteins from R. solanacaerum, using Multialign Viewer in Chimera X. Matched residues highlighted in gray were obtained using the Matchmaker tool to overlay the two 3D structures with default settings, and corresponds to structures that are fit iteratively with a cutoff of 2.0 A. In b and c, theNAD(P)H binding sites, catalytic sites and residues that are 100% conserved in all orthologs of are shown in green, blue and orange, respectively.

[0035] Figure 3. Lipids from CprA producing P. aeruginosa trigger autophagosomes in HeLa cells. Western blot analysis of LC3 and actin in Hela cells treated a, for 1 hour with 50 pg / mL of OMVs, b, for 6 hours with an equivalent amount of lipids extracted from 3.33 pg of OMVs from P. aeruginosa PAK or PAK EcprA OMVs’. In both panels, bacteria were cultivated in M63 supplemented with two different concentrations of MgCh: one (0.1 mM) described to activate the PmrAB two-component regulatory system, and one (2 mM) described to inactivate the PmrAB two-component regulatory system. Non-treated cells (NT) were used as a control.

[0036] Figure 4. (a-c) Liposomes made with bacterial lipids extracted from bacterial membranes of CprA-producing bacteria inhibits autophagy. Western blot analysis of LC3 and actin in Hela cells treated a, for 1 hour with 50 pg / mL of OMVs, b, for 6 hours with an equivalent amount of lipids extracted from 3.33 pg of OMVs from P. aeruginosa PAK or PAK EcprA OMVs’, c) for 6 hours with 50 pg of lipids extracted from P. aeruginosa PAK or PAK EcprA. Non-treated cells (NT) were used as a control. In the three panels, bacteria were cultivated in M63 supplemented with 0.1 mM MgCh.

[0037] Figure 5. HlyF and ortholog produced in Yarrowia lipolytica. Western blot analysis of LC3 and actin in Hela cells treated with lipids extracted from 2 chassis strains of Yarrowia lipolytica transformed with plasmid enabling the production of either HlyF, CprA or the ortholog from Ralstonia. Lipids extracted from a HlyF -producing E. coli was used as positive control. Non treated cells and lipids extracted for chassis 1 and 2 transformed with an empty JMP62 vector (not producing any ortholog) were used as negative control.

[0038] Figure 6. a, Western blot analysis for the detection of the N-terminal tagged proteins, b, Western blot analysis for the detection of LC3 and actin.

[0039] EXAMPLE:

[0040] Material and Methods

[0041] Bacterial strains and growth conditions. Bacteria were routinely grown in lysogeny broth (LB, Lennox). For OMVs production, bacteria were grown in Terrific broth (TB) (Gibco) or in M63minimal medium (Bio Basic) supplemented with 0.5% Bacto™ casamino acids (ThermoFisher), 0.2% D+glucose solution and MgCh (0.1 or 2 mM) (Sigma Aldrich). For plasmid maintenance in E. coli, antibiotics were used at the following concentrations: 50 mg / L kanamycin sulfate, 20 mg / L gentamicin, 10 mg / L tetracycline and 30 mg / L streptomycin. For plasmid maintenance in P. aeruginosa, antibiotics were used at 500 mg / L streptomycin, 50 mg / L tetracycline, 20 mg / L gentamicin for plasmid. 10 mM L-arabinose (L-Ara) (Sigma Aldrich) was added to the bacterial culture for induction of cprA under the arabinose inducible promoter and 6 mM L-rhamnose (Sigma Aldrich) was added to the bacterial culture for the induction of cprA under the rhamnose inducible promoter.

[0042] Plasmid construction. Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega Corporation, Charbonnieres-les-Bains, France) before amplifications by PCR. Expression plasmids used in E. coli were built from plasmid pK184 using the NEB HiFi cloning Kit (NEB), with oligonucleotides. The plasmid pAGO-15 was first constructed by the insertion of a PCR product within the EcoRI and Sa A restriction sites of pK184. The insert of pAGO-15 contains the intergenic region (432 pb) upstream the start codon of the hfyF ORF and a translational fusion of a 6-His and S-tag at the N-terminus of HlyF. The upstream intergenic region and the hlyF ORF were PCR-amplified with the genome of E. coli SP15 as a matrix, and the tags were amplified from the plasmid pET-30-Ek / LIC (Novagen) with primers. The three PCR products were assembled by NEB Hifi cloning and then digested by EcoRI and SaA for cloning in pK184. The other plasmids were built from pAGO-15, after its linearization and the exchange of the hlyF ORF with the different alleles of cprA from P. aeruginosa or the different CprA orthologs. The plasmid pAGO-15 was modified with the QuikChange site-directed mutagenesis method (Agilent Technologies) to obtain pAGO-16 harboring hlyF mutated at Y163F and K167A. The expression plasmids were built from pJN105 to obtain the genes under control of the ParaBAD promoter. The N-terminally tagged hlyF and cprA were amplified by PCR from pAGO-15 and pAGO-32 with the primers, digested with Sad and PsA and ligated in pJN105 digested with the same enzymes.

[0043] Mutants and complemented P. aeruginosa strains construction. The deletion mutants of P. aeruginosa PA ScprA, VMAEcprA and PAI SpmrAB were constructed by allelic exchange20as described by Bolard etal. (2019)21. Briefly, the region upstream and downstream of the target gene were PCR amplified using primers cprAupFor / cprAupRev andcprAdownFor / cprAdownRev respectively, for PAKAc / vd and PA14AcprA and primers PCR-ipmrABCl / C2 and PCR-ipmrABC3 / C4 respectively, for PA14A / wzr 8 (data not shown). For both mutants, the PCR products were either cloned into the pKNGlOl suicide vector22by one-step sequence and ligation-independent cloning (SLIC)23for PAKAcprA, or cloned into the pCR2.1-TOPO vector (ThermoFisher) and subcloned in pKNGlOl by restriction ligation with BamPGJApa for PA\4ApmrAB. The resulting plasmids pKNGAc / vd and pKNGA / wz maintained in E. coli CCI I8A / W' and E. coli HB101, respectively, were then mobilized into PAK or PA14 by triparental mating as previously described22. Transconjugants in which the double recombination events occurred, were analyzed by PCR analysis to confirm the gene deletion24. The resulting mutant PA14A / ?mrA8 was further transcomplemented with plasmid pME6012 and its derivatives21,25. The cis-complementation of PAKAcprA, PA\4AcprA and the construction of the overproducing strain PAO1 attB: '. PRha-cprA was performed by the insertion of cprA under a rhamnose inducible promoter in the attB chromosomic site. The cprA gene and His- and S-tags were amplified by PCR from the matrix pAGO-32 using cprARhaup and cprARhadown and cloned by SLIC into the miniCTXl-rhaSR- rAaBAD26. Transfer of M\mCP -PRha-cprA in P. aeruginosa PAKAcprA and PA\4AcprA strains was carried out by triparental mating, and then was selected for the insertion of PRha-cprA in the attB site.

[0044] Purification of bacterial OMVs. Briefly, after a 8 hours culture, the supernatant was recovered after centrifugation, sterilized by filtration, concentrated through a 100-kDa MWCO tangential flow filtration unit and then ultracentrifuged. Residual and surface proteins from the sample were digested with the Pronase, and OMVs were washed out by ultracentrifugation before undergoing an iodixanol density gradient ultracentrifugation, allowing the selection of fractions containing pure OMVs. The concentration of OMVs in the suspension was correlated with the protein concentration measured by BCA protein assay.

[0045] Preparation of liposomes from OMVs lipids. Lipids from P. aeruginosa OMVs were extracted using a method adapted from Bligh and Dyer27,with the solvent ratio 2:1 MeOH:CH2Cl2. This protocol has been described to recover only lipids, without protein or LPS contaminants, the latter being retained in the aqueous phase28. Following the drying the organic phase under N2, the lipid pellets were resuspended in DMEM IX without phenol red (ref 21063, Gibco). Well-calibrated - liposomes were then formed by lipid extrusion through 200 nm, 100 nm and 50 nm membranes, according to the manufacturer instruction about the Avanti Mini ExtruderExtrusion Technique (Avanti Polar Lipids Inc). Lipid concentrations in the liposome solution were quantified by the sulfo-phospho-vanillin (SPV) lipid assay, following the method of Izard and Limberger29with Triolein as reference (ASTM® Triolein Solution, ref 44896-U, SigmaAldrich).

[0046] Eukaryotic cell culture. HeLa cells (ATCC CCL-2), GFP-LC3 HeLa cells and HeLa-Difluo hLC3 cells (Invivogen) were cultured as previously described19. THP-1 cells and associated genetically invalidated cells (Invivogen, THP1-Null2, thp-kocasp4z, thp-konlrp3, thp-kogsdmdz) were cultured and maintained in RPMI supplemented with 10% heat-inactivated FCS at 37°C, 5% CO2. THP-1 cells were first pre-stimulated with 10 ng / mL of IFNy (Invivogen, rcyec-hifng) overnight and subsequently primed with 1 pg / mL of LPS (Invivogen, tlrl-eklps) for 3 hours. Cells were washed 3 times in PBS and then seeded at the density of 106cells / mL in 24 well plates. Different amounts of specified OMVs were then incubated with cells for 18 hours before analysis.

[0047] Cell lysis and IL-ip release determination. Cell culture supernatants were harvested and centrifuged at 2000 x gfor 10 minutes to remove cellular debris. LDH release, a marker of cell lysis, was assayed in 50 pL of the supernatant by using the Pierce kit (ThermoFisher, 88953) according to the manufacturer instructions. IL-ip release was assayed by ELISA on lOOpL of the supernatant, beforehand diluted by 2, by using the Invitrogen kit (88-7261).

[0048] Transmission Electron Microscopy. Negative staining of OMVs for TEM was performed according to standard procedures. Briefly, 5 pL of OMV samples, with equivalent volume / bacteria OD600nm, were added to carbon coated copper mesh grids and stained with 1% uranyl acetate for 1 minute. The grids were examined with a Jeol JEM- 1400 (JEOL Inc, Peabody, MA, USA) at 80 kV. Images were acquired using a digital camera (Gatan Orius, Gatan Inc, Pleasanton, CA, USA).

[0049] Cryoelectron microscopy (Cryo-EM). Isolated OMVs were visualized by cryoelectron microscopy. Briefly, 3 pL of sample were deposited onto glow-discharged lacey carbon grids and placed in the thermostatic chamber of a Leica EM-GP automatic plunge freezer, set at 20°C and 95% humidity. Excess solution was removed by blotting with Whatman n°l filter paper for 2.5 seconds, and the grids were immediately flash frozen in liquid ethane at -185°C. Imageswere acquired on a Talos Arctica (Thermo Fisher Scientific) operated at 200kV in parallel beam condition with a K3 Summit direct electron detector and a BioQuantum energy filter (Gatan Inc.). Energy-filtered (20 eV slit width) image series were acquired with Digital Micrograph software at a pixel size of 0.85Å between -1 and -1.5pm defocus.

[0050] Dynamic Light Scattering (DLS). OMVs were analysed by DLS using a Zetasizer Nano ZS (Malvern Instruments Ltd.) operating at a temperature of 25°C. The OMVs were diluted 10-fold in PBS for measurements in triplicates and analysed using a spectrophotometer MACRO cuvette in crystal PS (ref BSA001, Biosigma S.p. A).

[0051] Mouse model. Male, 8-week-old, C57B16J mice (ENVIGO, France) were infected intraperitoneally with 2.107CFU. Bacteria were cultivated in LB overnight and then 8 hours in M63 minimal medium (see above) with 0.1 mM MgCh for the wild type strain and the mutant strain and with 0.1 mM MgCh and 6 mM L-rhamnose (Sigma Aldrich) for the complemented strain. The severity of the clinical signs was evaluated blindly by scoring (data not shown).

[0052] Any animal presenting at least one clinical sign with a score of 3 led to humanely sacrifice the animal in accordance with decree N°2013-18 of February 1, 2013. All the experimental procedures were carried out in accordance with the European directives for the care and use of animals for research purposes and were validated by the local and national ethics committee. Protocol number 2023040715164643. 8 hours post-infection, tissue proteins were extracted from spleen with RIPA (0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 1% Igepal in Tris-buffered saline pH = 7.4) as previously described14. Clear lysates were processed for ELISA using commercial kits (Duoset R& D Systems, Lille, France) for Interleukin- ip (IL-ip). Data are expressed as picograms of cytokines per milligram of tissue protein.

[0053] Bioinformatics analysis. The tridimensional structure predictions of HlyF and CprA proteins from E. coli and P. aeruginosa were obtained using Alphafold 2 (Galaxy Version 2.3.1+galaxy2). The proteins were visualized using ChimeraX 1.3. The protein structure comparison was performed using the iPBA web-server30. For the phylogenetic tree of P. aeruginosa we constructed a Randomized Axelerated Maximum Likelihood tree (RAxML version 8.2.11) from a Mafft alignment of 1000 CDS extracted from each genome (www.bv-brc.org). The tree has been displayed and annotated with the online tool iTol (itol.embl.de / ). To analyze the allele distribution among P. aeruginosa isolates, the nucleotide sequences of cprAfrom the strains present in the phylogenetic tree were retrieved from the National Center for Biotechnology Information (NCBI). Then, the amino acid sequence corresponding to each allele was blasted against the P. aeruginosa database on the PubMLST.org website31and from the National Reference Center for Antibiotic Resistance of aeruginosa (NRC-RA Besangon, France). To analyze the presence of CprA orthologs in others bacterial core genome a pangenome analysis of each species was performed using reliable genomes from the NCBI genome database. Genome sequences of each species were obtained from the GenBank database and selected for analysis according to the following criteria: (i) keeping full strain genome representation instead of partial, (ii) excluding strains with assembly anomaly such as chimeric, contaminated, misassembled and (iii) filtering out strains with abnormal genome length, low-quality sequences, untrustworthy as types, unverified source organisms, many frameshifted proteins, abnormal gene to sequence ratio. The 3371 genomes selected, were annotated using prokka32and are submitted to a pangenome analysis using roary (with options: -cd 100 -i 80) for each bacterial species33. In this analysis, "core genes" are defined as those conserved in all isolates. The presence of a putative PhoP binding site upstream of the ORF of CprA orthologs was assessed using the virtual footprint tool on PRODORIC34, with PhoP matrix from S. enterica, E. coli and Y. pestis.

[0054] HlyF and ortholog produced in Yarrowia lipolytica. Gene for the production of either HlyF or its orthologues from Pseudomonas or Ralstonia, with a N-terminal His-tag, was optimized for Yarrowia lipolytica codon biais and cloned into a JMP62-type plasmid under the control of the strong constitutive 8UAS-TEF promoter (Genecust, France). Plasmids were digested with Notl for chromosomal insertion into 2 chassis strains of Yarrowia lipolytica modified with a Zeta platform. Chassis 1 strain is deleted for the 6 acyl-CoA oxidases of the beta-oxidation pathway (POX1-6) and the oleate A12-desaturase FAD2. Chassis2 strain is further deleted for the two acyl - CoA: diacylglycerol acyltransferases DGAT1 and DGAT2 and the phospholipid:diacylglycerol acyltransferase LRO1. 2 transformants (annotated T1 and T2) for each construction were grown in 50 mL of YT2D5(10 g / L yeast extract, 20 g / L bactotryptone and 50 g / L glucose) at 28°C during 48 h. Cell pellets were assessed for the production of the recombinant enzymes by SDS-PAGE and anti-HisTag western blot. 50 mg of lyophilised cells of Yarrowia lipolytica producing HlyF or its orthologues (1 transformant per construction) were extracted with the solvent ratio 1:2:1 MeOH:CHCl3:H2O. Following the drying of the organic phase under N2, lipid pellets were resuspended in 200µL DMEM 1X and 50 pL were loaded in24-wells microplate containing HeLa cells (5.104cells / mL). After a 5h incubation, cells were assessed for LC3 (forms I and II, as autophagy marker) and actin (as loading control) by SDS-PAGE and western blot. Lipids extracted from a HlyF -producing E. coli was used as positive control. Non treated cells and lipids extracted for chassis 1 and 2 transformed with an empty JMP62 vector (not producing any ortholog) were used as negative control.

[0055] Modified plasmids for the production of variant forms of HlyF and CprA. Modified plasmids for the production of variant forms of HlyF and CprA were synthesized by Genecust (France). Mono variants correspond to the WT amino acid replaced by an alanine. E. coli bearing the expression system for the production of HlyF WT, CprA WT and their variants were cultured 8h in 50 mL M63 supplemented with 0.1mM MgCl2. A fraction of the cell pellets were analysed for the production of N-terminal His-tagged proteins by SDS-PAGE and western blot. The remaining cell pellets were used to extract lipids (1:2:1 MeOH:CHCl3:H2O). Following the drying of the organic phase under N2, lipid pellets were resuspended in 200 pL DMEM and 30 pL were loaded in 24-wells microplate containing HeLa cells (5.104cells / mL). After a 5h incubation, cells were assessed for LC3 and actin by SDS-PAGE and western blots. Lipids extracted from a WT HlyF- or WT CprA-producing E. coli were used as positive control. Non treated cells and the catalytically inactive double variant HlyF Y163F / K167A were used as negative control.

[0056] Statistical analysis. All statistical analyses were performed with Prism 10.1.0 software. Data are represented as the mean ± SD. Significance was determined by a one-way ANOVA (analysis of variance) for IL-ip measurement in mice spleen, by a two-way ANOVA followed with Tuckeys’ multiple comparisons testing for IL-ip secretion, cell death in human monocytes and for mice clinical score, and by a Mantel-Cox test for mice survival. ****p < 0.0001, *** p < 0.001, ** p < 0.005, * p < 0.05, ns: not significant, p > 0.05.

[0057] Results

[0058] CprA exhibits structural homology with HlyF. CprA has been described as two forms: a full-length allele found in the PAK strain, and a truncated allele found in PAO1 due to an indel mutation that creates a premature stop codon at position 2456. CprA from PAK shares 50.13% amino acid residues identity with HlyF and is predicted to belong to the short-chain dehydrogenase / reductase (SDR) superfamily. The protein features a highly conserved catalyticsite and an NAD(P)H binding site as shown in Figure la. In PAO1, the truncated form of CprA retains both the catalytic and NAD(P)H binding sites but has lost one alpha helice and eleven beta sheet, which constitute the lower portion of the protein, resulting in a change in its conformation. Notably, HlyF and CprA exhibit similar predicted three-dimensional structures and share a comparable catalytic pocket with the NAD(P)H cofactor binding site located next to the catalytic site (Figure lb and 1c). The predicted structural similarity between HlyF and full-length CprA prompted us to investigate whether CprA shares similar properties with HlyF, such as the ability to produce OMVs that modulate autophagy in eukaryotic cells.

[0059] When produced in E. coli, the function of CprA is similar to that of HlyF. To determine whether structural homology leads to conserved function, we have cloned both the truncated (from PAO1) and full-length (from PAK) variants of cprA and expressed them in E. coli. We then purified OMVs from E. coli expressing these variants and tested their ability to modulate autophagy in HeLa cells, by assessing the accumulation of autophagosomes (data not shown)19. The marker of autophagosome LC3 was assessed by immunofluorescence using a fusion LC3-GFP or analyzed by western blotting to distinguish between the LC3-I (free) and the LC3-II (associated with autophagosomes) forms (data not shown). Treatment with OMVs derived from E. coli harboring the full-length CprA from the PAK strain resulted in a presence of LC3-GFP foci (data not shown), along with an accumulation of LC3-II protein, as evidenced by western blotting (data not shown). This phenotype closely resembles that observed in cells treated with OMVs produced by E. coli producing HlyF (i.e. the accumulation of autophagosomes, as reported in David et al. (2022)19). In contrast, OMVs produced by E. coli that produce the truncated form of CprA from PAO1 failed to induce the accumulation of LC3-II or GFP-LC3 foci. The observed outcome was comparable to the results seen in untreated cells or cells treated with OMVs produced by E. coli that produce an inactive form of HlyF with the two mutations in the catalytic domain Y167F and K164A (Figure 1, data not shown).

[0060] Therefore, the activity of the full-length form of CprA is similar to that of HlyF when produced in A. coli.

[0061] OMVs from CprA-producing P. aeruginosa trigger the accumulation of autophagosomes. To evaluate the ability of P. aeruginosa expressing cprA to induce autophagosomes accumulation through the production of specific OMVs, we used the wild-type PAK strain, its cprA gene-deleted mutant, the complemented mutant strain PAK EcprA atB PRha-cprA, and the PAO1strain transformed with a plasmid expressing either the full-length or the truncated form of cprA. referred to as cprAPAKor cprAPAO1respectively. OMVs were purified from culture supernatants (data not shown) and visualized using both transmission electron microscopy (TEM) and transmission electron cryomicroscopy (Cryo-TEM) and further characterized by dynamic light scattering (DLS). The PAK strain, with or without full-length CprA, yielded heterogeneous populations of spherical OMVs with a diameter of 140-150 nm ± 50 nm (data not shown), which exhibited a single lipid bilayer, confirming their classification as bona fide OMVs, as previously reported in the literature35,36. Upon treatment with OMVs isolated from P. aeruginosa producing full-length CprA, cells showed an accumulation of GFP-LC3 foci (data not shown), which is consistent with the increase in LC3-II compared to the untreated sample (data not shown). In contrast, cells treated with OMVs derived from P. aeruginosa producing the truncated form of CprA did not show GFP-LC3 foci or elevated levels of LC3-II. Thus, full-length CprA but not its truncated variant, allows E. coli and P. aeruginosa to produce of OMVs that induce accumulation of autophagosomes in eukaryotic cells.

[0062] Pseudomonas aeruginosa producing CprA produce OMVs that impede the autophagic flux.

[0063] Autophagy is a dynamic process that involves the formation and degradation of autophagosomes. Previous research has shown that OMVs produced by E. coli producing HlyF impede the autophagic flux at the lysosome-autophagosome fusion stage, leading to the accumulation of autophagosomes19. To determine whether OMVs derived from P. aeruginosa producing a full-length CprA can impede the autophagic flux at the lysosome-autophagosome fusion stage, we used HeLa-Difluo hLC3 cells to monitor the acidification of autophagosomes (data not shown). In cells treated with OMVs from P. aeruginosa producing a full-length form of CprA (PAK or PAO1 pJN cprAPAK), we observed a pronounced colocalization of GFP and RFP-positive puncta, similar to the positive control where cells were treated with chloroquine inhibiting the lysosomal fusion (data not shown). In contrast, HeLa-Difluo hLC3 cells treated with OMVs from P. aeruginosa producing the truncated CprA (PAO1 or PAO1 pJN cprAPAO1), showed a diffuse GFP and RFP labeling with no colocalization, similar to that observed in cells deprived by incubation in HBSS, known to induce a fully functional autophagic flux (data not shown). Previous work has demonstrated that the absence of acidification of autophagosomes in cells treated with OMVs from E. coli producing HlyF, is due to a fusion defect with lysosome19. Therefore, our results strongly suggest that OMVs from P. aeruginosa producingfull-length CprA, but not the truncated variant, impede the autophagic flux by preventing the fusion between the autophagosomes and the lysosomes.

[0064] The production of CprA by P. aeruginosa results in OMVs with a greater capacity to activate the non-canonical inflammasome pathway. The non-canonical inflammasome pathway is induced by the activation of the NLRP3 inflammasome by cytosolic lipopolysaccharide (LPS). Previous research has shown that OMVs from bacteria producing HlyF were more prone to activate the non-canonical inflammasome pathway than OMVs purified from bacteria producing an inactive form of HlyF19. It was hypothesized that the expression of cprA could affect the ability of OMVs to activate the non-canonical pathway of NLRP3 inflammasome. Human monocytic THP-1 wild-type (WT) cells were used along with THP-1 cells knocked out for NRLP3 (THP1NRLP3KO), an intracellular sensor that is part of the caspase-1 activating complex, and for Caspase-4 (THP1CASP4KO) or Gasdermin-D (THP1GSDMDKO), two essential components of the non-canonical inflammasome pathway (data not shown). The cells were exposed to OMVs from P. aeruginosa PAK WT, the mutant PAK cprA and the complemented strain (PAK cprA attB:: PRha-cprA'). Althought the effect was smaller with the OMVs from the mutant PAK

[0065]

[0066] OMVs from the 3 strains induced THP-1 cell lysis (measured by LDH release) and IL-ip release (measured by ELISA) in a Caspase-4 and GSDMD-dependent manner. However, no cell death was observed in a NLRP3 -dependant manner. These results indicate that P. aeruginosa OMVs activate the non-canonical inflammasome pathway (data not shown). Exposure of cells to PAK WT and PAK cprA attB:: PRha-cprA OMVs resulted in a significant increase in cell lysis and IL-ip release compared to PAK scprA. This process was found to be dependent on Caspase-4 and GSDMD (data not shown). Our findings indicate that that OMVs from P. aeruginosa, which produce CprA, are much more prone to activate the non-canonical inflammasome pathway. This effect could be explained by the blockage of the autophagic flux, which inhibits the primary negative feedback mechanism of non-canonical inflammasome activation, as observed with OMVs from HlyF -producing E. coli19.

[0067] Role of the PmrAB two-component system in CprA-mediated OMVs production and autophagic disruption. Previous studies have shown that the PmrAB two-component system plays a regulatory role in the transcriptional activation of cprA, but the role and enzymatic activity of CprA in the bacteria was unclear at the time7,21. Given the functional similarities between the CprA allele of PA14 and PAK (data not shown), we chose to evaluate the role ofPmrAB in CprA-mediated OMVs production in PA14, a more virulent strain that causes disease in a wide range of organisms. To test whether PmrAB boosts the toxicity of OMVs, we performed a comparative analysis of the effects of OMVs obtained from the wild-type P. aeruginosa PA14 strain, its ΔpmrAB-deficient isogenic mutant harboring either the empty vector (PA14 spmrAB + pME6012) or the vector encoding the wild-type pmrAB allele (PA14 spmrAB + pABWT)21. As PmrAB is known to be activated by a low concentration of Mg2+, we cultivated the strains in a medium with a low concentration of MgCl2(0.1 mM)7. Western blot analysis showed an accumulation of LC3-II protein levels in cells exposed to OMVs from the wild-type PA14. In contrast, the strain that lacked the PmrAB did not show any accumulation of LC3-II, similarly to the untreated cells. Complementation with the wild-type pmrAB allele partially restored the ability of the mutant strain to produce OMVs that induce LC3-II accumulation (data not shown). These findings highlight the crucial role of a functional PmrAB two-component system in producing OMVs that impede the autophagic flux when environnemental conditions activate the two-component system.

[0068] PmrB gain-of-function mutations increase the production of OMVs that block autophagic flux. Exposure to colistin may select for pmrB mutants that confer a gain of polymyxin resistance, as observed in clinical isolates11’21,42. This phenomenon is attributed to the elevated mutation rate that occurs in this locus upon colistin selection43. These mutations are referred to as “gain-of-function” mutations because they activate the PmrAB regulon in the absence of environmental signals11. To investigate whether a pmrAB allele described as gain-of-function allele could also induce the production of OMVs that block the autophagic flux, we treated Hela cells with OMVs from PA14 lApmrAB complemented with either the wild-type pmrAB allele (pABWT) or with the a pmrAB harbouring the deletion ΔL172 in PmrB (pAB16.2), identified as a pmrAB gain-of-function allele21. OMVs were produced by cultivating bacteria in the minimum medium M63 supplemented with either an activating (0.1 mM) or an inhibiting (2 mM) concentration of MgCl2. We observed a clear accumulation of the LC3-II protein in HeLa cells treated with OMVs obtained from the strain complemented with the pmrAB gain-of- function allele (i.e. magnesium non-responsive allele), regardless of the concentration of MgCl2(data not shown). In contrast, OMVs from the strain complemented with the wild-type pmrAB allele (i.e. magnesium -responsive allele) blocked the autophagy flux only when the bacteria are cultivated in a MgCh concentration activating the PmrAB two-component system. The toxicity of OMVs on cells was abolished when the bacteria were cultivated with a high concentrationof MgCl2, inactivating PmrAB. This shows that pmrAB gain-of-function allele enable the constitutive production of toxic OMVs, regardless of environmental factors.

[0069] CprA is a virulence factor of P. aeruginosa in a mouse sepsis model. Subsequently, the role of CprA in the virulence of P. aeruginosa was evaluated in a mouse model of infection. Mice were intraperitoneally infected with 2.107CFU of PA14 WT, PA14 cprA mutant, or the complemented PA14 cprA attB:: PRha-cprA. The clinical scores for mice infected with the mutant strain were significantly lower than those infected with the WT or complemented strain at both 4 and 8 hours post-infection (data not shown). Between 20 and 24 hours post-infection, the WT and complemented strains caused mortality rates of 94% and 100%, respectively. In contrast, infection with the PAM cprA mutant strain resulted in a statistically significant delay in survival kinetics, with a median of 32 hours post-infection (data not shown). The lower clinical scores and the related mortality rates observed in the PAM cprA mutant compared to PAM with cprA (WT or complemented strain), may be explained by a lower level of inflammation exerted by a strain lacking cprA, as observed in THP-1 cells (data not shown).

[0070] To verify this assessment, the levels of the pro-inflammatory cytokine IL-ip induced by the non-canonical pathway of NLRP3 inflammasome were evaluated in mice spleen 8 hours postinfection. A reduction in the inflammatory response was observed in the spleen of mice infected with the PAM scprA mutant strain in comparison to the-wild type or complemented strain. This was demonstrated by the observation of reduced levels of interleukin IL-ip (data not shown). These results demonstrate that CprA contributes to the virulence of P. aeruginosa and is responsible for a stronger inflammatory response. This is likely responsible for the observed clinical scores and associated survival kinetics.

[0071] The cprA gene is ubiquitously present in P. aeruginosa. Analysis of available genomes in the databases indicated that the cprA gene and its genetic environment are part of the core genome of P. aeruginosa. This is true for the PAO1, PAK, PAM strains, as well as the more phylogenetically distant PA7 strain. The genetic organization upstream and downstream of cprA includes essential genes, such as those encoding subunits of cytochrome C oxidase which is an enzyme necessary for aerobic respiration, and the aerotaxis receptor Aer (data not shown). Out of the 5,286 genomes obtained from the PubMLST database (2,797 genomes) and the collection of the French National Reference Center for Antibiotic Resistance P. aeruginosa strains (2,489 genomes), only 17 (0.32%) displayed a truncated variant of CprA. Among these,14 had a variant identical to the strain PA01 (data not shown). Twelve major alleles were identified, and their distribution was not dependent on the P. aeruginosa clades, unlike the well-known type III secretion system exotoxins ExoU (Clade B) and ExoS (clade A), and the exolysin ExlA (most of the clade C) (data not shown). The most common allele is found in CprAATCC27853, found in 81 % of the isolates (data not shown). This allele is present in the most prevalent epidemic high-risk clones such as STI 11, ST175, ST233, ST235, ST244, ST257, ST308, ST357, and ST65444. The CprA allele from the outlier strain PA7 is notably the most genetically divergent (data not shown) compared to the other alleles. We assessed the capacity of the most frequent allele, CprAATCC2785, the allele with the highest rate of SNPs CprAPA7, and the CprAPAKand CprAPA14alleles, which have already been shown to be functional in this study, to produce toxic OMVs. The two E. coli hosting the constructs CprAATCC27853and CprAPA7led to the production of OMVs blocking autophagy, just as does CprAPA14and CprA77177(data not shown). We also confirmed the production of autophagy -blocking OMVs in clinical strains isolated from patients at the University Hospitals of Toulouse with cystic fibrosis, ear-nose-throat infections, and infections due to medical devices (data not shown). After sequencing the cprA gene in these isolates, we identified the CprAATCC27853allele in all of them, which is consistent with the prevalence of this allele among P. aeruginosa. These results show that all P. aeruginosa strains, with the exception of a few strains harboring a truncated CprA allele such as PAO1, are potentially capable of producing OMVs that inhibit autophagy.

[0072] CprA and HlyF represent a novel family of virulence determinants in Gram-negative bacteria. Given the relative taxonomic distance between E. coli and P. aeruginosa, it was of interest to ascertain whether CprA and HlyF orthologs were found in other bacteria. A number of cryptic SDRs, similar to CprA and HlyF, have been identified in the genome of Gramnegative bacteria. The potential orthologs exhibit a protein size ranging from 360 to 385 residues and a minimum of 35% amino acid identity, including the key residues of the NAD(P)H binding site (TG. TGF. G) and the catalytic site (YT. SK), which are typical of SDRs. CprA / HlyF orthologs have been found mainly in bacteria belonging to the class of the Gammaprote obacteria, and especially the orders of Pseudomodales and Enterobacterales. Orthologs are also found in the Betaproteobacteria class, especially in the Burkholderiales order (data not shown). The majority of these orthologs are encoded on the bacterial chromosome, with a similar GC% to the rest of the genome, suggesting that they belong to the core genome (Figure 2a). This is particularly true for 18 bacterial species for which severalgenomes could be used for a pangenome analysis (data not shown). Although the number of core genes varied among species, more than 93% of isolates in each species possess an HlyF ortholog, with the exception of D. dadantii and P. agglomerans, indicating that the genes are highly conserved among the genome. The absence of an HlyF ortholog in a minor portion of genomes could be explained by sequencing or assembly errors in retrieved genomes. We also observed a larger amount of P. agglomerans missing the HlyF ortholog, certainly because the orthologs is encoded by a large plasmid (110 to 500 kbp), such as those from A. coli, E. albertii, S. enterica and P. vagans (data not shown). To investigate the roles of these putative SDRs, we purified OMVs produced by E. coli expressing different orthologous proteins from pathogens that are of interest in terms of human, mammal and plant health such as S. marcescens, K. pneumoniae, Y. pestis, and R. solanacearum (data not shown). Strikingly, we consistently observed an accumulation of LC3-II protein, similar to the results obtained with OMVs from E. coli expressing hlyF or cprA (data not shown). All validated orthologs, including the two most distant forms (HlyF from E. coli SP15 and HlyF / CprA ortholog from R. solanacearum GMI1000), are functional and maintain three-dimensional structural similarity and conserved residues (Figure 2b, 2c, 2d). Furthermore, all of these orthologs possess a protein domain classified as fatty acyl CoA reductases (FARs) in the National Library of Medicine's conserved domain database (CDD)45. All these orthologs, including HlyF and CprA, are characterized by the FAR-N, SDRe domain (accession: cd05236), but lack the FAR-C superfamily domain (C-terminal domain of FAR, accession: cd0971). Fatty acyl-CoA is reduced to fatty alcohols by FARs, which can catalyze both saturated and unsaturated C16 or C 18 fatty acids46. The homology of CprA, HlyF, and their orthologs to FARs suggests that these SDRs may modify the bacteria's outer membrane composition by targeting lipids. This leads us to the hypothesis that these SDRs could modify the lipid composition of the bacterial outer membrane vesicles.

[0073] Liposomes made with bacterial lipids extracted from OMVs of CprA-producing bacteria, inhibits autophagy. We extracted lipids from the OMVs of P. aeruginosa PAK expressing or not cprA, using a method adapted from Bligh and Dyer27. Following liposomes formation trough lipid extrusion, HeLa cells were treated with a lipid amount equivalent to the dose of 3.33pg protein in OMVs (Figure 3a and 3b). We observed an accumulation of LC3-II protein in cells treated with OMVs or liposomes from P. aeruginosa PAK, but not from its isogenic mutant in cprA, suggesting that lipids extracted from OMVs are as toxic to eukaryotic cells aswhole OMVs. Although the lipids responsible for the anti -autophagic activity of OMVs have not yet been identified, we confirmed here that the component responsible for this activity is present in the lipidic fraction (see also Figure 4a, 4b and 4c wherein liposomes made with bacterial lipids extracted from bacterial membranes of CprA-producing bacteria). This result, together with the in silico analysis of the conserved enzymatic domain of HlyF and CprA, strongly supports the hypothesis that this new family of SDRs modifies lipid(s) of the bacterial membrane leading to the production of OMVs and liposomes with anti -autophagic activities.

[0074] HlyF and ortholog produced in Yarrowia lipolytica induce a lipid production blocking the autophagic flux. Lipids were extracted from 50 mg of lyophilised cells of Yarrowia lipolytica producing HlyF or its orthologues (1 transformant per construction) with the solvent ratio 1:2:1 MeOH:CHCl3:H2O. Following the drying of the organic phase under N2, lipid pellets were resuspended in 200µL DMEM 1X and 50 pL were loaded in 24-wells microplate containing HeLa cells (5.104cells / mL). After a 5h incubation, cells were assessed for LC3 (forms I and II, as autophagy marker) and actin (as loading control) by SDS-PAGE and western blot.

[0075] Lipids extracted from a HlyF -producing E. coli was used as positive control. Non treated cells were used as negative control and lipids extracted for chassis 1 and 2 transformed with an empty JMP62 vector (not producing any ortholog). These results demonstrated here that HlyF and ortholog produced in Yarrowia lipolytica induce a lipid production blocking the autophagic flux and that the HlyF expression in Yarrowia lipolytica induces a lipids synthesis with the same autophagy-blocking activity as the HlyF expression in E. coli (Figure 5).

[0076] Variants of HlyF and CprA were produced in E. coli. Western blot analysis of the cell pellet with anti-his tag showed that variants HlyF D367A, CprA D383A, HlyF K369A and CprA K385A are well produced. Cell pellets were then extracted to recover lipids. Such lipids were assessed for their potential to induce autophagy blockade. The lipids extracted from variants HlyF D367A and CprA D383A do not trigger autophagy blockade anymore whereas lipids extracted from variant HlyF K369A and CprA K385A reproduce a WT phenotype. These results indicate that the D is essential for activity (Figures 6a and 6b).TABLE:

[0077] Table 1. Non-exhaustive list of HlyF orthologs

[0078] E. cloacae ATCC13047 (CP001918) ECL_04112 / ADF63646.1

[0079] K pneumoniae SB4496 (NZ_CAAHGC010000001.1) SB04496_04318 / VGP43912.1

[0080] K aerogenes ATCC13048 GCA_003417445.1

[0081] Enterobacter aerogenes KCTC2199 (CP002824.1) EAE_01945 / AEG95324.1

[0082] S. marscesens SM39 (AP013063.1) SM39_2772 / BAO34761.1

[0083] Y pestis CO92 (AL590842.1) YPO1559 / CAL20204.1

[0084] Y pestis CO92 (NC_003143) YPO_RS08785 / WP_002211905.1

[0085] Y. pseudotuberculosis IP32953 (BX936398.1) YPTB1570 / CAH20809.1

[0086] Y. enterocolitica 8081 (AM286415.1) YE2757 / CAL12791.1

[0087] P. aeruginosa PAK(CP020659.1) Y880_0993 / ARI01029.1

[0088] P. aeruginosa PAK(LR657304.1) PAKAF_03592 / VUY45701.1

[0089] P. aeruginosa PA14 (NC_008463) cprA / WP_003140184.1

[0090] UCBPP-PA14 (CP000438.1) PA14_44311 / ABJ15632.1

[0091] P. aeruginosa PA7 (CP000744.1) PSPA7_3771 / ABR86037.1

[0092] P. aeruginosa ATCC27853 (CP015117.1) A4W92_03795 / AMX86105.1

[0093] R. solanacearum GMI100 (AL646052) RScl462 / CAD15164.1

[0094]

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Claims

CLAIMS:

1. A method of producing fatty alcohols in a medium, said medium comprising HlyF protein, or an ortholog of HlyF protein, and fatty acyl-CoA.

2. A method for producing fatty alcohols, the method comprising the step of expressing or overexpressing hemolysin F (hlyF) gene or an ortholog of hlyF gene in an expression system, said hlyF gene or ortholog of hlyF gene encoding respectively a hlyF protein or an ortholog of hlyF protein.

3. The method according to any of claims 1 or 2, wherein the ortholog of HlyF protein is a short-chain dehydrogenase / reductase (SDR) comprising aNAD(P)H binding site and a catalytic site.

4. The method according to claim 3, wherein the ortholog of HlyF protein exhibits a protein size ranging from 360 to 385 residues, exhibits at least 35% amino acid identity with HlyF protein, includes a residue TGXTGFXG (SEQ ID NO:2) in said NAD(P)H binding site and a residue YTXSK (SEQ ID NO:3) in said catalytic site and includes a residue MXXDXX (SEQ ID NO:4), preferentially MXXDXK (SEQ ID NO:5) in its C-ter region.

5. The method according to any of claims 1 to 4, wherein the ortholog of HlyF protein possesses a fatty acyl CoA reductase (FARs) domain and lacks the FAR-C superfamily domain.

6. The method according to any of claims 2 to 5, wherein the ortholog of hlyF gene is CprA gene, encoding CprA protein.

7. The method according to any of claims 2 to 6, wherein the expression system is a heterologous production system or a cell-free expression system.

8. The method according to any of claims 2 to 7, wherein the expression system is a bacteria or a yeast.

9. The method according to the claim 8 wherein the yeast is Yarrowia lipolytica.

10. The method according to any of claims 2 to 7, wherein the expression system is a Gram-negative bacterium.

11. The method according to claim 10, wherein the Gram-negative bacterium is selected from the list consisting in Escherichia coli, Salmonella enterica Kentucky, Cedecea neteri, Escherichia albertii, Enterobacter clocae, Klebsiella michiganensis, Klebsiella pneumoniae, Raoultella ornithinolytica, Klebsiella aerogenes, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter sakazakii, Serratia marcescens, Serratia odorifera, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia pestis, Dickeya zeae, Dickeya dadantii, Pantoea ananatis, Pantoea agglomerans, Pantoea vagans, Pseudomonas oryziphila, Pseudomonas mosselli, Pseudomonas aeruginosa, Pandoraea pnomenusa, Pandoraea apista, Pandoraea sputorum, Pandoraea commovens, Ralstonia pickettii, Ralstonia solanacearum, Collimonas fungivorans or Cupriavidus brasilensis.

12. The method according to any of claims 10 or 11, wherein the Gram-negative bacterium is Pseudomonas sp.

13. The method according to any of claims 10 to 12, wherein the Gram -negative bacterium is Pseudomonas aeruginosa.

14. A mixture of fatty alcohols obtained with the method according to any of claims 1 to

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