Modified yeast strains to produce long chain dicarboxylic acids

Abstract

The present invention relates to the field of producing long chain dicarboxylic acids, i.e. dicarboxylic acids having a chain length of 16 to 24 carbon atoms. Indeed, the present invention discloses genetically modified yeast strains which lack particular genes or gene products, and / or express or over-express particular genes, capable to produce significant amounts of long chain dicarboxylic acids instead of sophorolipids which are produced by their wild-type counterparts. Long chain dicarboxylic acids are valuable precursors for a variety of products such as high-grade polymers, plasticizers, coatings, corrosion inhibitors, adhesives, perfumes, or antibiotics.

Description

[0001] Modified yeast strains to produce long chain dicarboxylic acids Field of the invention 5 The present invention relates to the field of producing long chain dicarboxylic acids, i.e. dicarboxylic acids having a chain length of 16 to 24 carbon atoms. Indeed, the present invention discloses genetically modified yeast strains which lack particular genes or gene products, and / or express or over-express particular genes, capable to produce significant amounts of long chain dicarboxylic acids instead of sophorolipids which are 10 produced by their wild-type counterparts. Long chain dicarboxylic acids are valuable precursors for a variety of products such as high-grade polymers, plasticizers, coatings, corrosion inhibitors, adhesives, perfumes, or antibiotics. Background art 15 Long-chain aliphatic dicarboxylic acids (LCDA) are versatile, biodegradable, chemical intermediates of different chain length (>C14) and saturation, usable as precursors for high-grade polymers like polyesters (PE), polyamides (PA), and polyurethanes (PU), lubricants, coatings, corrosion inhibitors, perfumes, adhesives and macrolide antibiotics. 20 Today, mainly short- and medium-chain dicarboxylic acids are used. Dicarboxylic acids with longer chains (> C14) are used less frequently because they are less available and more expensive. This is due to their challenged production processes. 25 The longest dicarboxylic acid that is commercially available by chemical synthesis from fossil resources is dodecanedioic acid. The reaction starts with cyclic trimerization of butadiene, which is catalyzed using nickel or other metals, e.g. titanium. The obtained cyclododecatriene is then hydrogenated and sequentially oxidized with oxygen and nitric acid, respectively, to dodecanedioic acid (Streck and Hartig, 1990). 30 Alternative chemical production of dicarboxylic acids from renewable resources, such as fats and oils, has received more attention lately. In the review by Stempfle et al. (2016) several laboratory-scale methods that do allow the production of long and odd chain dicarboxylic acids are described. Methods such as ozonolysis, alkaline oxidation and cross-metathesis reactions are interesting alternative routes to the multistep syntheses by using a selective terminal functionalization of fatty acid derivatives from 5 plant oils that already contain the linear long-chain structure. Although these routes all show promising results, they also show similar disadvantages comprising expensive catalysts, harsh reaction conditions and the formation of unwanted side products. An industrially relevant chemical synthesis starting from plant oils is the self- metathesis of oleic acid to produce octadecenedioic acid. Two moles of oleic acid are 10 converted to one mole of 9-octadecene and one mole of 9-octadecenedioic acid. This reaction is catalyzed by a second generation, highly complex, Ruthenium-based Grubbs catalyst and has to be performed at a relatively mild 45°C under an inert atmosphere, e.g. nitrogen gas. The main disadvantage of this process is the maximal conversion yield of 50 % as half of the substrate will be converted to unwanted 15 octadecene. As this is an equilibrium reaction, a mixture of all three compounds will be obtained at the end of the process resulting in a diƯicult downstream processing (Ngo et al.2006). Besides relying in chemical synthesis methods, one can also produce dicarboxylic 20 acids and LCDA using biotechnological routes deploying yeast cell factories. The current biotechnological production consists of using a Candida tropicalis strain which converts alkanes, fatty alcohols and fatty acids to long-chain dicarboxylic acids. This process can be performed under mild conditions (1 atm and 30°C), it utilizes yeast as a less expensive catalyst and there is the possibility to produce unsaturated 25 dicarboxylic acid chains. The only side product is the yeast biomass which can be separated easily and used as cattle feed or for bio-gas production. Therefore, this process has received a lot of attention in the last 40 years and a few companies produce long-chain dicarboxylic acids in a fermentative way. Nippon Mining co. were among the first to patent a fermentative production process in 1982 (Patent US4339536). Later on, 30 the genetically engineered strains created by Picataggio et al. (1992) were applied. Note that the parental C. tropicalis strain ATCC 20336 was reclassified as C. viswanathii and that a mixed nomenclature is used (Arie et al.2000). Many eƯorts have been made to optimize the production process of dicarboxylic acids in C. tropicalis / viswanathii. This includes optimizing the medium, nitrogen concentration, aeration, feeding strategy of substrates such as glucose, glycerol, 5 carbon sources, the hydrophobic source such as alkanes, oils, fatty acids, or fatty acid derivatives like fatty acid esters. Also pH seems to be an important parameter. The production of long chain dicarboxylic acids has to be carried out at a pH above 7.5 (Lin et al.2000). At a pH higher than 7.5, there is a high transmembrane pH gradient since the intracellular pH is around 6.5 (Liu et al.2003). As a reaction the yeast cells will 10 rapidly secrete the formed dicarboxylic acids. When the medium pH is below 7.5, this secretion is much less likely to occur which will cause the accumulation of dicarboxylic acids within the cell. Hence, in most research papers, a constant pH between 7.5 and 8 is kept (Lu et al.2009). Liu et al (2004) went even further and established an optimal pH control strategy where the pH was gradually increased from 6.75 to 8.1. A severe 15 drawback linked to the cultivation at higher pH is the occurrence of soap formation and hence sever foaming problems during the fermentation process. Indeed, NaOH or KOH solutions need to be added to the fermentation broth the maintain the high pH upon secretion of dicarboxylic acids (which will obviously acidity the medium). At this high pH, the Na+and K+ions will lead to saponification (=Na / K-salt or soap formation) of the 20 triacylglycerides and free fatty acids derived from the triacylglycerides. The resulting soap formation in combination with aeration and stirring which causes excessive foaming resulting eventually in reactor overflow and inferior processes. Just as many other members of the Candida-yeast-clade, C. tropicalis / viswanathii is considered an opportunistic pathogen and is in Europe classified as a class two risk 25 group organism, meaning that for large-scale applications, safety precautions need to be taken, leading to elevated production costs. For biosafety and IP reasons, a lot of attention is given to the search for alternative microorganisms. Yet, most of them lack the excellent and dedicated production machinery to obtain commercial relevant yields or are also considered class two risk group organisms (e.g. C. guilliermondii; Table 1). 30 Also the oleaginous yeast Yarrowia lipolytica has been deployed as an LCDA producer. This yeast has the advantage of being Generally Regarded As Safe (GRAS), but again product numbers are inferior compared to C. tropicalis / viswanathii (Table 1). Another strategy is the extended metabolic engineering of platform organisms, like Saccharomyces cerevisiae and Escherichia coli, who by nature do not produce LCDAs. Hitherto this has not resulted in good microbial cell factories (Table 1). 5 Table 1: Most relevant medium- and long-chain dicarboxylic acid (DA) producers and some characteristics (based on the highest reported yields). Strain Substrate Product Amount (g / l) Reference C. tropicalis / methyl laurate C12:0 DA 66 Picataggio et al., 1992 C. viswanathii methyl myristate C14:0 DCA 210 oleic acid C18:1 DA 100 Zibek et al., 2007 C. maltosa tridecane C13:0 DA 15 Hara et al., 2001 C. cloacae lauric acid C12:0 DA 10 Green et al., 2000 C. guilliermondii oleic acid C18:1 DA na Werner et al., 2017 C. aaseri dodecane C12:0 DA na Hilmi Ibrahim et al., 2020 C. sorbophila methyl laurate C12:0 DA 92.5 Lee et al., 2018 Wickerhamiella sorbophila Y. lipolytica oleic sunflower C18:1 DA 23 Nicaud et al., 2006 oil S. cerevisiae Lauric acid (C12) na na Zimmer et al.1995 E. coli C12-fatty acid C12:0 DA 0.16 Sathesh-Pradu & Lee, C14-fatty acid 0.41 2015 Na: not available Note that most research focusses on obtaining medium-chain DA (like C12 and C14) 10 and that in general the obtained amounts for LCDA production such as C18:1 DA is lower compared to medium chain DA (see for instance C. tropicalis example in Table 1). Moreover, hitherto no non-pathogenic strain is able to produce LCDA at amounts comparable to the opportunistic C. tropicalis / viswanathii strain. Finally, as far as we know, no organism (natural or modified) has been reported to produce LCDAs with 20 15 carbon atoms or more at significant amounts. Also chemical synthesis of these very long chain dicarboxylic acids seems troublesome, although few reports describing academic eƯorts are available (Goldbach et al., 2016). To conclude, LCDAs are compounds of commercial interest. Yet, their application is hampered by ineƯicient chemical production processes, or biotechnological processes that come with restraints such as the pathogenic nature of the best producers and low yields of engineered non-pathogenic strains. Moreover, the fermentation pH is a key 5 parameter for optimal LDCA production and has to be kept around 8. For this purpose, often NaOH or KOH is added. The free fatty acids either supplied as such or derived from the triacylglycerides from vegetable oil or fatty acid esters will react with the Na+or K+ions and form a soap which causes excessive foaming resulting in potential reactor overflow and inferior processes. Finally, fewer attention has been given to the 10 development C16-C24 LCDA production processes compared to C12, although there is commercial interest in these compounds. The present invention overcomes these limitations by oƯering a non-pathogenic yeast strain engineered to produce LCDA in high amounts: up to 99.6 g / l are obtained in a 15 fermenter. Moreover, the fermentation is conducted at an optimal pH of 5.8, in this way avoiding saponification events and related foam formation that obstruct the production process. 20

[0002] Brief description of figures Fig.1: Graphical representation of the expected LCDA biosynthesis from fatty acids and its competing pathways in S. bombicola. Enzymes are indicated in capital letters (P450, cytochrome P450 monooxygenase; CPR, cytochrome P450 reductase; FAO1, fatty 5 alcohol oxidase; ADH, alcohol dehydrogenase; FALDH, fatty aldehyde dehydrogenase; FAA1, fatty acyl-CoA synthase; POX1, acyl-CoA oxidase; MFE2, multifunction enzyme type 2; POT, 3-ketoacyl-CoA thiolase; UGTA1, glucosyltransferase 1; UGTB1, glucosyltransferase 2). P450s could be involved in multiple enzymatic reactions within the ω-oxidation pathway. The three metabolic engineering strategies, blocking the 10 precursor loss at branch points, are indicated with crosses. Fig.2: Characterization of the genetically engineered strain carrying pox1 and ugta1 deletions cultivated in shakeflasks for 8 days. (A) cell growth. (B) glucose concentration in the culture medium over the cultivation time (C) the pH change during the cultivation. 15 Three replicates (n = 3) were carried out. Error bars indicate the standard deviation of the mean (SD). Fig.3: LCDA production in the pox1 and ugta1 double deletion mutant in shakeflask after 8 days. (A) LCDA detection on a TLC plate. Reference LCDAs (saturated C12-C16) 20 were used to compare the position of the synthesized LCDAs. (B) LCDA production profile of the mutant and the concentrations. Three replicates (n = 3) were carried out. Error bars indicate the standard deviation of the mean (SD). Fig. 4: Optimization of the bioprocess for LCDA production in the Δpox1Δugta1 S. 25 bombicola strain. (A) Cell growth comparison (CFU) at diƯerent pHs. (B) Glucose consumption at diƯerent pHs. (C) Time course LCDA production at diƯerent pHs during the bioprocess. Fig.5: Characterization of the genetically engineered strain carrying pox1, ugta1 and faa1 deletions compared to the parental strain with pox1 and ugta1 deletions in shake flasks for 8 days. (A) Cell growth comparison in CGQ. (B)final DCW. (C) pH profile during the cultivation. Three replicates (n = 3) were carried out. Error bars indicate the standard 5 deviation of the mean (SD). Statistical significance was calculated using the unpaired two-tailed t-test and p-values are indicated above as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001. Fig.6: LCDA production in the triple deletion strain Δpox1Δugta1Δfaa1 compared the 10 parental double deletion strain Δpox1Δugta1 in shakeflask after 8 days or 12 days. (A) DiƯerence in appearance of the culture liquid after sampling and sedimentation of the cells. (B) LCDA chain length profile and quantities for the two strains. Three replicates (n = 3) were carried out. Error bars indicate the standard deviation of the mean (SD). Statistical significance was calculated using the unpaired two-tailed t-test and p-values 15 are indicated above as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001. Fig.7: The bioprocess for LCDA production in the triple knock-out S. bombicola strain (Δpox1Δugta1Δfaa1) at pH 5.8. (A) Monitoring the cell growth (DCW). (B) Glucose concentration in a fed-batch manner. (C) Time course LCDA production during the 20 bioprocess. Fig. 8: C22:0 LCDA production in CYP52 family gene overexpression strains (Δpox1Δugta1 background). Three replicates (n = 3) were carried out. Error bars indicate the standard deviation of the mean (SD). Statistical significance was calculated using 25 the unpaired two-tailed t-test and p-values are indicated above as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001. Fig.9: Characterization of the genetically engineered strains overexpressing fatty acid elongase genes and comparison of the fatty acid elongation activities. The three fatty 30 acid elongase overexpression strains were derived from the E3 strain (Δpox1Δugta1 background). A. Cell growth comparison in CGQ for 24 h. B. Comparison of DCW after 8 days of shakeflask cultivation in Lang medium. C. Final pH of the culture broth. D. LCDA chain length profile and quantities for the strains. E. Comparison of longer LCDAs (≥C20) titers. F. Comparison of chain length specific longer LCDA production. Three replicates 5 (n = 3) were carried out. Error bars indicate the standard deviation of the mean (SD). Statistical significance was calculated using the unpaired two-tailed t-test and p-values are indicated above as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001. Fig.10: The eƯect of faa1 deletion in Ylelo2 overexpression strain on the cell growth and 10 the longer LCDA (≥C20) production. All engineered strains were derived from the E3 strain (Δpox1Δugta1 background). A. Cell growth monitoring in CGQ for 24 h. B. Comparison of DCW after 8 days of shakeflask cultivation in Lang medium. C. Final pH of the culture broth. D. LCDA chain length profile and quantities for the strains. E. C16- C18 LCDA titers. F. Comparison of longer LCDAs titers. G. Comparison of specific types 15 of longer LCDA titers. Three replicates (n = 3) were carried out. Error bars indicate the standard deviation of the mean (SD). Statistical significance was calculated using the unpaired two-tailed t-test and p-values are indicated above as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001. 20 Fig.11: The eƯect of the combination of Sbelo1 overexpression and faa1 deletion on the cell growth and the longer LCDA (≥C20) production. The engineered strains were derived from the E3 strain (Δpox1Δugta1 background), except for the triple deletion strain (^pox1^ugta1^faa1), which is referred to as ^3. A. Cell growth monitoring in CGQ for 24 h. B. Comparison of DCW after 8 days of shakeflask cultivation in Lang medium. C. Final 25 pH of the culture broth. D. LCDA chain length profile and quantities for the strains. E. C16-C18 LCDA titers. F. Comparison of longer LCDAs titers. G. Comparison of specific types of longer LCDA titers. Three replicates (n = 3) were carried out. Error bars indicate the standard deviation of the mean (SD). Statistical significance was calculated using the unpaired two-tailed t-test and p-values are indicated above as ns (not significant), 30 *p < 0.05, **p < 0.01, ***p < 0.001. Description of the invention The yeast Starmerella bombicola is well-known for its production of sophorolipids, a biological detergent that is being commercialized by several companies. The yeast possesses excellent abilities to deal with lipidic or hydrophobic substrates. For 5 sophorolipid production, high concentrations of fat or oil (= triacylglycerides) are added and these are readily hydrolyzed by lipases, taken up by the cell and incorporated into sophorolipids to come to high product concentrations and productivity. Surprisingly, this dedicated targeting to the sophorolipid biosynthetic pathway can be re-shuttled to other non-natural processes. For this, first sophorolipid synthesis needs to be disabled 10 (Fig.1). While this traditionally is done by disabling the first specific enzymatic step of the sophorolipid biosynthetic pathway: terminal or subterminal oxidation of a fatty acids by the cytochrome P450 monooxygenase CYP52M1, we decided to keep this step and disable the second enzyme of sophorolipid synthesis: the UDP-glucose dependent glucosyltransferase I. Surprisingly, the native activity of the CYP52M1 enzyme - 15 normally also subterminal hydroxylating fatty acids, a process not contributing to LCDA synthesis - and probably other CYP52 enzymes of S. bombicola - there are seven more genes detected in the yeast genome – can convert the provided fatty acids to LCDAs. It is not clear if the full conversion is governed by the CYP52 enzymes or if FAD, ADH or FALDH enzymes are involved as well! 20 To avoid loss of the fatty acid substrate and the produced LCDAs in the beta-oxidation, this pathway was disabled by knocking-out the first enzymatic step (pox1; Fig.1). Surprisingly, LCDA production was further increased by in addition disabling the fatty acyl-CoA synthase (faa1; Fig. 1). This is indeed an unexpected outcome as the completely opposite action was required to achieve increased LCDA production in 25 Yarrowia lipolytica: in a patent from DuPont, one of the endogenous faa genes (YLACOS - 6P) was overexpressed (so not at all knocked-out!) and is shown to result in increased LCDA production (US20190144897A1; Zhu et al., 2019). In this way the LCDA titer in a shake flask increased from about 1 g / l to 2.5 g / l after 8 30 days of incubation (Δpox1Δugta1Δfaa1) compared to the parental double deletion strain Δpox1Δugta1. After 12 days of incubation 5.5 g / l was obtained. This means that one additional single mutation could increase the final LCDA-titres 5 till 6- fold in a shake flask and that we have a powerful strain at hand to explore for industrial application. The engineered S. bombicola yeast strain (Δpox1Δugta1Δfaa1) was taken to a 1L fermenter set-up in a 0.7 L working volume. Further optimization of the pH parameter in a fermenter set-up was conducted and it was found that pH 5.8 is the 5 optimal value for engineered S. bombicola strains. Hence, the production experiment was conducted at pH 5.8. The fermentation ran for 30 days to obtain high LCDA concentrations. The dry cell weight (DCW), glucose and LCDA concentration in the fermenter were determined. The maximum amount of LCDAs measured is 99.9 g / l (28d). Maximum productivity was reached between day 16 and 18 (0.64 g / l / h). A 10 concentration close to 100 g / l was never obtained for a non-pathogenic yeast! Hence, the present invention relates in first instance to a modified yeast strain comprising a non-functional or dysfunctional UGTA1 and FAA1 enzyme and / or not containing a ugta1 and faa1 gene, combined with a non-functional or dysfunctional β- oxidation. 15 The present invention thus relates to modified yeasts strains which in addition to comprising a non-functional and / or dysfunctional UGTA1 enzyme additionally comprise a non-functional and / or dysfunctional FAA1 enzyme and / or in which the ugta1 and faa1 genes are removed and / or disabled, combined with a non-functional or dysfunctional β-oxidation and which strains produce long chain dicarboxylic acids. 20 As explained below, β-oxidation comprises four reactions. β-oxidation enzymes in yeast comprise of several Pox, Mfe2 and Pot enzymes and the corresponding genes are several pox, mfe2 and pot genes. Any of the genes encoding the responsible enzymes can be removed and / or disabled and any of the enzymes taking part in the reactions can be rendered non-functional or dysfunctional to result in a non-functional or 25 dysfunctional β-oxidation. Furthermore, the present invention relates to a modified yeast strain comprising a non- functional or dysfunctional UGTA1, POX1 and FAA1 enzyme and / or not containing a ugta1, pox1 and faa1 gene. The present invention thus relates to modified yeasts strains which in addition to comprising a non-functional and / or dysfunctional UGTA1 enzyme additionally comprise a non-functional and / or dysfunctional POX1 and FAA1 enzyme and / or in which the ugta1, pox1 and faa1 genes are removed and / or disabled and which strains 5 produce long chain dicarboxylic acids. The term ‘non-or dysfunctional’ means in general an enzyme or a fragment or a variant thereof which is not functioning ‘normally’, and / or, has no (non-functional) or an impaired activity (dysfunctional). The term thus refers to an enzyme which is: a) not 10 functional because it is not present, b) still present but non-functional or c) still present but with a weakened or reduced activity, whereby a weakened or reduced activity is an activity that is significantly less (p < 0.05) than 90%, 80%, 70%, 60% or 50%, 40% or 30%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5% such as less than 4%, 3%, 2% or 1% of the activity of the corresponding 15 wild-type enzyme. The fact that dysfunctional genes can trigger the same effect as knockout or disabled genes is established knowledge in molecular biology and genetics, and has been demonstrated for numerous genes and enzymes. We illustrate this phenomenon by focusing on small mutations occurring in the human POX counterpart, named ACOX1 (Acyl-coenzyme A oxidase). More than 20 mutations in the 20 human acox1 gene have been identified in patients with peroxisomal acyl-CoA oxidase deficiency (pseudoneonatal adrenoleukodystrophy). These include point mutations and small insertions / deletions that disrupt enzyme function, leading to accumulation of very long-chain fatty acids and severe neurodegenerative symptoms (among others illustrated by Ferdinandusse et al.2007). The same effect is observed and studied in 25 detail in other organisms as well, like in the roundworm Caenorhabditis elegans. Also for faa1 gene ample examples of dysfunctional genes harboring point mutations, while still being present in the genome, are available. This was already described in Saccharomyces cerevisiae (normal budding yeast) in 1977. 30 Situation a) wherein said enzyme or a fragment or a variant thereof is not functional because it is not present, situation b) is still present but non-functional or situation c) is still present but with a weakened or reduced activity, can be obtained through any known means to avoid, reduce and / or silence the transcription and / or translation of the nucleic acid sequence encoding said enzyme or through any known means to impair enzyme activity. For example, but not limited to, by knock out; by insertion of a nucleic 5 acid fragment containing a marker gene or any other nucleotide fragment in the target gene resulting impaired transcription or translation of the nucleic acid sequence encoding said enzyme; through the usage of CRISPR; through homologous recombination; through siRNA; through CRISPRi; through the use of riboswitches; through recombineering; through ssDNA mutagenesis; through RNAi, miRNA, or 10 asRNA; through mutating the enzyme or the nucleic acid sequence encoding said enzyme; through transposon mutagenesis; by disruption of (the function of) a necessary regulator / activator protein; through interference with the cellular synthesis of the target enzyme or of an activator / regulator; through the use of one or more aptamers; through the use of one or more ribozymes; through the use of antibodies, 15 amino acids, peptides or any small molecules that interfere with transcription, translation, the synthesis of an active enzyme or enzyme activity; through the use of an oligoribonucleotide sequence such a dsRNA used to initiate RNA interference (RNAi) or an anti-sense nucleic acid; through the introduction of point mutations; through the usage of truncated, modified or mutated enzymes; through the usage of inhibitors or 20 antibodies; through mutation (spontaneous, induced and / or directed, point mutation, deletion, frameshift, insertion or any other type of mutation); … or any other means known to a skilled person. The enzymes UGTA1, POX1 and FAA1 and the genes ugta1, pox1 and faa1 are well- known in the art. 25 The ugta1 gene is a gene unique to S. bombicola and other sophorolipid producing yeasts or fungi. The corresponding enzyme UgtA1 is part of the dedicated sophorolipid biosynthetic pathway where it governs the second catalytic step using UDP-glucose as a donor for the glucosylation of terminal and subterminal hydroxylated fatty acids with 30 in general a chain length of 16 to 18 carbon atoms (Saerens et al., 2011; Van Bogaert et al., 2013). Variant sequences occur in other sophorolipid producing yeasts such as in Starmerella riodocensis; here a sequence sharing 83% amino acid (AA) similarity and 72% AA identity is retrieved on the contig with GenBank reference NRDT01000043.1 (position 56086..57474, complement). Also in Starmerella kuoi a gene encoding for an UGTA1 enzyme is retrieved (GenBank reference NRDS01000039.1, position 5 33536..34909), as well as in Starmerella floricola (GenBank reference JAKTRP010000011.1, position 37579..38934). The later enzyme has an AA similarity and identity of 80% and 65% respectively, and the NA identity is 61%. The UGTA1 amino acid (AA) sequence (SEQ ID N°1) is: 10 MSPSSHKPLILACGLPLSGHIMPVLSLVHGLTDDGYEATVVTGRAFEQKVRDVGADFVPLEG NADFDDHTLDDLVPGRKDMAPSFDRTVQDVEHMMVATLPEQFAAIQRAFKKLSASGRPVVL VSEVLFFGAHPISLGAPGFKPAGWICLGVLPLLIRSDHTLGLDNDRSPEAHAKKLAMNHALE HQIFVKATAKHKEICRELGCTEDPKFIWEHSYIAADKFLQLCPPSLEFSRDHLPSNFKFAGSTP KHRTQFTPPSWWGDVLSAKRVIMVTQGTFAVSYKHLIVPTLEALKDEPDTLTVAILGRRGAKLP 15 DDVVVPENARVIDYFNYDALLPHVDALVYNGGYGGLQHSLSHSVPVVIAGDSEDKPMVASR AEAAGVAIDLKTGLPTVEQIKEAVDSIIGNPKFHEASKKVQMELESHNSLKILEESIEEIASHDFG LLTKSDEETEDIPVKGPALAVSS The ugta1 nucleotide (NT) sequence (SEQ ID N°2) is: 20 ATGAGCCCTTCATCACACAAACCCCTGATTCTCGCTTGCGGCTTGCCTCTTTCAGGCCAT ATAATGCCCGTTTTGAGTCTGGTACACGGCCTTACGGACGACGGATACGAAGCTACTGTT GTGACAGGCAGAGCGTTTGAACAAAAAGTTCGAGATGTGGGTGCAGACTTTGTTCCTTTA GAAGGGAACGCAGATTTTGATGACCACACCTTAGACGATCTGGTCCCGGGCCGTAAAG ACATGGCCCCAAGCTTCGATCGTACAGTTCAAGATGTGGAGCACATGATGGTAGCTACT 25 CTTCCTGAGCAGTTTGCCGCTATTCAGAGGGCTTTCAAAAAGCTCAGCGCAAGCGGCCG CCCTGTCGTTCTTGTCAGTGAAGTGCTGTTTTTCGGTGCACACCCTATCAGCCTCGGTGC TCCTGGTTTCAAACCCGCTGGCTGGATTTGTTTAGGGGTTTTGCCTCTTTTGATCCGCAGT GATCATACCTTAGGACTTGACAACGACAGGAGCCCCGAAGCACATGCAAAGAAACTCG CTATGAACCACGCTCTTGAGCACCAAATTTTCGTTAAAGCCACTGCTAAGCACAAGGAAA 30 TCTGCCGAGAGTTAGGTTGCACTGAAGATCCCAAATTTATCTGGGAGCACAGTTACATTG CTGCAGACAAGTTCCTGCAGCTGTGCCCGCCTTCTCTTGAGTTCAGCAGAGACCATCTG CCTAGCAACTTCAAATTCGCCGGCTCAACGCCCAAGCACCGAACTCAATTCACCCCTC CTTCCTGGTGGGGGGATGTTCTGAGTGCCAAGCGAGTCATCATGGTCACTCAAGGAACT TTTGCTGTCAGTTACAAGCATCTTATTGTGCCTACTCTTGAGGCCTTGAAGGACGAGCCTG ACACTTTAACAGTAGCCATATTGGGCCGCCGCGGTGCCAAGCTACCGGATGATGTTGTG GTTCCTGAGAATGCTCGCGTGATCGACTACTTCAACTACGATGCTCTACTTCCTCACGTT 5 GATGCTCTTGTCTACAATGGTGGATATGGCGGACTTCAGCACAGCTTAAGCCACTCTGTT CCAGTTGTTATTGCTGGTGACTCTGAAGACAAGCCAATGGTGGCATCGAGAGCTGAGGC CGCTGGCGTGGCAATTGATTTGAAAACTGGCTTGCCTACAGTGGAGCAAATCAAAGAAG C TGTTGATTCGATAATTGGAAATCCGAAATTCCACGAAGCCTCGAAGAAGGTTCAAATGGA 10 GTTGGAAAGCCACAACTCCTTGAAAATTCTTGAGGAAAGCATCGAGGAAATCGCCAGCC ATGACTTTGGTCTTTTGACCAAGAGTGACGAGGAAACTGAAGATATACCTGTCAAAGGGC CGGCCTTAGCGGTGAGTTCTTAG β-oxidation is a catabolic process by which fatty acid molecules are broken down. The 15 feed of free fatty acids is first converted to acyl-CoA by fatty acyl-CoA-synthetases before they enter the actual β-oxidation pathway, and is then transported into the peroxisomes of the yeast or fungal cells. β-oxidation is a cyclic pathway, which comprises four reactions. After every cycle an acetyl-CoA molecule and an acyl-CoA lacking two carbon atoms are obtained, this until 20 in the final cycle two acetyl-CoA molecules are released. POX enzymes are governing the first step in β-oxidation; they display acyl-CoA dehydrogenase activity and remove two hydrogens between carbons 2 and 3 (Hiltunen et al., 2003). In some yeast species, multiple acyl-CoA dehydrogenase genes or POX genes occur, 25 which are all functionally translated into isozymes. C. maltosa for example has two such genes. In the genome of Y. lipolytica, even five POX genes were found some of them show activity against short-chain fatty acids (with a chain length of less than 6 carbon atoms), others against long-chain fatty acids (with a chain length of more than 18 carbon atoms) and some are active against molecules of all chain lengths (Wang et 30 al., 1999). According to the teaching of US-patent 9738913 B2 and others, in order to completely shut down the β-oxidation pathway in C. tropicalis, a quadruple mutant has to be created: pox4 and pox5 on both allelic positions as C. tropicalis is a diploid yeast species (Picataggio et al., 1991). However, only one gene has been identified in Saccharomyces cerevisiae and other conventional yeasts and also S. bombicola only harbors one pox gene). 5 The POX1 AA sequence (SEQ ID N°3) is: MIDSNECSSITFEGKEYDTFTEPPILMKQERAKTSFPTMEVTHFLNGGEKETAKFEAVKRQIEM SPVFDNSDYYDVNGDYKKCRERTMQKVAAIAEIVTDGRDEHEITSYLSAISIVDSQSYTRIGVH FGLFLSGVRSGGTPEQFNYWVEQGAAGLRNFFGCFCMTEMGHGSNVAGLETTATYDENTE 10 EFVINTPSTAATKWWIGGLAHTATHGLVYARLLVKGKDYGVKQFVVPLRDRSNWNLLPGIGI GDIGKKLGRDGIDNGWVQFSNVRIPRLFMLMKYSRVSANGNVDNKAPAQLAYGALIGGRVT MVRDSYTWASRYLTIAIRYAAVRRQFAQGSTVETKLLDYTYHQRRLLPRLAYAYAMNAASRHL QSVYANTTQTLANTNPKNKAAMEAAVGEAKALFALSAGLKAFSTWGTLAMIDECRQACGGH GYSAYNGFIDWTAFAVQVTWEGDNNVLALSTGRALIGRHLEAKAGPVKDDQLGSGDISQPS 15 VLIRGWNLVATKATEQATTQYKKLEKSGLEVDKIWEKLSQLRFKVARISTRNFLVKSFFEEVEK NASPAIKQVLTELATLFALWSIEEEASVFLEFKFITAEDLSQINLLVDEYCGKIREQAIGLTDSFN WSDYFINAPIGNYDGDVYRNYFRKITDRNPRSETHAPYFLSVMQPFFKRTFEDDPDLSSLEEE EREINEE 20 The pox1 NA sequence (SEQ ID N°4) is: ATGATTGATTCAAACGAGTGCAGCAGCATAACCTTTGAGGGCAAGGAGTATGACACTTTCAC AGAGCCGCCTATCCTCATGAAGCAGGAGAGGGCGAAGACCTCCTTCCCTACAATGGAGG TCACTCACTTTCTGAACGGGGGTGAGAAGGAGACCGCCAAGTTTGAGGCTGTCAAGCGC CAGATTGAGATGTCACCGGTGTTTGACAACTCGGACTACTACGATGTTAACGGCGACTACA 25 AGAAGTGCCGTGAGCGAACTATGCAGAAGGTTGCTGCCATCGCAGAGATTGTCACAGATG GCCGCGACGAGCACGAGATCACCTCATATCTGTCTGCTATCTCAATCGTAGACTCTCAATC ATACACGCGTATTGGCGTTCATTTTGGTTTGTTTTTGTCCGGGGTACGATCAGGCGGAACTC CAGAACAATTTAACTACTGGGTTGAGCAGGGCGCTGCGGGCCTTCGGAACTTCTTCGGCT GCTTCTGCATGACTGAAATGGGCCATGGATCCAACGTCGCTGGTTTGGAGACAACCGCCA 30 CATACGATGAGAACACTGAGGAGTTCGTCATCAACACCCCTTCAACCGCAGCCACTAAGT GGTGGATTGGAGGCCTTGCACACACTGCCACTCATGGCCTTGTGTACGCGCGTTTACTTGT GAAGGGCAAGGACTACGGCGTCAAGCAGTTTGTCGTTCCATTGCGCGACCGCAGCAACT GGAATCTGCTCCCTGGCATCGGAATTGGCGACATTGGAAAGAAGCTGGGCAGAGACGGT ATCGACAATGGATGGGTGCAGTTCAGCAACGTGCGAATCCCGCGTCTTTTCATGTTGATGA 35 AATACAGCAGGGTCTCTGCTAACGGCAATGTTGACAACAAGGCGCCTGCACAGCTCGCTT ATGGCGCGCTTATTGGCGGAAGAGTCACCATGGTCCGCGATAGTTATACCTGGGCGTCGC GGTACCTCACTATCGCCATTCGGTATGCTGCTGTCCGAAGACAGTTTGCTCAGGGTTCTACT GTGGAGACTAAACTTCTTGACTATACTTACCACCAGCGCCGCTTGCTCCCACGACTCGCCT ATGCTTATGCCATGAACGCTGCTTCGCGTCATTTGCAATCAGTTTATGCTAATACCACTCAGA CCCTGGCGAACACAAACCCGAAAAACAAGGCAGCCATGGAGGCTGCAGTTGGTGAAGC 5 TAAGGCACTTTTCGCTTTAAGCGCTGGTCTCAAGGCCTTTTCTACTTGGGGCACTCTTGCCA TGATCGATGAATGTCGCCAAGCCTGTGGTGGACATGGATACTCCGCCTACAATGGTTTTATC GACTGGACCGCATTTGCCGTCCAGGTCACTTGGGAGGGCGACAATAACGTTCTTGCCTTG AGCACTGGACGTGCCCTCATCGGCCGCCACCTCGAGGCTAAGGCGGGCCCTGTTAAGG ACGATCAGCTCGGATCAGGAGATATTTCGCAGCCCAGCGTCCTCATTCGCGGCTGGAAC 10 CTTGTGGCCACTAAGGCTACTGAGCAGGCCACGACCCAGTACAAGAAGTTGGAGAAGTC CGGCCTCGAAGTCGATAAGATCTGGGAGAAGCTCTCGCAGTTGCGCTTCAAGGTCGCTCG TATCAGCACGCGCAACTTCCTCGTAAAGTCTTTCTTCGAGGAGGTCGAAAAGAATGCTTCG CCAGCCATAAAGCAGGTGCTCACAGAACTGGCCACCTTATTTGCCCTATGGTCTATTGAGG AAGAGGCTTCGGTTTTCCTCGAGTTCAAATTCATTACTGCTGAGGATCTCTCGCAGATCAAC 15 CTCCTGGTTGACGAGTACTGTGGCAAAATACGTGAGCAGGCCATTGGCCTAACTGACAGTT TCAACTGGTCCGACTACTTTATCAATGCGCCCATTGGGAACTACGACGGTGACGTTTACCG CAACTACTTCCGCAAGATCACTGACCGTAACCCGAGATCGGAGACTCACGCTCCTTACTT CCTCTCTGTCATGCAGCCATTCTTTAAGAGAACGTTTGAGGACGACCCGGATCTTTCTAGTC TAGAGGAGGAAGAGAGAGAGATCAACGAGGAGTAA 20 Variant sequences with a different nucleotide and amino acid sequence, but fulfilling the same function are present in other yeast species. The invention also comprises those pox and POX sequences and their modifications. 25 The second step of the β-oxidation pathway which involves a hydration step and the third step of the β-oxidation pathway which involves a second dehydrogenation, are performed by one and the same enzyme in eukaryotic organisms: the so-called multifunctional enzyme. Contrary to mammals, yeasts only possess MFE2 and until now there is no evidence for the presence of isozymes in any yeast or fungal species. 30 The MFE2 AA sequence of S. bombicola (SEQ ID N°5) is: MAENLRYDGKVVVVTGAGGGLGKAYALFFGARGASVVVNDLGGTLNGGDGNSRVADGVV KEIEALGGKAAANYDSVENGDKIVETAIKAFGTVHIIINNAGILRDVSLKKMTDKDFNFVQSVH VFGSYAVTRAAWPYFKQQKFGRVINTASAAGLYGNFGQANYSAAKSALVGFTETLAKEGAKY 35 NITANVIVPLAASRMTETILPPDILEKLKPELIVPVVGYLVHENTAESNGIYESAAGVVTKVRWQ RGAGVQFRADDSFTPAAVLNKFEEINDNFEPAEYPSGPKDLLAAFENGKNLPSNEQGSTPVS FENQVVIVTGAGGGIGQQYALMLGKLGAKVVVNDLGNADATVELIKKAGGTAVADKHNVTD GEAVVKTALDNFGAIHAVINNAGIIRDRGILKMTPDLWNAVQQVHLFGSFSVTKAAWPHFQK QKYGRVVNTTSTSGIYGNFGQTNYSAAKAGLIGFTKTVALEGAKYNILCNCVAPTAGTAMTAD VFPQDMLETLKPRYIAPITVLLASEHSPDTGKVYEAGAGWIGRTRWQRTSGVMIPGITVEKVK QNWQKITDFDDGKATNFESASEANMYIFNMAAEGEDQGSEGGESEASASGEYSYDDKTIILY 5 NLGVGASEKQLNYTFENNQDFQPVPSFGTIPLFSAPFPFDEVVPNFNPMKLLHGEQYLELKK WPIAPEATLKTTGKLLDLADKGKAAVAMVEYISVDKNSGEPVFLNVMSTFLRGSGGFGGEKN FKDHGPITAANKPPAREPDYIAKYKTTDNQAAIYRLSGDYNPLHIDPEFAAVGGFDRPILHGLA SFGISSRLLVEKYGVFKNIKVRFSGHVFPGETLQVSAWKEGPKVIFETTVLERNTKAITAAAIELA DDGKSKL 10 The mfe2 NA sequence of S. bombicola (SEQ ID N°6) is: ATGGCGGAGAATCTTAGGTACGACGGCAAAGTTGTCGTTGTCACTGGCGCAGGAGGCG GACTTGGAAAAGCCTACGCGCTGTTCTTTGGTGCTCGTGGTGCATCAGTTGTTGTGAATG ATCTCGGAGGCACATTGAATGGTGGTGACGGTAACTCTAGAGTTGCTGATGGAGTTGTAA 15 AAGAAATTGAGGCTCTTGGAGGCAAAGCAGCGGCAAATTACGATAGCGTCGAGAATGGT GACAAGATCGTTGAAACCGCTATCAAAGCATTCGGCACGGTACACATTATCATCAATAAC GCAGGAATTCTTCGTGACGTCAGTCTAAAAAAGATGACGGATAAGGATTTCAACTTTGTCC AGTCTGTCCACGTCTTTGGCTCGTACGCGGTTACGAGGGCTGCTTGGCCTTATTTCAAAC AACAGAAGTTCGGTCGTGTGATCAACACCGCAAGCGCAGCTGGTCTATATGGCAACTTT 20 GGCCAGGCCAATTATTCTGCGGCTAAATCCGCTTTGGTGGGCTTTACTGAAACTTTGGCT AAAGAGGGCGCCAAATACAATATCACCGCTAACGTTATTGTTCCACTGGCGGCGTCGCG CATGACTGAGACCATTCTTCCTCCTGACATTCTAGAGAAGCTGAAGCCCGAGCTCATCGT TCCTGTTGTCGGATACCTCGTTCATGAGAATACAGCAGAGAGCAATGGAATCTACGAAAG TGCTGCTGGCGTTGTAACCAAGGTGAGATGGCAGCGTGGAGCTGGTGTACAGTTCAGG 25 GCTGATGACTCGTTCACTCCCGCTGCAGTGTTGAACAAATTCGAAGAAATCAACGACAA CTTTGAGCCAGCAGAGTACCCCAGTGGGCCCAAGGATCTTCTAGCTGCCTTTGAAAATG GCAAGAATCTGCCTTCGAATGAGCAGGGAAGCACTCCAGTAAGTTTCGAGAACCAGGTC GTTATCGTTACTGGTGCGGGAGGCGGAATTGGACAGCAATATGCTCTCATGCTCGGTAA GTTGGGAGCGAAGGTTGTTGTGAATGACCTTGGCAACGCTGATGCTACCGTGGAATTGAT 30 CAAGAAGGCTGGAGGAACGGCTGTGGCGGATAAGCACAATGTCACCGATGGTGAGGC AGTTGTGAAGACTGCTCTAGACAATTTCGGTGCTATCCATGCGGTTATCAACAATGCCGG TATCATTCGTGATCGTGGCATTCTCAAGATGACGCCCGATCTCTGGAATGCTGTTCAACA GGTTCATCTATTCGGTTCCTTTTCAGTCACCAAAGCTGCATGGCCTCATTTCCAGAAGCA GAAATACGGACGTGTGGTCAACACGACTTCAACCTCTGGAATCTACGGAAATTTCGGAC 35 AGACAAACTACTCAGCGGCAAAGGCCGGTCTCATTGGCTTCACCAAGACTGTGGCACTA GAAGGTGCCAAGTACAACATTCTTTGCAACTGCGTTGCCCCTACAGCAGGAACTGCTAT GACTGCTGATGTGTTCCCTCAAGATATGCTGGAGACGTTGAAGCCAAGGTACATTGCGC CAATCACTGTCCTGCTTGCTAGTGAGCACTCGCCCGACACCGGTAAGGTCTACGAAGC AGGTGCTGGCTGGATTGGCCGCACGCGTTGGCAGAGAACTTCGGGTGTCATGATTCCT 40 GGTATCACAGTGGAAAAGGTTAAGCAAAATTGGCAGAAAATCACCGATTTCGATGACGGG AAGGCTACCAACTTTGAGTCCGCCTCCGAAGCAAACATGTACATCTTCAACATGGCAGC TGAGGGCGAAGACCAAGGCTCTGAAGGTGGCGAATCTGAGGCTTCAGCAAGCGGCGA ATATTCTTACGACGACAAGACGATCATTTTGTACAACTTGGGAGTTGGTGCGAGCGAGAA GCAGCTCAATTATACTTTTGAAAACAATCAGGATTTCCAGCCAGTGCCGAGTTTCGGCAC CATCCCGCTCTTCAGCGCTCCATTCCCATTTGATGAAGTTGTGCCCAATTTCAATCCAATG AAGCTCCTTCATGGAGAGCAATATTTGGAGTTGAAGAAGTGGCCCATTGCCCCAGAGGC 5 AACGTTGAAGACCACGGGCAAGCTTCTCGATCTTGCAGACAAGGGCAAAGCTGCTGTA GCGATGGTGGAATATATCTCTGTCGATAAGAATTCTGGTGAGCCTGTGTTCCTCAACGTCA TGTCAACATTCTTGAGAGGCTCCGGAGGTTTCGGGGGTGAGAAGAATTTCAAGGACCAT GGCCCCATCACAGCAGCCAACAAGCCACCGGCTCGCGAGCCCGACTATATCGCCAA GTACAAGACCACGGACAACCAGGCTGCAATCTATCGACTATCAGGAGACTACAACCCT 10 CTTCACATTGATCCTGAGTTTGCTGCCGTTGGCGGATTCGATCGTCCGATTCTTCACGGC CTTGCGTCTTTCGGAATCTCATCAAGATTGTTGGTTGAAAAGTATGGCGTTTTCAAGAACAT CAAGGTAAGATTCTCGGGCCATGTGTTCCCTGGTGAGACTCTGCAAGTTTCCGCTTGGAA GGAAGGTCCCAAGGTGATTTTTGAGACGACGGTGCTGGAGCGTAACACCAAAGCCATTA CTGCAGCAGCAATTGAGCTGGCTGATGATGGTAAGTCTAAGCTGTGA 15 Variant sequences with a different nucleotide and amino acid sequence, but fulfilling the same function are present in other yeast species. The invention also comprises those mfe2 and MFE2 sequences and their modifications. 20 The final step involves the cleavage of the 3-ketoacyl-CoA by the enzyme beta- ketothiolase (also known as thiolase). This reaction breaks the bond between the carbons 2 and 3, producing acetyl-CoA and a new acyl-CoA that is two carbon atoms shorter than the original molecule. The shortened acyl-CoA then re-enters the beta- 25 oxidation cycle for further processing. In yeast, the thiolase enzyme is referred to as the POT enzyme (Fig.1). The POT AA sequence of S. bombicola (SEQ ID N°7) is: MNRLQNMQEQLSLNEAGAADKLFAKHADDVVIIAAHRTAITKAKKGGFRNTTSGEILARLLRA 30 QIDKVGIDPKIIDSLVVGNVCNPGAGVNEHRAAQLVAGIPHTTPFMAINRQCSSGLMAVNTIY NEIKSGQIDIGIGAGVESMSTQYGSNMMDGFPARFSSDAAGQKCLIPMGITSENVASEFGVS RPDQDAFAAASYNKAEKAQAAGLFKEEILPIYGIIEDDEGNETDQLVSEDDGIRKGVTPESLSKI RAAFKEDGTTTAGNSSQVSDGAGSVILARRSVAQKLGLPIIGKFVHCRTLGVPPEVMGIGPAV AIPAVLKDLGLTVDDVDVFEINEAFASQALYSVRKAGIDINKVNPKGGAIAFGHPLGATGARQ 35 VSTLLTELHRTNKKVGVTSMCIGTGMGAASVIVAE The pot NA sequence of S. bombicola (SEQ ID N°8) is: ATGAACAGACTACAGAACATGCAAGAGCAGCTGAGCCTCAACGAGGCTGGTGCCGCC GACAAGCTTTTTGCCAAGCACGCCGATGACGTCGTCATCATCGCTGCGCACCGTACAG CCATCACGAAGGCAAAGAAAGGTGGCTTTCGCAACACTACGAGTGGGGAGATCCTCGC 5 TCGTCTACTCAGAGCTCAGATTGACAAAGTTGGCATCGACCCGAAGATTATCGACAGTCT GGTTGTGGGCAACGTTTGTAACCCCGGAGCCGGTGTGAATGAGCATCGTGCTGCCCAG CTTGTCGCTGGAATTCCACACACCACTCCTTTCATGGCTATCAACCGTCAGTGCTCCTCT GGCTTGATGGCCGTGAACACCATTTACAACGAAATCAAGTCTGGTCAGATTGATATTGGTA TTGGCGCTGGTGTCGAGAGTATGTCTACACAGTATGGCTCTAACATGATGGATGGCTTTC 10 CTGCGCGTTTTTCGTCGGATGCTGCTGGTCAAAAGTGTCTCATTCCCATGGGCATCACCT CTGAGAATGTTGCTTCAGAGTTTGGCGTCAGCCGCCCGGACCAGGATGCGTTTGCTGCT GCTTCGTACAACAAGGCCGAAAAGGCACAGGCTGCCGGCCTTTTTAAGGAGGAAATTCT CCCCATTTATGGTATAATTGAGGATGATGAGGGCAATGAAACCGACCAACTCGTGTCCGA AGACGATGGCATCCGCAAGGGCGTAACCCCTGAGTCTCTCAGCAAGATTCGCGCTGCC 15 TTCAAGGAGGATGGTACCACTACTGCCGGTAACTCTTCTCAGGTCAGCGATGGCGCAG GCTCCGTGATTCTTGCTCGTCGTTCAGTCGCTCAGAAGTTGGGGCTCCCGATCATTGGC AAGTTCGTGCATTGCCGTACCCTTGGTGTTCCCCCTGAGGTGATGGGCATCGGCCCCG CTGTCGCCATTCCTGCTGTGTTGAAGGATCTCGGCCTCACCGTCGATGACGTTGATGTCT TCGAGATCAACGAGGCGTTCGCGTCACAAGCGCTTTACTCCGTCCGCAAGGCTGGCAT 20 CGACATCAACAAGGTGAATCCTAAGGGTGGTGCTATCGCCTTCGGCCATCCTCTCGGTG CCACCGGCGCGCGCCAAGTCTCTACCCTTCTCACAGAGCTACATCGCACCAACAAGAA GGTCGGTGTAACGTCAATGTGCATCGGTACCGGCATGGGCGCCGCTAGTGTCATCGTT GCGGAGTAA 25 Variant sequences with a different nucleotide and amino acid sequence, but fulfilling the same function are present in other yeast species. The invention also comprises those pot and POT sequences and their modifications. 30 Fatty acids are inputs for various cellular processes and are mostly used in the form of acyl-CoAs. The activation of free fatty acids with coenzyme A is catalyzed by fatty acyl- CoA synthetases (FAA). In baker’s yeast there are four orthologues of acyl-CoA synthetases identified, each with a diƯerent source and fatty acid length preference (Johnson et al., 1994). In contrast, in in S. bombicola, it looks like FAA1 is the only and main fatty acyl-CoA synthetase (Jezierska et al., 2019). The FAA1 AA sequence (SEQ ID N°9) is: 5 MFSVQVDKPQRAGETGSIRNSKAADKPSTCPAGTDIKTVHELVSYGIETFGDSNFLGQREFIKL HTETREVTKKVDGEDKKVKKDWQYFEMGKFDWETYRELEQTRKKLGSAMVKCGIKPGEGKM HLYAKTCREWMQTAVACASQNITLVTAYDTLGIEGLRESINQTETSGILLDKGNLSNLSKVLQDA PSIKFVVYRDSEGELSDANKKEIESFAGYNGGIKVYSYTEFLKLGEENPVEYHAPKADDICCIMY TSGSTGPPKGVTLLHSTVVAGVAGATGNVTRKSVSSTDVFLAILPLAHIFEFTAELAVFYWGSAI 10 GYGSPKTITDTNMRNCKGDMRELQPTILVGVPAVFEAIKKGISGQISKAPVVSQKVFWGAFKLK QTLLSLHLPVPLLDSVIFKKIKDATGGRLRFVMNGGAPVSAGTRTFINVLLAPLIMGYGLTETNA MCAILNPLNLDMDSTGEIVTSVTIKLVDVPDTGYFAKNNQGEIWVKGPAVSPGYWKNEKETKE AYTEDGWFKTGDIGELTKNGKIRIVDRKKNLIKTLNGEYVAVERLESLYRSNKYVSNICVFADSS HAKPVAIVCPVESAIKDLCSAKGLQYSESVIHEKSINGPVLESLHATAKECNLRPVEWLAGVAL 15 SDEEWTPQNGFVTSAQKVQRKKIGTEYKKQLDEIFKNA The faa1 NA sequence (SEQ ID N°10) is: ATGTTCTCCGTGCAAGTTGACAAGCCCCAGCGAGCGGGCGAAACTGGATCCATCCGCAA TTCTAAAGCTGCAGACAAGCCATCTACCTGTCCAGCAGGCACTGACATCAAAACTGTGCAC 20 GAACTGGTCAGCTATGGTATTGAGACTTTTGGTGACTCGAACTTTCTAGGTCAGCGAGAGTT CATTAAACTTCATACTGAGACTCGTGAAGTCACTAAGAAAGTCGATGGAGAGGATAAGAAAG TCAAGAAGGATTGGCAGTACTTTGAGATGGGAAAGTTTGACTGGGAAACATATCGCGAGCT CGAGCAGACGAGAAAGAAGCTGGGCTCCGCCATGGTCAAGTGCGGAATCAAGCCGGGC GAGGGGAAGATGCATTTGTACGCCAAGACTTGTCGTGAGTGGATGCAAACTGCCGTTGCTT 25 GCGCAAGCCAGAATATCACTCTAGTAACTGCATATGATACGTTGGGCATCGAGGGCCTTCG TGAGAGTATCAATCAAACTGAAACAAGCGGTATTCTGCTAGACAAGGGCAATCTCTCCAAC CTTTCCAAGGTCCTGCAAGACGCACCATCGATTAAGTTTGTCGTATATCGAGACTCAGAAGG CGAGCTCAGCGACGCTAACAAGAAAGAAATAGAAAGTTTTGCTGGTTATAATGGCGGCATC AAAGTCTATTCTTACACCGAGTTTTTAAAGCTTGGAGAAGAGAATCCTGTCGAATACCACGC 30 CCCCAAGGCTGATGATATCTGCTGCATTATGTACACCTCGGGCTCCACTGGCCCACCAAA GGGTGTAACGCTCCTTCACAGCACTGTAGTTGCTGGTGTCGCCGGTGCTACCGGAAATGT CACAAGAAAGTCGGTCAGCTCGACCGATGTGTTCCTTGCCATATTGCCTCTGGCCCATATTT TCGAATTCACTGCTGAACTCGCCGTCTTCTATTGGGGCTCAGCCATCGGCTATGGCTCTCC GAAGACGATCACAGACACAAACATGCGTAATTGCAAAGGTGACATGCGAGAGCTGCAGCC 35 GACTATCCTTGTTGGCGTGCCCGCTGTTTTCGAGGCTATCAAGAAGGGAATCAGTGGTCAG ATATCCAAAGCCCCTGTAGTCAGCCAGAAGGTGTTTTGGGGAGCTTTCAAGCTCAAACAAA CTTTATTGTCTTTGCACCTCCCTGTCCCTCTTCTGGACTCTGTGATTTTCAAGAAGATTAAGGA CGCAACAGGCGGTCGTCTGCGCTTCGTGATGAACGGAGGCGCGCCTGTCTCTGCTGGC ACTCGCACGTTCATCAACGTCCTACTTGCTCCCCTCATTATGGGCTATGGACTTACTGAGAC 40 CAACGCGATGTGTGCTATTCTCAACCCCCTGAACCTTGATATGGATTCTACCGGTGAGATTG TTACAAGTGTTACGATAAAGCTTGTAGATGTACCTGACACTGGCTACTTTGCCAAGAACAATC AAGGCGAGATCTGGGTGAAGGGTCCAGCAGTTAGTCCTGGTTACTGGAAGAACGAGAAGG AAACCAAGGAGGCATATACCGAAGATGGATGGTTTAAGACCGGCGACATAGGTGAGCTTA CCAAGAACGGCAAAATACGCATTGTTGACCGTAAGAAGAACCTCATTAAGACGTTGAACGG TGAGTATGTCGCTGTGGAGCGCCTTGAGTCCTTGTACCGCTCCAACAAGTACGTCAGCAAC 5 ATCTGCGTATTTGCCGACAGTTCTCACGCCAAGCCGGTTGCAATTGTCTGCCCCGTAGAGT CCGCCATCAAAGATCTTTGTTCCGCGAAAGGTCTGCAATACAGCGAGAGTGTTATCCACGA GAAGTCTATAAACGGGCCCGTTCTTGAATCTCTCCATGCCACGGCCAAGGAGTGCAACTTA CGTCCTGTTGAGTGGCTCGCAGGAGTTGCCCTCAGCGATGAGGAGTGGACTCCTCAGAAT GGATTTGTCACTAGCGCACAGAAGGTCCAGCGAAAGAAGATTGGAACGGAGTACAAGAAG 10 CAGTTGGACGAAATTTTTAAGAACGCGTAA Variant sequences with a different nucleotide and amino acid sequence, but fulfilling the same function are occurring in other yeast species. The invention also comprises those faa and FAA sequences and their modifications. 15 Said yeast strains are well-known in the art and are for example described by: Starmerella bombicola (previously Candida) (Spencer et al., 1970), Starmerella apicola (Gorin et al., 1961) (previously Candida), which was initially identified as T. magnolia, Wickerhamiella domericqiae (Chen et al., 2006), Pseudohyphozyma bogoriensis sp. 20 (previously Rhodotorula or Candida) (Tulloch et al., 1968), Starmerella batistae (Konishi et al., 2008) (previously Candida), Starmerella (previously Candida)floricola (Imura et al., 2010), Starmerella (previously Candida) batistae, Starmerella (previously Candida) riodocensis, Starmerella stellata (previously Candida) and Candida sp. NRRL Y-27208 (Kurtzman et al., 2010), Starmerella kuoi (Kurtzman, 2012) (previously Candida), 25 Starmerella cerana (Kumar et al., 2023), Rhodotorula muciliginosa and Candida rugosa (Chandran and Das, 2011), Cyberlindnera samutprakarnensis (Poomtien et al., 2013), Cryptoococcus sp. VITGBN2 (Basak and Das, 2014), Lachancea thermotolerans (Mousavi et al., 2015), Candida gropengiesseri, Candida magnoliae, Candida antarctica, Pseudozyma antarctica, Candida lipolytica and any other sophorolipid 30 producing strains or strain with (putative) sophorolipid-synthesizing capacities described in literature or not. As illustrated by the S. cerana example from the above paragraph, novel sophorolipid producing yeast are till date still described. Indeed, even already described species are not always examined for sophorolipid synthesizing capacities, concentrations are below the detection limits and / or the cultivation conditions are not inducing sophorolipid biosynthesis. This was illustrated by the work of Kurtzman et al. (2012) who screened previously described yeast species from the Starmerella-clade for sophorolipid synthesis and found novel producers. 5 The present invention further relates to the usage of a modified strain as described above to produce long chain dicarboxylic acids and further relates to the usage of a modified strain as described above wherein said long chain dicarboxylic acids have a chain length of 16 till 24 carbon atoms. Moreover, the present invention relates to a method to produce long chain dicarboxylic 10 acids comprising: ^ providing oil (such as rapeseed oil or used cooking oil), ^ providing a modified strain as described above, ^ bringing said oil and said modified strain in a fermenter, ^ adjusting the pH of said mixture of oil and strain in said fermenter to about 15 5.8, ^ allowing the production of long chain dicarboxylic acids by said strain for a certain period of time. With the term ‘oil’ is meant: oils or fats derived from plants or animals, waste streams 20 or recycling processes. These fats and oils preferentially contain C16 and C18 saturated and / or unsaturated fatty acids besides fatty acids of other chain length. Non- limiting examples of plant-derived or vegetable oils are canola oil, rapeseed oil, safflower oil, soybean oil, sesame oil, almond oil, avocado oil, olive oil, palm oil, palm kernel oil, palm oil, sunflower oil, peanut oil, cottonseed oil, walnut oil, grape seed oil, 25 flaxseed oil, cacao butter, corn oil, neem oil and rice bran oil. Non-limiting examples of animal-derived fats or oils are beef tallow; butterfat from cow, goat, sheep, buffalo or yak pork lard, cod liver oil, fish oil. Waste streams coming from the oil production process (like watery streams from equipment cleaning, secondary extractions or pressings, tannery fleshing waste oil, etc.) can be used as well. Finaly, recovered oils 30 from recycling and collection processes like used cooking oils, grease trap waste are included as well. These can be derived from households, wastewater treatments, waste treatment plants and several food, oil and / or fat processing industries. With the term ‘fermenter’ is meant: any standard fermenter or bioreactor known by the person skilled in the art to run aerobic microbial fermentation processes in liquid medium in a controlled and sterile way. The core of such fermenter is a vessel with a 5 volume ranging from half a liter to hundred cubic meters and this vessel is made from glass (small scale) or stainless steel (large scale). The vessel can be double walled for temperature control reasons. The vessel contains the microbial production medium with the microorganism. There is a stirrer and aeration system present. There are several liquid inlets and at least one liquid outlet (like for sampling). There is at least 10 one gas inlet and at least one gas outlet. There is monitoring of gas influx and optional outflux, pH and dissolved oxygen. One can control temperature, pH, dissolved oxygen and if needed other parameters such as substrate feeding rate, harvesting and foam. 15 With the terms ‘adjusting the pH to about 5.8 is meant adding KOH or NaOH solutions or similar basic aqueous solutions until a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5,8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or 6.6 is measured with the pH probe present in the fermenter. With the terms ‘allowing the production of long chain dicarboxylic acids by said strain 20 for a certain period of time’ is meant ‘allowing the fermentation to take place for 6 till 30 days, preferentially taking at least 8, 10, 12, 14, 16 or 18 days.. As mentioned above, CYP52M1 is the key enzyme for C16-C18 LCDA production and is essential in the strains described above. The involvement of the other CYP52 enzymes 25 was explored by individual overexpression: CYP52E3 seems to be of importance for the creation of longer chain LCDAs (like C20, C22 and C24) which are of industrial relevance for the creation of novel coatings and lubricants. CYP52E3 is a cytochrome P450 monooxygenase enzyme previously reported to be present in S. bombicola. Members of the CYP52 enzyme family are supposed to 30 hydroxylate fatty acids and / or alkanes. CYP52E3 has no important role in sophorolipid synthesis, but the gene is induced upon the growth on the alkane hexadecane (Van Bogaert et al., 2009). The CYP52E3 AA sequence (SEQ ID N°11) is: MNINFSDVLVLGGISVSFLLAYQAIYFYFIYSPRAKKLGCALPPVFFSFPLGIPEVIRLVNAWFND DLLEYFTFKFEEFQRKTGFQSVAGQLWIGTIEPENIKTMLATSFKDYSLGFRYEAMYGLLGNGIF TLSGEGWKHSRALLRPQFSREQVSHLESMRTHINMLINNHFKGGKVVDAQVLFHNLTIDTATE 5 FLFGESTNTLDPALAQHGFPGPKGLVTGEQFAEAFTSALELLSVRVMAGAAWFLVWTPKFWR SCKVCHNFIDYFVFKALATPMEKDQEADRYVFIRELTKETSDPRVIRDQALNILLAGRDTTAALL SFTTYYLGAYPEVYDELREAVIADFGKEDAEPPTFEQLKQCKVLQNVIREVLRLHPNVPLNFRE AITDTKFPTGGGPNGDQPVFVPKGQKVFYATYVMQRNEGLWGPDSTTFRPDRWNESREAIA SGWDYIPFNGGPRICLGQQFALTEASYTLVRICQEFSRIEVLHPDVITSRNVMKQRMRLTNSSS 10 GGVIAKFIR The cyp52e3 NA sequence (SEQ ID N°12) is: ATGAACATTAATTTCTCTGACGTGCTCGTGCTAGGAGGCATCAGCGTGAGCTTTTTGCTCG CCTACCAGGCGATTTACTTTTATTTCATTTACTCGCCACGAGCCAAAAAGCTCGGTTGCG 15 CTCTTCCACCGGTCTTCTTCTCTTTCCCACTCGGAATACCGGAGGTCATACGTCTTGTGA ACGCCTGGTTCAACGATGATCTCCTTGAGTATTTCACCTTCAAATTCGAGGAGTTCCAGC GCAAAACCGGATTCCAATCAGTCGCTGGGCAACTATGGATTGGGACTATTGAGCCCGAG AACATCAAGACTATGCTCGCTACTTCATTTAAAGACTACTCCCTAGGCTTCCGTTACGAG GCCATGTACGGCCTTCTCGGAAATGGCATTTTCACTCTCAGTGGTGAGGGCTGGAAGCA 20 CAGCCGCGCTTTGTTGCGTCCGCAATTTAGTCGTGAGCAAGTCTCTCACCTTGAATCAAT GCGCACACACATCAATATGTTGATCAACAACCACTTCAAGGGTGGCAAAGTCGTCGATG CTCAGGTTTTGTTCCACAATCTAACCATTGATACTGCTACCGAATTCCTATTCGGAGAGAG CACCAACACTCTTGACCCTGCTCTTGCTCAGCATGGATTCCCTGGACCTAAGGGTCTTGT AACCGGTGAGCAGTTTGCTGAGGCTTTTACCTCTGCTCTCGAATTGCTTTCTGTGCGAGTT 25 ATGGCCGGCGCCGCATGGTTCCTCGTTTGGACCCCCAAATTCTGGCGCTCATGCAAAG TCTGCCACAACTTCATTGATTACTTCGTTTTCAAGGCTCTGGCCACTCCTATGGAGAAGGA CCAGGAAGCTGATCGCTACGTCTTTATTCGAGAACTCACAAAGGAGACCTCTGACCCAC GGGTCATCCGCGACCAGGCCCTCAACATCCTCTTGGCTGGTCGTGATACCACTGCGGC ACTTCTCAGCTTCACCACCTACTACCTTGGTGCCTACCCTGAGGTCTACGATGAGCTTCG 30 CGAGGCTGTTATTGCGGACTTCGGCAAGGAAGATGCTGAGCCCCCTACGTTTGAGCAG CTTAAGCAGTGCAAGGTGCTACAGAACGTCATTCGGGAAGTTTTGCGATTGCACCCGAAT GTGCCCCTCAACTTCCGCGAGGCCATTACCGATACTAAGTTCCCCACAGGAGGCGGC CCGAATGGAGACCAGCCCGTTTTCGTTCCCAAGGGACAGAAAGTGTTTTACGCCACCTA CGTCATGCAGCGAAATGAGGGTCTCTGGGGTCCTGACTCCACAACATTCCGCCCTGAC CGCTGGAACGAGTCAAGAGAGGCCATCGCATCCGGATGGGACTACATTCCTTTCAACG GCGGCCCTCGTATTTGCCTGGGTCAGCAGTTCGCTCTCACAGAGGCGAGCTACACGCT CGTGCGTATCTGCCAAGAGTTCTCCAGGATTGAGGTTCTCCACCCTGATGTTATTACCTC CAGGAACGTGATGAAACAGCGCATGCGTTTGACCAACTCTTCCAGCGGCGGCGTCATA 5 GCGAAGTTCATTCGCTAG CYP52M1 and CYP52E3 are cytochrome P450 monooxygenase enzymes; a group of enzymes consisting of various families and subfamilies mainly facilitating the oxidation of various substances across a wide range of organisms such as bacteria, yeasts, 10 plants, and animals. Several cytochrome P450 monooxygenase enzymes are capable of terminal hydroxylation of fatty acids, including fatty acids with a chain length of 20 carbon atoms or more. These can be – as demonstrated above – members of the CYP52 family as found in S. bombicola and also in other yeasts such as Y. lipolytica or C. maltosa, but can also be retrieved in other families such as, but not limited to, CYP2 15 and CYP4 found in mammals, CYP86 and CYP94 from plants and CYP102 and CYP153 from bacteria. Hence, the current invention refers to the use of cytochrome P450 monooxygenase enzymes or systems able to hydroxylate fatty acids with a chain length of 20 carbon atoms or more for the generation of LCDAs with a chain length of 20 carbon atoms or more. The term ‘systems’ refers to the fact that in some cases specific 20 electron delivering enzymes - like CPR depicted in Fig.1 - need to be co-expressed, while some enzymes – like CYP102 – are self-sufficient and harbor a CPR or electron- providing unit in the protein structure. The share of very-long chain LCDAs can be further increased by (over)expression of 25 endogenous and heterologous elongases. In a first test, we deployed the endogenous elongase1 gene. As the name suggest, elongases elongate long-chain fatty acids to very-long chain fatty acids. This by adding two carbon units using malonyl-CoA in each cycle of elongation. In S. cerevisiae, 3 fatty acid elongases (ScELO1-3p) with specific substrate preferences 30 were characterized (Toke and Martin, 1996; Oh et al., 1997). Tailoring the length of fatty acids has among others been applied in the oleaginous yeast Y. lipolytica (Rigouin et al., 2018; Wang et al., 2022). The genome contains two genes encoding fatty acid elongases (YlELO1 and YlELO2). YlELO1, is responsible for the elongation of relatively shorter fatty acids (e.g., C14-16) while YlELO2 prefers C18 fatty acids (VLFAs; Rigouin et al., 2018). The Sbelo1 NA sequence (SEQ ID N°13) is: 5 ATGACCAACCTTGCATCGGAGTTTGAGCAGGCCGCAAAGGTTGCGTTTCACGGTATGCC GTCCAGCTTGCCGAATGCTGTTGACATCCGAGTCCCTACTGCGGACCAGCCCTTTGGC ATCGCACTGTGGCCCATATTCCGATACTGTGCTTACGCCTTGACCAACTATGACGTTGAG TCGTTCAAGTACGAATACAACTCCGATGTGCCTCTCTCGAAGATCCCTCATGTCCTTCTC GCGATCCTTTCTTACTACATCATCATCTTCGGAGGTCGTTGGTTGTTGAAGAGTGTGCGCC 10 CCATAAAATTTGGCTTGATCTTCAAGCTTCACAACTTACTTCTCACATTGTTGAGTGGTGGC TTACTGGCTTTGCTCGTCGAACAGACGTTTCCCATCATCGTGCACCACGGCGTGTTTTTC TCTATTTGCAGCGCGCAAGCCTGGACTCAAAAGATTGAGCTCATTTACTACCTCAATTATT TGACCAAGTATTACGAGTTGATCGACACTATCTTCATGGTGTTCCGCAAACGACCTTTGAC TTTCCTTCACACGTATCACCACGGTGCTACTGCCTTGCTTTGCTTCAGCCAGCTGAACGG 15 GAAAACATCTGTCAGCTACGTTCCCATCTCGCTTAACCTTGCCGTTCACGTGGTGATGTA TTTCTACTACTTCCTGTCTGCGTGTGGGATCCGTCCATGGTGGAAGACCTGGGTCACTCG ATTCCAGATTATCCAGTTCGTCATTGATCTTGGCTTTGTTTACTTCGCAACCTATACCCACT TCGTCAGCCGTCTTAACCTGAACTTGCCCAACATGGGAACTTGTGCTGGCGAAGAGTCA GCTGCTATCGATGGGTGCTTGATTTTGTCTTCTTACTTGGTTCTGTTCATCTCGTTCTACATC 20 AAGTCTTACATTGTTGCTCCGGCCGCACGCAAGGCAGCCCGGGAAGAGCAAAAGGCC GAAGCCGCGCCTAAGAGCCCCAGCGTAAGGGAGGAGACCAAAGCAGCTCTCAATTCT GCTGCGCCCGTATCAGCTTCTCCCCGCAAGCGAGCAACTAGGAGTCGCAAGGCCTAA The SbELO1 AA sequence (SEQ ID N°14) is: 25 MTNLASEFEQAAKVAFHGMPSSLPNAVDIRVPTADQPFGIALWPIFRYCAYALTNYDVESFKYE YNSDVPLSKIPHVLLAILSYYIIIFGGRWLLKSVRPIKFGLIFKLHNLLLTLLSGGLLALLVEQTFPII VHHGVFFSICSAQAWTQKIELIYYLNYLTKYYELIDTIFMVFRKRPLTFLHTYHHGATALLCFSQL NGKTSVSYVPISLNLAVHVVMYFYYFLSACGIRPWWKTWVTRFQIIQFVIDLGFVYFATYTHFV SRLNLNLPNMGTCAGEESAAIDGCLILSSYLVLFISFYIKSYIVAPAARKAAREEQKAEAAPKSPS 30 VREETKAALNSAAPVSASPRKRATRSRKA The Ylelo1 NA sequence (SEQ ID N°109) is: ATGCTCTCGTCAATCTCGCCCGACCTATACTCGTCCTTCTCGTTCAAAAACTCGCTCGCC GAGGCCATGCCCTCCGTGCCACACGAACTCATCAACTCAAAAACACTCTCATGGATGTA CAATGCCTCTCTGGACATTCGGGTTCCTCTGACTATCGGAACCATCTACGCCGTCTCCG TGCACCTGACCAACTCATCTGAACGAATCAAGAAACGCCAGCCCATTGCCTTTGCCAAG 5 ACCGCACTCTTCAAGTGGCTCTGTGTCCTCCACAATGCAGGTCTGTGTCTCTACTCAGCA TGGACCTTTGTCGGTATCCTCAACGCCGTCAAACACGCCTACCAAATCACAGGAGACAG CTCCGCCCCCTTCTCCTTCAACACCCTCTGGGGATCGTTTTGTTCACGTGACTCCCTCT GGGTCACCGGCCTCAACTACTACGGATACTGGTTCTATCTGTCCAAATTCTACGAAGTGG TGGACACCATGATCATCCTCGCAAAGGGAAAACCGTCCTCAATGCTCCAGACATACCAC 10 CACACCGGCGCCATGTTCTCCATGTGGGCCGGCATCCGATTCGCCTCTCCCCCCATCT GGATCTTTGTGGTTTTCAACTCCCTCATCCACACAATCATGTACTTTTACTACACCCTCAC CACCCTCAAGATCAAGGTTCCCAAGATCCTCAAGGCATCTCTGACCACCGCCCAGATC ACCCAGATTGTCGGAGGTGGCATCCTGGCTGCCTCCCACGCCTTTATTTATTACAAGGA CCACCAGACTGAGACCGTCTGTTCTTGTCTCACTACCCAGGGTCAGTTTTTCGCTCTCGC 15 CGTCAATGTCATCTATCTGAGTCCTCTGGCCTATCTCTTTATTGCCTTCTGGATTCGATCTT ACTTGAAGGCCAAGTCCAACTAG The YlELO1 AA sequence (SEQ ID N°110) is: MLSSISPDLYSSFSFKNSLAEAMPSVPHELINSKTLSWMYNASLDIRVPLTIGTIYAVSVHLTNS 20 SERIKKRQPIAFAKTALFKWLCVLHNAGLCLYSAWTFVGILNAVKHAYQITGDSSAPFSFNTL WGSFCSRDSLWVTGLNYYGYWFYLSKFYEVVDTMIILAKGKPSSMLQTYHHTGAMFSMWA GIRFASPPIWIFVVFNSLIHTIMYFYYTLTTLKIKVPKILKASLTTAQITQIVGGGILAASHAFIYYKD HQTETVCSCLTTQGQFFALAVNVIYLSPLAYLFIAFWIRSYLKAKSN 25 The Ylelo2 NA sequence (SEQ ID N°111) used for expression in S. bombicola is: ATGAGCGCCGTCCCTATTGAATTCAACGTCCCCTCCGTGGACCGACCCTTTGGTATCTA CCTCTGGGCCATCTTTGACCAGGCCTGGGAGAAGCTTTTCGGCTGGCCCGCGTCCTCT TTCATTTTCGTGCGAAATGACCCCAACATCCCCTTTTCCTCTACCCCTCCCGTGATCATTG CCATCATTGTGTACTACATTGTCATCTTTGGCGGCCGAGAGGTGATGCGAAACCTGTCTC 30 CCATCCGACTCAACTGGCTCTTCCAGATCCACAACATCTTCCTCACCCTTCTGTCCGGT ATGCTCCTCCTCCTCCTCGTTGAGCAGCTCTTCCCCATCATTGTCCGACAGGGTATCCT CTACGCCATCTGCGACTACGGATCTTGGACTCAGCCCATTGTCTTCTGCTACTACCTCAA CTACCTGACCAAGTACTTTGAGCTGATCGACACCGTTTTCCTTGTGCTGCGAAAGAAGAA GCTGACTTTCCTCCACACCTACCACCATGGTGCCACTGCTCTTCTGTGCTACACCCAGC TCATTGGTAAGACCTCGGTCTCTTGGGTCCCCATCACCCTTAACCTGTTTGTCCACGTTG TCATGTACTTCTACTACTTCCTGGCTGCGCGAGGTATCCGAGTGTGGTGGAAGGAGTGG 5 GTCACCCGGCTCCAGATCATCCAGTTCGTTATCGATCTTGGATTTGTCTACTTTGCCTCTT ACACCTACTTCACCTCTACCTACTGGCCCTGGATGCCCAACATGGGCTCTTGTGCCGG CGAGGAGTTTGCTGCTATTTACGGCTGTGGTCTGCTGACCTCTTACCTCTTCCTCTTCATC GCCTTCTACATCAACTCTTACCGAAAGCCCTCTTCCAAGGGACCTTCCAAGCCTGTTGTT GCTGTCGATGGCCCTGTTGGCGGCGTCAACGCCCAGACTGGTGCTTCTCGAGGCCAG 10 ACCACTACCCGATCTCGACGAGCATAA The YlELO2 AA sequence (SEQ ID N°112) used for expression in S. bombicola is: MSAVPIEFNVPSVDRPFGIYLWAIFDQAWEKLFGWPASSFIFVRNDPNIPFSSTPPVIIAIIVYYI VIFGGREVMRNLSPIRLNWLFQIHNIFLTLLSGMLLLLLVEQLFPIIVRQGILYAICDYGSWTQPI 15 VFCYYLNYLTKYFELIDTVFLVLRKKKLTFLHTYHHGATALLCYTQLIGKTSVSWVPITLNLFVHV VMYFYYFLAARGIRVWWKEWVTRLQIIQFVIDLGFVYFASYTYFTSTYWPWMPNMGSCAGEE FAAIYGCGLLTSYLFLFIAFYINSYRKPSSKGPSKPVVAVDGPVGGVNAQTGASRGQTTTRSRR A 20 In addition, the present invention also relates to the usage of a modified yeast strain comprising an over-expressed elongase gene and / or an over-expressed CYP52E3 gene to produce long chain dicarboxylic acids. Overexpression of an endogenous gene or expression of a heterologous gene refers to a any genetic modification technique known by the person skilled in the art wherein a 25 gene of interest is manipulated to produce higher-than-normal levels of its corresponding mRNA and protein product within a host organism. Non-limiting examples of how overexpression can be achieve include: modification of the promoter type, strength, inducibility and / or regulation (here both an endogenous or heterologous promoter can be used), modification of the gene copy number, post-translational 30 modifications that enhance protein stability and / or activity, co-express chaperones or foldases to improve protein folding and reduce aggregation, use of enhancer elements to augment transcriptional activity, codon optimization to optimize codon usage for the host organism in this way enhancing translational efficiency and protein yield, use RNA stabilizing elements, such as AU-rich elements (ARE) removal or the inclusion of stabilizing sequences in the 3’ UTR, to increase mRNA stability and longevity, attach fusion partners or tags to enhance solubility and / or stability, utilize signal peptides to 5 direct the protein to specific cellular compartments, improving folding and processing and / or increased concentration at the site of action, optimize culture parameters, or combinations thereof. The invention further relates to the usage of a modified yeast strain as described above 10 wherein said yeast strain is a yeast strain selected from the yeast strains Starmerella (Candida) bombicola, Starmerella (Candida) apicola, Starmerella (Candida) batistae, Starmerella (Candida) magnolia, Candida gropengiesseri, Starmerella (Candida) floricola, Starmerella (Candida) riodocensis, Starmerella (Candida) stellata, Starmerella (Candida) kuoi, Starmerella cerana, Rhodotorula muciliginosa, Candida 15 rugosa, Cyberlindnera samutprakarnensis, Cryptoococcus sp. VITGBN2, Lachancea thermotolerans, Candida sp. NRRL Y-27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis sp., Wickerharmiella domericqiae, Candida antarctica, Pseudohyphozyma antarctica, Candida lipolytica and a sophorolipid-producing strain or potential sophorolipid-producing strain of the Starmerella clade and beyond. 20 The invention further relates to the usage of a modified strain as described above wherein said long chain dicarboxylic acids are enriched in compounds having a chain length of 20, 22 or / and 24 carbon atoms. 25 Examples C18 LCDA production 1.1 Introduction 30 A non-pathogenic and industrially relevant yeast, Starmerella bombicola has high potential to be a good platform organism to produce LCDAs, since it has been proven to produce high amounts of fatty acid-derived products, such as sophorolipids and hydroxyl fatty acids (De Graeve et al., 2019; Pekin et al., 2005). The main route to lose the fatty acid input for LCDA production is energy generation through β-oxidation. Moreover, our desired products, LCDAs, are also degraded in this pathway. In order to establish LCDA production, we decided to disable β-oxidation by deleting the 5 pox1 gene in the S. bombicola genome of the ura3 negative strain by homologous recombination. The open reading frame of the pox gene was successfully replaced by the nourseothricin resistance marker gene (nat1) cassette and it was confirmed with colony PCR. Note that also other individual genes of the β-oxidation could have been disabled to achieve a similar eƯect. 10 A second phase in our strain development relates to avoiding fatty acid substrate loss in sophorolipid synthesis (see Fig. 1). Sophorolipid biosynthesis is initiated by cytochrome P450 monooxygenase, CYP52M1 coupled with its redox partner, cytochrome P450 reductase (CPR) creating a hydroxylated fatty acid, which is then further processed by glucosyltransferase 1 (UGTA1), which conjugates a hydroxyl fatty 15 acid with UDP-glucose (Saerens, et al., 2011; Van Bogaert et al., 2009). For sequential strain development, the open reading frame of ugta1 was deleted by homologous recombination with the ura3 marker cassette. The pox and ugta1 double deletion strain was evaluated for LCDA production capacities. 20 1.2 Materials and methods 1.2.1 Strain construction For the gene deletion construct of pox1 and ugta1, 1 kb long upstream and downstream regions of the target open reading frame were amplified via highfidelity PCR. The gene fragments and a selection marker gene cassette (e.g., nat1 and ura3) were assembled 25 through CPEC (Quan & Tian, 2009). The gene deletion construct was amplified to introduce into S. bombicola competent cells via electroporation (2.5 kV; 200 Ω; (Saerens et al., 2011)). The successful homologous recombination events at both the 5’ and 3’ of the cassette was confirmed with colony PCR. The used primer or oligonucleotide sequences and their names are listed in Table 2. Table 2: Names and sequences of primers or oligonucleotides used in the context of Example 1. For pox1 deletion plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 15: TP2406_pJet_pox_R AGGAGTTAGGAATTGACTACCAGACCAAGGCTTGCTGAAAAACTCGAGCC SEQ ID N° 16: P668_pox_up_F CCTTGGTCTGGTAGTCAATTCCTAAC SEQ ID N° 17: TP2407_pox_up_R TTTTTAAAACTTTGATTTCGACAATCGGGG SEQ ID N° 18: TP2409_nat_pox_F CCCCGATTGTCGAAATCAAAGTTTTAAAAAGACATCCGATGTGTAGTTAATCA SEQ ID N° 19: TP2410_nat_pox_R GATTCAAATAATGCCAAGTTTGCTCTTCATATACGCTTTGCAGGTGGTGTTG SEQ ID N° 20: TP2408_pox_down_F ATGAAGAGCAAACTTGGCATTATTTGAATC SEQ ID N° 21: P2085_upPOXdown_rev CAGCGAGCCTCGACCCATTATGC SEQ ID N° 22: TP2405_pJet_pox_F GTCGAGGCTCGCTGGATCTTTCTAGAAGATCTCCTACAATATTCTCAG For amplification of pox1 deletion construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 16: P668_pox_up_F CCTTGGTCTGGTAGTCAATTCCTAAC SEQ ID N° 23: P669_pox_down_R CCAGCGAGCCTCGACCCATTATG For pox1 deletion yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 24: TP2504_PoxKO_chk_F CGCAGAGTAAAGCGCGTAC SEQ ID N° 25: P1773_nat1_check_rv_B TCGGTGGTGAAGGACCCATC SEQ ID N° 26: P2365_nat1_sequencing_1 ATCGAGGCACTGGATGGGTCCTTCAC SEQ ID N° 27: TP2619_PoxKO_chk_R2 CTTTTGGCGGCTCGAATTCC For ugta1 deletion plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 28: TP1928_pUC19_R AAGCTTGGCGTAATCATGGTC SEQ ID N° 29: TP2801_Sbugta1_UF_F AACAGCTATGACCATGATTACGCCAAGCTTCTGTTTCCATCCATTTCCGCTGAGTG TAAATATCC SEQ ID N° 30: TP2802_Sbugta1_UF_R CAGAATTCGAACACTGCCATCATGGTTCAATATATGGCCTGAAAGAGGCAAGCCG SEQ ID N° 31: TP2960_ura_fw for BB TTGAACCATGATGGCAGTGTTCG SEQ ID N° 32: TP2915_ura rv CACTATACACATCGTCATCAACTC SEQ ID N° 33: TP2803_Sbugta1_DF_F GCCATGGAGTTGATGACGATGTGTATAGTGAATCGTACGATCAAATCAGATCAG SEQ ID N° 34: TP2804_Sbugta1_DF_R ACGACGGCCAGTGAATTCGAGCTCGGAACCACTCCTAGAAAAGAAATTGACCAG SEQ ID N° 35: TP2574_pUC19_BB_F GGTTCCGAGCTCGAATTCAC For amplification of ugta1 deletion construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 36: TP2816_ugtA1KOfw CTGTTTCCATCCATTTCCGC SEQ ID N° 37: TP2817_ugtA1KOrv ACTCCTAGAAAAGAAATTGACCAG For ugta1 deletion yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 38: P1101_ugta1KO-F CTCGGGTAACTTTAGCCATC SEQ ID N° 39: P295_ura3seqRev GGGCGTTCCTCCTCTGATGTTC SEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 41: P2036_ugta1KO-R TTCCTCCTTCCTTGCCTCATTCC 1.2.2 Shakeflask experiment and growth monitoring Engineered strains were grown on 3C plates (100 g / l glucose, 10 g / l yeast extract, 1 g / l urea, 20 g / l agar) to adapt to a high glucose condition before pre-culture in 5 ml Lang medium (Lang et al., 2000; pH 5.8) for 2 days at 30 °C and 200 rpm. For monitoring cell growth and the production of LCDAs, the pre-culture was diluted to a starting OD600of 0.5 in a 250 ml volume non-baƯledflask containing a total of 50 ml Lang medium at 30 °C and 200 rpm. The cell growth was monitored by determining colony forming units (CFU) after spreading 10-6diluted cell cultures on 3C agar plates and incubation at 30 °C for 3 days. In addition, the pH change during the cultivation was monitored. After 48 hours of cell growth, the production phase was initiated by supplementing with 37.5 g / l of rapeseed oil. 1.2.3 Glucose quantification 5 To monitor the glucose consumption during the shake flask cultivation and fermentation, cells were moved by centrifugation at 13,000 rpm, for 10 min to harvest the supernatant. The glucose concentration was quantified using an HPLC-RID (Shimadzu) equipped with a RezexTMROA-Organic Acid H+LC Guard column (50 × 7.8 mm; Phenomenex; 03B-0138-K0).5 mM sulfuric acid was used as the eluent atflow rate 10 0.1 ml / min. Glucose was used for the calibration curve. 1.2.4 Thin layer chromatography (TLC) For detection and quantification of the produced LCDAs, 8 ml of the cell culture after 8 days of cultivation was vigorously mixed with the same volume of ethyl acetate. 15 Continuously the mixture was shaken at 200 rpm for 1 h. The ethyl acetate phase was harvested at 7,690 × g for 20 min and air-dried before detection through TLC and quantification in HPLC-MS. For LCDA detection on TLC plates, the running solvent (n- heptane / dimethyl ether / acetic acid (50:50:1, by vol) was used. The plates were stained with 0.4 g / l of bromocresol green dissolved in ethanol to detect LCDAs. 20 1.2.5 LCDA detection and quantification For quantification of the produced LCDAs, dried LCDAs were dissolved in 100% (v / v) ethanol. The LCDA profile was analysed using a UPLC-MS (Shimadzu) equipped with a Kinetex® 2.6 µm Polar C18100 Å, LC column (100 × 2.1 mm; Phenomenex; 00D-4759- 25 AN). DiƯerent LCDAs were separated using an acetonitrile / water gradient (0.1% formic acid) 0-1 min, 25%, 1-8 min, 100%, 8-10 min 25%,flow rate 0.35 ml / h-1. To quantify the LCDAs, commercial LCDAs (C12:0, Merck, D1009; C14:0, Merck, D221201; C16:0, Merck, 177504; C18:0, TCI, O0222; C22:0, Merck, 306673) were used for the calibration curves. 1.2.6 Fermentation process and optimization The fermentation processes were performed in a 1 l fermenter with a working volume of 0.7 l (SciVario® twin 1 l vessel; Eppendorf) in the medium described by Lang et al. (pH 5 5.8). A late stationary phase liquid culture was inoculated at 10 % (v / v). To prime the cells to an oil-containing environment, 0.5% (v / v) rapeseed oil was added. After the growth phase of 48 h, rapeseed oil was continuously fed at 0.3 ml / h until the end of the fermentation process. The stirring rate was held at 600 rpm and the airflow rate and temperature were maintained at 42 l / h (1 vvm) and 30°C, respectively. For testing 10 diƯerent pH conditions during the production phase, pH was adjusted to pH 3.5, 4.6, 5.2, 5.8, and 8.0 by the addition of 25% (w / v) NaOH. In the case of pH 8.0, pH was adjusted to pH 5.8 during the growth phase, and then gradually increased to pH 6.5 (48 h), pH 7.0 (72 h), pH 7.5 (96 h), pH 7.7-8.0 (120 h). For fed-batch fermentation, glucose was supplemented to maintain the concentration above 40 g / l. If necessary, the 15 harvested cell culture was acidified with 5 M HCl before the LCDA extraction and secondary extraction were performed. 1.3 Results 1.3.1 Creation and evaluation of the Δpox1Δugta1 S. bombicola strain 20 The pox1 gene was deleted by replacing it with the nourseothricin resistance marker gene (nat1) cassette by homologous recombination as described in the 1.2.1 Strain construction. Next, the ugta1 gene in the genome was replaced by the ura3 cassette as explained in Section 1.2.1 Strain construction. Uracil auxotrophic colonies were subjected to colony PCR and we continued with a colony showing the correct amplicon 25 sizes. In essence, the knock-outs of pox1 and ugta1 were successfully executed in one strain. 1.3.2 Evaluation of the Δpox1Δugta1 strain in shake-flask cultivations To understand the cellular physiology of the novel strain cell growth a shakeflask cultivation experiment was conducted as explained in section 1.2.2 Shakeflask experiment. Glucose consumption, and pH change over the cultivation time (Fig.2) 5 were monitored as well as LCDA production (Fig.3). As the cell growth entered the stationary phase, the glucose consumption speed was increased. Approximately the half amount of the initial glucose (120 g / l) was left after 8 days of cultivation (Fig.2B). From the beginning of the cultivation, pH decreased from pH 5.8 to pH 3 (Fig.2C). LCDAs from the cell culture were extracted and subjected to TLC with reference LCDAs. 10 A strong LCDA signal (lower band) was detected when comparing the height of the band with the bands of reference LCDAs (Fig.3A). We analysed the LCDA production profile and quantified the prominent LCDAs, mainly C16 and C18 LCDAs. The total amount of LCDAs produced in the pox1 and ugta1 double deletion mutant in shakeflask after 8 days reached 1 g / l (Fig.3B). In conclusion, the engineered strain is capable of the high 15 bioconversion of fatty acids into LCDAs using the endogenous ω-oxidation pathway. 1.3.3 Fermenter experiments The Δpox1Δugta1 strain was taken to a fermenter set-up in a 0.7 L working volume. The same medium as in shakeflasks was applied. Since pH is a crucial factor for high 20 production of sophorolipids S. bombicola, diƯerent pHs (e.g., pH 3.5, pH 4.6, pH 5.2, pH 5.8, pH 6.2, pH 8) were tested. Note that optimal pH for sophorolipid production is 3.5 (Gobbert et al., 1984). For optimal dicarboxylic acid and LCDA production in C. tropicalis, pH should be higher than 7.5 (see before). Surprisingly, it turned out that a diƯerent pH strategy compared to both established production processes need to be 25 followed for LCDA production in S. bombicola: optimal LCDA production was achieved at pH 5.8 (Fig.4). After 366 hours or 14 days of cultivation 9.9 g / l LCDAs were obtained (Fig.4C). This is a promising amount for a non-pathogenic yeast in which further genetic improvement steps are still possible. Other pH values of 4.6, 5.2 and 6.4 were evaluated in the same fermenter set-up, but 30 LCDA yields were inferior compared to pH 5.8 ranging from 2.3 to 3.2 g / l after 14 days of cultivation. The major LCDA constituents produced by the Δpox1Δugta1 S. bombicola strain were C18:1, C18:0; C18:2, C16:0, C16:1 and C16:2 dicarboxylic acids. A detailed analysis of the composition is given in Example 2, Fig.6. 5 2.1 Introduction In order to further increase the LCDA producing capacities, we decided to also knock- out the faa1 gene. This gene is responsible for converting free fatty acids to acyl-CoA; in 10 this way shuttling them to various cellular processes such as triacylglyceride build-up, integration in cell-membranes or protein lipidations (Fig.1). Again, by blocking this pathway, increased fatty acid influx is available for LCDA synthesis. Nevertheless, such knock-out could have a negative eƯect on cell viability and hence also negatively influence LCDA production. Surprising, and despite an eƯected growth, the 15 Δpox1Δugta1Δfaa1 strain was able to clearly outperform its parental Δpox1Δugta1 strain regarding LCDA production. 2.2 Materials and methods 2.2.1 Strain construction 20 Before knocking out the faa1 gene, the ura3 marker needs to be recovered. Hence, the ura3 gene at the ugta1 locus, which was used as a selection marker for the deletion of ugta1, was removed by homologous recombination with an amplified gene fragment containing the combined 1 kb long upstream and downstream regions of the ugta1 locus. The absence of the ura3 gene was verified on SD agar plates (6.7 g / l of yeast 25 nitrogen base without amino acid, 0.77 g / l of complete supplement mixture, 20 g / l of glucose and 20 g / l of agar) supplemented with 1 g / l 5-fluoroorotic acid (5-FOA). The marker recycling was additionally confirmed with colony PCR. For the faa1 gene deletion construct, 1 kb long upstream and downstream regions of the target open reading frame were amplified via PCR. The gene fragments and a selection marker gene 30 cassette (ura3) were assembled through CPEC (Quan & Tian, 2009). The gene deletion construct was amplified to introduce into S. bombicola competent cells via electroporation. The transformants were selected on SD-ura agar plates (6.7 g / l of yeast nitrogen base without amino acid, 0.77 g / l of complete supplement mixture without uracil, 20 g / l of glucose and 20 g / l of agar). The successful homologous recombination event was confirmed with colony PCR. The used primer or oligonucleotide sequences and their names are listed in Table 3. Table 3: Names and sequences of primers or oligonucleotides used in the context of Example 2. For ura3 recycling at the ugta1 locus plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 28: TP1928_pUC19_R AAGCTTGGCGTAATCATGGTC SEQ ID N° 42: TP2855_ugtA1 UF_pUC 30_fw AACAGCTATGACCATGATTACGCCAAGCTTCTGTTTCCATCCATTTCCGC SEQ ID N° 43: TP2856_ugtA1 UF_DF 30_rv TCTTCCCTGATCTGATTTGATCGTACGATTTATATGGCCTGAAAGAGGCAA GC SEQ ID N° 44: TP2857_ugtA1 DF_fw AATCGTACGATCAAATCAGATCAGG SEQ ID N° 45: TP2858_ugtA1 DF_pUC 30_rv ACGACGGCCAGTGAATTCGAGCTCGGAACCACTCCTAGAAAAGAAATTG SEQ ID N° 35: TP2574_pUC19_BB_F GGTTCCGAGCTCGAATTCAC For amplification of ura3 recycling at the ugta1 locus construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 36: TP2816_ugtA1KOfw CTGTTTCCATCCATTTCCGC SEQ ID N° 37: TP2817_ugtA1KOrv ACTCCTAGAAAAGAAATTGACCAG For ura3 recycling at the ugta1 yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 38: P1101_ugta1KO-F CTCGGGTAACTTTAGCCATC SEQ ID N° 41: P2036_ugta1KO-R TTCCTCCTTCCTTGCCTCATTCC For faa1 deletion plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 46: TP100_FAA1up_cpec_pJet_F CTCGAGTTTTTCAGCAAGATTCCAGTTGCAGCTGTGTTAC SEQ ID N° 47: TP101_FAA1up_cpec_ura3_R CGAACACTGCCATCATGGTTCAACGGAGAACATACTCGTTTCG SEQ ID N° 48: TP102_ura3_cpec_FAA1up_F TACGAAACGAGTATGTTCTCCGTTGAACCATGATGGCAGTGTTCG SEQ ID N° 49: TP103_ura3_cpec_FAA1down_R GATGCTTGGGGGTTGATGACTCACTATACACATCGTCATCAACTC SEQ ID N° 50: TP104_FAA1down_cpec_ura3_F GTTGATGACGATGTGTATAGTGAGTCATCAACCCCCAAGCATCATAC SEQ ID N° 51: TP105_FAA1down_cpec_pJET_R GTAGGAGATCTTCTAGAAAGATGATTGGCTAGGCTATGGTTTATCG SEQ ID N° 52: TP106_pJET_cpec_FAA1down_F TAAACCATAGCCTAGCCAATCATCTTTCTAGAAGATCTCCTACAATATTC SEQ ID N° 53: TP107_pJET_cpec_FAA1up_R GTAACACAGCTGCAACTGGAATCTTGCTGAAAAACTCGAGCCATCC For amplification of faa1 deletion construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 54: TP3525_faa1_fw TCCAGTTGCAGCTGTGTTAC SEQ ID N° 55: TP3526_faa1_rv GATTGGCTAGGCTATGGTTTATCG For faa1 deletion yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 56: TP3523_faa1_region_fw ACCTGTTTGGAGTTGCTGC SEQ ID N° 57: TP3642_ura3-seq-rv GGGCGTTCCTCCTCTGATGTTC SEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 58: TP3524_faa1_region_rv GGAGAATCACTACATACGGCTG 2.2.3 Shakeflask experiment and growth monitoring Engineered strains were grown on 3C plates to adapt to a high glucose condition before pre-culture in 5 ml Lang medium (pH 5.8) for 2 days at 30 °C and 200 rpm. For monitoring cell growth and the production of LCDAs, the pre-culture was diluted to a starting OD600 of 0.5 in a 250 ml volume non-baƯledflask containing a total of 50 ml medium described by Lang et al. at 30 °C and 200 rpm. The cell growth was monitored by determining colony forming units (CFU) after spreading 10-6diluted cell cultures on 3C agar plates and incubation at 30 °C for 3 days. In addition, the pH change during the cultivation was monitored. After 48 hours of cell growth, the production phase was initiated by supplementing with 37.5 g / l of rapeseed oil. To compare the biomass between the parental strain (Δpox1Δugta1) and the triple deletion strain (Δpox1Δugta1Δfaa1), the dry cell weight after 8 days was measured. Furthermore, the cell growth of the pox1, ugta1 and faa1 triple deletion strain (Δpox1Δugta1Δfaa1) in 20 ml of Lang medium (OD600 of 0.5) at 30°C and 200 rpm was monitored in Cell Growth Quantifier (CGQ; Scientific Bioprocessing) to compare with the growth of the parental strain. TLC, analysis of glucose and LCDA concentration, and fermentation process were conducted as described for example 1. 2.3 Results 2.3.1 Creation and evaluation of the Δpox1Δugta1Δfaa1 S. bombicola strain As one can learn from Fig.1, additional deletion of faa1 could further channel fatty acids toward LCDA synthesis. To establish this third deletion, wefirst recycled the ura3 marker present at the former ugta1 locus. This strain was successfully created, and the ura3-negative double deletion strain was used to introduce the faa1 deletion. Colony PCR on both the 5’ and 3’ integration sites confirmed that we obtained correct triple deletion strains (Δpox1Δugta1Δfaa1). To understand the cellular physiology of the novel strain, we determined the dry cell weight (DCW) and pH profile during shakeflask fermentation (Fig.5). As the strain significantly grew slower compared to the parental strain at 16 h when the growth rate (µ = (log10Backscatter t2-log10Backscatter t1) / (t2-t1)) of the parental strain reached the µmax. At 16 h, for the triple deletion strain, a value of 0.032 ± 0.007 h-1was obtained, while this was 0.063 ± 0.002 h-1for the parental strain (Fig.5A). Furthermore, there was a significant reduction of the dry cell weight of the triple knock-out strain compared to the parental strain after 8 days of cultivation (Fig.5B). This clearly illustrated the growth deficiency caused by the faa1 mutation. 5 The high LCDA concentrations become visible in the culture broth by turning it into a turbid mixture (Fig.6A). Despite the aƯected growth, the triple knock-out strain clearly produced more LCDAs, especially if a longer incubation time to compensate for the slower growth was applied. C16 and C18 LCDAs were the dominant chain lengths in the LCDA mixture (Fig 6B). Upon similar cultivation time (8 days), a 2.5-fold increase 10 compared to the parental strain was observed, and even higher titres were obtained upon longer incubation time (12 days): the titre was improved from approximately 1 g / l for the parental strain to over 5 g / l: a very significant increase that could have a great contribution to the development of an industrially relevant process (Fig.6B). 15 2.3.2 Fermenter experiments with the Δpox1Δugta1Δfaa1 S. bombicola strain Since the additional deletion of faa1 significantly improved the total LCDA titer in shake flasks, the next step was to take the triple knock-out strain was to a fermenter 1 l set-up in a 0.7 l working volume while maintaining the pH at 5.8, as shown by us to be the optimal pH for LCDA production in S. bombicola. The bioprocess ran for 30 days to 20 obtain high LCDA concentrations. The dry cell weight (DCW), glucose and LCDA concentration in the fermenter were monitored (Fig.7). For stable cell activity during the long bioprocess, additional glucose was supplemented to maintain the glucose concentration above 40 g / l in the fermentation broth (Fig.7B). The maximum titer of LCDAs measured was 99.9 g / l (28 d). Note that due to the high 25 concentration and the continuous rapeseed oil feeding during the production phase, there were some deposits in the fermenter (walls, probes, lid), hampering correct sampling. Hence, we performed a total extraction of the broth at the end (30 d), which yielded 117.8 g / l. Maximum productivity was reached between day 16 and 18 (0.64 g / l / h; Fig.7C). During the entire bioprocess, a total of 147.86 mL of rapeseed oil was added 30 and in thefinal extraction, 38.64 mL was recovered. This means about 74% of the rapeseed oil was converted to LCDAs. The total amount of glucose added was 159 g, of which 108.5 g was consumed (68%). In conclusion, the engineered strain from Example 2 more eƯiciently converts fatty acids from oil to LCDAs at the optimal pH condition compared to the engineered strain from Example 1 and produces LCDAs in amounts 5 relevant for industrial processes. Example 3: Creation and evaluation of a S. bombicola strain over-expressing CYP52E3 and elongase-1 for increased production of ≥C20 LCDAs 3.1 Introduction 10 For both Δpox1Δugta1 and Δpox1Δugta1Δfaa1 S. bombicola strains limited amounts of longer LCDAs (≥C20) can be detected (Fig.6). S. bombicola, CYP52M1 is considered to have optimal activity towards C16 and C18 fatty acids, with some hydroxylating capacity towards longer chain fatty acids as well (Van Bogaert et al., 2009; Van Bogaert et al., 2014). Nevertheless, there are 7 additional CYP52 family genes annotated in the 15 S. bombicola genome, but it is not clear if and how they are expressed and what substrate range they can hydroxylate. We wanted to investigate if we can unluck this hidden power for the hydroxylation of very long-chain fatty acids (≥C20), hence resulting in longer LCDAs. To further change the profile of fatty acid pool and hance also thefinal LCDAs, the 20 abundant fatty acids (e.g., C16-18) supplied as feed stock can be elongated by fatty acid elongases. In this example, we relied on the endogenous elongase 1 from S. bombicola itself. 3.2 Materials and Methods 25 3.2.1 Cloning and strain engineering We started from the strain described in Example 1: Δpox1Δugta1 S. bombicola strain. Before overexpressing the endogenous CYP52 genes, the ura3 marker needs to be recovered. This was done as described in Example 2, Section 2.2.1. The endogenous CYP52 family genes were individually amplified via PCR and assembled with fragments of 1 kb long upstream and downstream regions of the CYP52M1 locus in plasmids. The gene overexpression constructs were integrated into the genome so that the CYP52M1 gene was replaced by the respective CYP52 family 5 genes and the CYP52 genes were put under the control of the CYP52M1 promoter, that has a high activity in the stationary phase (Van Bogaert et al., 2009). For overexpression of the endogenous gene encoding a putative fatty acid elongase (ELO1) at the pox1 locus, the open reading frame of the gene was amplified from the genome. The gene overexpression was put under the control of the constitutive and 10 strong gapd (glyceraldehyde-3-phosphate dehydrogenase) promoter and trpC (Aspergillus nidulans tryptophan synthase) terminator (Nakayashiki et al., 2005; Van Bogaert et al., 2008; Li et al., 2016). To insert the overexpression cassette at the pox1 locus, where the nourseothricin resistance gene (natR) cassette is located in the E3 strain, the hygromycin B resistance gene (hygR) cassette was used for a counter- 15 selection on YPD agar plates containing either 500 μg / l of hygromycin B (Merck, Darmstadt, Germany, H3274) or 600 μg / l of nourseothricin (Jena Bioscience, Jena, Germany, AB-102l). The hygromycin B resistance gene expression was under the control of native eno (enolase) promoter (Li et al., 2016) and TK terminator (Herpes simplex virus tyrosine kinase). Only transformants showing hygromycin B resistance were 20 subjected to colony PCR to verify the correct genome modification. The used primer or oligonucleotide sequences and their names are listed in Table 4. Table 4: Names and sequences of primers or oligonucleotides used in the context of Example 2. For CYP52M1 deletion plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 28: TP1928_pUC19_R AAGCTTGGCGTAATCATGGTC SEQ ID N° 59: TP2910_pUC19_cyp52m1_fw AACAGCTATGACCATGATTACGCCAAGCTTATGGCTCAACAATTGCGGCTGGTG SEQ ID N° 60: TP2911_CYP52m1 UF_rv ATATGTACTTTTCAATATGATAAACGGAG SEQ ID N° 61: TP2957_CYP52m1-ura-fw TCTCCGTTTATCATATTGAAAAGTACATATTTGAACCATGATGGCAGTGTTCG SEQ ID N° 32: TP2915_ura rv CACTATACACATCGTCATCAACTC SEQ ID N° 62: TP2916_ura_cyp52m1 DF_fw GCCATGGAGTTGATGACGATGTGTATAGTGGTTTCTTAGCCTCCCATGGAAG SEQ ID N° 63: TP2917_pUC19_cyp52m1 DF_rv ACGACGGCCAGTGAATTCGAGCTCGGAACCAATTGTTCGATGGATAGCTTTG SEQ ID N° 35: TP2574_pUC19_BB_F GGTTCCGAGCTCGAATTCAC For amplification of CYP52M1 deletion construct Name Oligonucleotide sequence (5’→3’)SEQ ID N° 64: TP2959_Sbcyp52m1_fwATGGCTCAACAATTGCGGCTGGSEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For CYP52M1 deletion plasmid yeast colony PCR SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCC SEQ ID N° 39: P295_ura3seqRev GGGCGTTCCTCCTCTGATGTTC SEQ ID N° 40: P1506_Ura3_promstudy_seq1 CCCAATATGCAGGTGGAGTG SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For CYP52E3 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 60: TP2911_CYP52m1 UF_rv ATATGTACTTTTCAATATGATAAACGGAG SEQ ID N° 67: TP3404_CYP52E3-fw TCTCCGTTTATCATATTGAAAAGTACATATATGAACATTAATTTCTCTGACGTGC SEQ ID N° 68: TP3405_tPRY2-CYP52E3-rv CTGGCCGCGTCGGACCGAGTGATGAGCTGTCTAGCGAATGAACTTCGCTATG SEQ ID N° 69: TP3403_tPRY2-BB-fw ACAGCTCATCACTCGGTCC For amplification of CYP52E3 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 64: TP2959_Sbcyp52m1_fw ATGGCTCAACAATTGCGGCTGG SEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For CYP52E3 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCCSEQ ID N° 70: TP3432_CYP52E3-seq-rvAGAAGTGCCGCAGTGGTATCSEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For CYP52E4 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 60: TP2911_CYP52m1 UF_rv ATATGTACTTTTCAATATGATAAACGGAGSEQ ID N° 71: TP3406_CYP52E4-fwTCTCCGTTTATCATATTGAAAAGTACATATATGTTTATTGGACTCTCAGACGSEQ ID N° 72: TP3407_tPRY2-CYP52E4-rv CTGGCCGCGTCGGACCGAGTGATGAGCTGTCTAGCGGATGAACTTTGCCATTAC SEQ ID N° 69: TP3403_tPRY2-BB-fw ACAGCTCATCACTCGGTCC For amplification of CYP52E4 overexpression construct Name Oligonucleotide sequence (5’→3’)SEQ ID N° 64: TP2959_Sbcyp52m1_fwATGGCTCAACAATTGCGGCTGGSEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For CYP52E4 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCC SEQ ID N° 73: TP3433_CYP52E4-seq-rv TGTTTAGAGCCTGGTCACGG SEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For CYP52E5 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 60: TP2911_CYP52m1 UF_rv ATATGTACTTTTCAATATGATAAACGGAG SEQ ID N° 74: TP3408_CYP52E5-fw TCTCCGTTTATCATATTGAAAAGTACATATATGTTTATTGGACTCTCAGACGCCCTTG SEQ ID N° 75: TP3409_tPRY2-CYP52E5-rv CTGGCCGCGTCGGACCGAGTGATGAGCTGTCTAGCGGATGAACCTTGCCATTACSEQ ID N° 69: TP3403_tPRY2-BB-fwACAGCTCATCACTCGGTCC For amplification of CYP52E5 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 64: TP2959_Sbcyp52m1_fw ATGGCTCAACAATTGCGGCTGG SEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For CYP52E5 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCC SEQ ID N° 76: TP3434_CYP52E5-seq-rv CCATAACTCGGACACTCAGC SEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For CYP52E6 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 60: TP2911_CYP52m1 UF_rv ATATGTACTTTTCAATATGATAAACGGAG SEQ ID N° 77: TP3410_CYP52E6-fw TCTCCGTTTATCATATTGAAAAGTACATATATGATTATTGATCTTTCAGACGCGSEQ ID N° 78: TP3411_tPRY2-CYP52E6-rvSEQ ID N° 69: TP3403_tPRY2-BB-fwCTGGCCGCGTCGGACCGAGTGATGAGCTGTCTAACGAATGAATCTCGCCATCACAGCTCATCACTCGGTCCFor amplification of CYP52E6 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 64: TP2959_Sbcyp52m1_fw ATGGCTCAACAATTGCGGCTGG SEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For CYP52E6 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCC SEQ ID N° 79: TP3435_CYP52E6-seq-rv AGACTTGCTCACGGCTGAACTG SEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For CYP52N1 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’)SEQ ID N° 60: TP2911_CYP52m1 UF_rvATATGTACTTTTCAATATGATAAACGGAGSEQ ID N° 80: TP3412_CYP52N1-fw TCTCCGTTTATCATATTGAAAAGTACATATATGATTCTTTATGCTGTGCTGGGCG SEQ ID N° 81: TP3413_tPRY2-CYP52N1-rv CTGGCCGCGTCGGACCGAGTGATGAGCTGTTTAACTCAGTTTCACTCGGACGCC SEQ ID N° 69: TP3403_tPRY2-BB-fw ACAGCTCATCACTCGGTCC For amplification of CYP52N1 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 64: TP2959_Sbcyp52m1_fw ATGGCTCAACAATTGCGGCTGG SEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For CYP52N1 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCC SEQ ID N° 82: TP3436_CYP52N1-seq-rv GACTCGCCGAATAGAAAGTG SEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For CYP52N2 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 60: TP2911_CYP52m1 UF_rv ATATGTACTTTTCAATATGATAAACGGAG SEQ ID N° 83: TP3414_CYP52N2-fw TCTCCGTTTATCATATTGAAAAGTACATATATGATTTTTTATGCTGTGCTTGGSEQ ID N° 84: TP3415_tPRY2-CYP52N2-rvSEQ ID N° 69: TP3403_tPRY2-BB-fwCTGGCCGCGTCGGACCGAGTGATGAGCTGTTTAACTCAGTTTTACTCGGACACAGCTCATCACTCGGTCCFor amplification of CYP52N2 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 64: TP2959_Sbcyp52m1_fw ATGGCTCAACAATTGCGGCTGGSEQ ID N° 65: TP2961_Sbcyp52m1_rvAATTGTTCGATGGATAGCTTTGGFor CYP52N2 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCC SEQ ID N° 85: TP3437_CYP52N2-seq-rv GGTTCGACAAGTTTGCCAAG SEQ ID N° 40: P1506_Ura3_promstudy_seq_1 CCCAATATGCAGGTGGAGTG SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For CYP52N3 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’)SEQ ID N° 60: TP2911_CYP52m1 UF_rvATATGTACTTTTCAATATGATAAACGGAGSEQ ID N° 86: TP3416_CYP52N3-fw TCTCCGTTTATCATATTGAAAAGTACATATATGATTTTTTATGCTGTGCTTGG SEQ ID N° 87: TP3417_tPRY2-CYP52N3-rv CTGGCCGCGTCGGACCGAGTGATGAGCTGTTTAACTCAGTTTTACTCGGAC SEQ ID N° 69: TP3403_tPRY2-BB-fw ACAGCTCATCACTCGGTCC For amplification of CYP52N3 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 64: TP2959_Sbcyp52m1_fw ATGGCTCAACAATTGCGGCTGG SEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For CYP52N3 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 41: P2036_UGTA1KO_cass_fw TTCCTCCTTCCTTGCCTCATTCC SEQ ID N° 88: TP3438_CYP52N3-seq-rv ATCTCAATGCCCTTGCTGTC SEQ ID N° 40:P1506_Ura3_promstudy_seq_1CCCAATATGCAGGTGGAGTGSEQ ID N° 66:P2253_check_downCYP52M1TCGACTCGCCAAATTCCATCGGPlasmid construct for ura3 recycling after CYP52E3 overexpression at the CYP52M1 locus Name Oligonucleotide sequence (5’→3’) SEQ ID N° 28: TP1928_pUC19_R AAGCTTGGCGTAATCATGGTC SEQ ID N° 89: TP3698_CYP52E3-UF-BB-fw AACAGCTATGACCATGATTACGCCAAGCTTCGAGGCTGTTATTGCGGACTTC SEQ ID N° 90: TP3492_Tpry2-BB-rv AGGAGGCTAGCTTGAAGATG SEQ ID N° 91: TP3699_CYP52-UF-DF-fw CTGAACTCACCATCTTCAAGCTAGCCTCCTGTTTCTTAGCCTCCCATGGAAGAAAC SEQ ID N° 63: TP2917_pUC19_cyp52m1 DF_rv ACGACGGCCAGTGAATTCGAGCTCGGAACCAATTGTTCGATGGATAGCTTTG SEQ ID N° 35: TP2574_pUC19_BB_F GGTTCCGAGCTCGAATTCAC For amplification of ura3 recycling construct after CYP52E3 overexpression at the CYP52M1 locus Name Oligonucleotide sequence (5’→3’)SEQ ID N° 92: TP3700_CYP52E3-UF-fwCGAGGCTGTTATTGCGGACTTCSEQ ID N° 65: TP2961_Sbcyp52m1_rv AATTGTTCGATGGATAGCTTTGG For yeast colony PCR to check ura3 recycling after CYP52E3 overexpression at the CYP52M1 locus Name Oligonucleotide sequence (5’→3’) SEQ ID N° 92: TP3700_CYP52E3-UF-fw CGAGGCTGTTATTGCGGACTTC SEQ ID N° 66: P2253_check_downCYP52M1 TCGACTCGCCAAATTCCATCGG For Sbelo1 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 93: TP4010_Pgapd-BB-fw CCAATGGCAGTGGCTTACCACSEQ ID N° 94: TP4011_Pgapd-BB-rvCGTGCGATGGAGTGGTAAGCCACTGCCATTGGTTTTTAAAACTTTGATTTCGACAATCSEQ ID N° 95: TP4012_Sbole1-fw ATGACCAACCTTGCATCGGAGTTTG SEQ ID N° 96: TP4013_Sbelo1-Pgapd-rv CTGCTCAAACTCCGATGCAAGGTTGGTCATCTCGATTGTGTAGAGTTGTTTTTGTT GSEQ ID N° 97: TP4014_ttrpC-BB-fwGATCCACTTAACGTTACTGAAATCATCSEQ ID N° 98: TP4015_ttrpC-Sbelo1-rv TTTGATGATTTCAGTAACGTTAAGTGGATCTTAGGCCTTGCGACTCCTAGTTGC SEQ ID N° 99: TP3869_eno promoter-ttrpC- TTGTGAGACCCAAACTCGTATTCTGAGTCTAACCCAGGGGCTGGTGACGGAATTT rv TC SEQ ID N° 100: TP3870_eno promoter-fw AGACTCAGAATACGAGTTTGGG SEQ ID N° 101: TP3871_eno promoter-rv TTCTAATAGATGTTTGTCTGTGCAG SEQ ID N° 102: TP3872_eno promoter- hygR-fw AGAGCCTGCACAGACAAACATCTATTAGAAATGAAAAAGCCTGAACTCACC SEQ ID N° 103: TP3873_pox-BB-hygR-rv GATTCAAATAATGCCAAGTTTGCTCTTCATATGAACAAACGACCCAACACCG SEQ ID N° 104: TP3874_pox-BB-fw ATGAAGAGCAAACTTGGCATTATTTG For amplification of Sbelo1 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 105: TP4048_pox-UF-fw CCTTGGTCTGGTAGTCAATTCC SEQ ID N° 106: TP4049_pox-DF-rv CAGCGAGCCTCGACCCATTATGC For Sbelo1 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 24: TP2504_PoxKO_chk_F CGCAGAGTAAAGCGCGTAC SEQ ID N° 107: TP4076_Sbelo1-seq-rv CCAGTAAGCCACCACTCAACSEQ ID N° 108: TP3926_hygR-seq_fwATATGCGCGATTGCTGATCCSEQ ID N° 27: TP2619_PoxKO_chk_R2 CTTTTGGCGGCTCGAATTCC 3.2.2 Shakeflask experiment and LCDA extraction Engineered strains were grown on 3C plates to adapt to a high glucose condition before pre-culture in 5 ml Lang medium (pH 5.8) for 2 days at 30 °C and 200 rpm. For the production of LCDAs, the pre-culture was diluted to a starting OD600 of 0.5 in a 250 ml volume non-baƯledflask containing a total of 50 ml culture in Lang medium (120 g / l glucose; starting pH 5.8; (Lang et al., 2000)) at 30 °C and 200 rpm. After 48 hours of cell growth, the production phase was initiated by supplementing with 37.5 g / l of rapeseed oil. After 8 days of the cultivation, 8 ml of the cell culture was vigorously mixed with the 5 same volume of ethyl acetate. Continuously the mixture was shaken at 200 rpm for 1 h. The ethyl acetate phase was harvested at 7,690 × g for 20 min and air-dried. 3.2.3 LCDA detection and quantification See Example 1. 10 3.3 Result 3.3.1 Identification of CYP52 enzymes able to contribute to the production of LCDAs with a chain length of 20 or more carbons In total 8 endogenous CYP52 enzymes were subjected to test their ability to generate LCDAs with 20 or more carbon atoms. Overexpression was established in the 15 Δpox1Δugta1 strain described in Example 1. CYP52E3 overexpression significantly improved the production of C22:0 LCDAs compared to the amounts from either the parental strain or the CYP52M1 deletion strain (ΔM1). In essence, the endogenous CYP52E3 is highly active in the hydroxylation of C22:0 fatty acids (Fig.8). 20 3.3.2 Overexpression of the S. bombicola elongase in the CYP52E3 overexpression strain For the high production of longer LCDAs (≥C20) from rapeseed oil, which contains high amounts of C16 and C18 fatty acids, it is necessary to modify the fatty acid pool and create higher amounts of fatty acids longer than 20 carbon atoms. Therefore, we aimed 25 to identify the endogenous genes encoding fatty acid elongases in S. bombicola. Unlike S. cerevisiae and Y. lipolytica, only one copy of fatty acid elongase (elo1) was found in S. bombicola genome through a multiple sequence alignment and comparison of the highly conserved motifs within the ELO domains. The open reading frame of the elo1 gene was overexpressed at the pox1 locus in the CYP52E3 strain (E3) as described in section 3.2.1. To investigate the eƯect of the putative fatty acid elongase gene (elo1) overexpression, 5 we quantified the LCDAs produced in the parental strain (E3) and the overexpression strain. Importantly, the proportion of longer LCDAs (≥C20) was deeply analysed to observe whether the putative ELO1 eƯiciently elongates the abundant fatty acids (e.g., C16-18) in rapeseed oil. There was no significant diƯerence between the two strains in terms of the total LCDA titre. However, the percentages of the longer LCDAs were 10 significantly increased in the elo1 overexpression strain (Table 5). C22:0 LCDA, for example, was significantly produced (43% of the total LCDA). Furthermore, the produced amount of C24:0 LCDA made up 10% of total LCDA. As a result, the percentages of C16 and C18 LCDAs were significantly reduced. In the case of C18:0 LCDAs, only 5% contributed to the total LCDA amount compared to the parental strain 15 (17%). In summary, the combinatorial overexpression of two endogenous genes, CYP52E3 and elo1, significantly improved production of LCDA with a chain length of 20 carbons or longer. Table 5: Relative percentage of LCDAs in the strain overexpressing the cyp52E3 gene 20 (E3) and the strain both overexpressing the cyp52E3 gene and the elongase 1 gene (E3+Sbelo1). Both strains have the Δpox1Δugta1 parental background. LCDAs Percentage [%] E3 E3+Sbelo1 C12:0 2.38 ± 0.27 0.13 ± 0.14 C14:2 6.62 ± 0.23 7.25 ± 0.36 C14:1 18.61 ± 0.44 10.01 ± 5.26 C14:0 2.80 ± 0.08 0.41 ± 0.28 C16:2 0.66 ± 0.33 0.54 ±0.48 C16:1 3.32 ± 0.82 2.80 ± 0.58 C16:0 7.18 ± 0.62 2.67 ± 0.29 C18:1 2.28 ± 0.10 3.64 ± 0.57 C18:0 16.86 ± 0.65 5.16 ± 0.28 C20:1 0.34 ± 0.03 1.52 ± 0.49 C20:0 11.11 ± 0.73 12.29 ± 0.60 C22:0 25.99 ± 0.54 43.21 ± 3.25 C24:0 1.84 ± 0.21 10.39 ± 2.55 Example 4: Evaluation of other elongase genes in modified S. bombicola strains for increased production of ≥C20 LCDAs 4.1 Introduction The two elongase enzymes from Y. lipolytica YlELO1 and YlELO2 (GenBank reference numbers are XP_501125.1 and XP_505094.1 and seq ID N° 109-112) will be evaluated in the E3 overexpression strain (Δpox1, Δugta1, ΔCYP52M1::pCYP52M1-CYP52E3- Tpry2, in brief referred to as E3)in a similar way as the endogenous elo1 gene. 4.2 Materials and methods 4.2.1 Strain construction The open reading frame of the heterologous elongase genes were overexpressed at the pox1 locus in the CYP52E3 strain (E3) as described in section 3.2.1. The open reading frames of Ylelo1 and Ylelo2 were amplified from the Y. lipolytica genome (ATCC 20460). In order to obtain an intron-free nucleotide sequence of Ylelo2, 5’ end-extended primer ( SEQ ID N° 118) was designed for PCR. The used primer or oligonucleotide sequences and their names are listed in Table 6. Table 6: Names and sequences of primers or oligonucleotides used in the context of Example For Ylelo1 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 93: TP4010_Pgapd-BB-fw CCAATGGCAGTGGCTTACCAC SEQ ID N° 94: TP4011_Pgapd-BB-rv CTCGATGGAGTGGTAAGCCACTGCCATTGGTTTTTAAAACTTTGATTTCGACAATCGG SEQ ID N° 113: TP4092_Ylelo1-BB-rv GTATAGGTCGGGCGAGATTGACGAGAGCATCTCGATTGTGTAGAGTTGTTTTTGTTG SEQ ID N° 114: TP4090_Ylelo1-fw ATGCTCTCGTCAATCTCGCCC SEQ ID N° 115: TP4116_Ylelo1-new-rv CTAGTTGGACTTGGCCTTCAAG SEQ ID N° 116: TP4117_Ylelo1-new-BB- CGATCTTACTTGAAGGCCAAGTCCAACTAGGATCCACTTAACGTTACTGAAATCATC fw SEQ ID N° 99: TP3869_eno promoter-ttrpC-rv TTGTGAGACCCAAACTCGTATTCTGAGTCTAACCCAGGGGCTGGTGACGGAATTTTC SEQ ID N° 100: TP3870_eno promoter-fw AGACTCAGAATACGAGTTTGGG SEQ ID N° 101: TP3871_eno promoter-rv TTCTAATAGATGTTTGTCTGTGCAG SEQ ID N° 102: TP3872_eno pro-hygR-fw AGAGCCTGCACAGACAAACATCTATTAGAAATGAAAAAGCCTGAACTCACC SEQ ID N° 103: TP3873_pox- BB-hygR-rv GATTCAAATAATGCCAAGTTTGCTCTTCATATGAACAAACGACCCAACACCG SEQ ID N° 104: TP3874_pox- BB-fw ATGAAGAGCAAACTTGGCATTATTTG For amplification of Ylelo1 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 105: TP4048_pox- UF-fw CCTTGGTCTGGTAGTCAATTCC SEQ ID N° 106: TP4049_pox- DF-rv CAGCGAGCCTCGACCCATTATGC For Ylelo1 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 24: TP2504_PoxKO_chk_F CGCAGAGTAAAGCGCGTAC SEQ ID N° 115: TP4116_Ylelo1-new-rv CTAGTTGGACTTGGCCTTCAAG SEQ ID N° 108: TP3926_hygR-seq_fw ATATGCGCGATTGCTGATCC SEQ ID N° 27: TP2619_PoxKO_chk_R2 CTTTTGGCGGCTCGAATTCC For Ylelo2 overexpression plasmid construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 93: TP4010_Pgapd-BB-fw CCAATGGCAGTGGCTTACCAC SEQ ID N° 94: TP4011_Pgapd-BB-rv CTCGATGGAGTGGTAAGCCACTGCCATTGGTTTTTAAAACTTTGATTTCGACAATCGG SEQ ID N° 117: TP4094_Ylelo2ex-BB-rv GACGTTGAATTCAATAGGGACGGCGCTCATCTCGATTGTGTAGAGTTGTTTTTGTTG SEQ ID N° 118: TP4093_Ylelo2ex-fw ATGAGCGCCGTCCCTATTGAATTCAAC SEQ ID N° 119: TP4114_Yelo2ex-new-rv TTATGCTCGTCGAGATCGGGTAGTG SEQ ID N° 120: TP4115_Ylelo2ex-new- CAGACCACTACCCGATCTCGACGAGCATAAGATCCACTTAACGTTACTGAAATCATC BB-fw SEQ ID N° 99: TP3869_eno promoter-ttrpC-rv TTGTGAGACCCAAACTCGTATTCTGAGTCTAACCCAGGGGCTGGTGACGGAATTTTC SEQ ID N° 100: TP3870_eno promoter-fw AGACTCAGAATACGAGTTTGGG SEQ ID N° 101: TP3871_eno promoter-rv TTCTAATAGATGTTTGTCTGTGCAG SEQ ID N° 102: TP3872_eno pro-hygR-fw AGAGCCTGCACAGACAAACATCTATTAGAAATGAAAAAGCCTGAACTCACC SEQ ID N° 103: TP3873_pox- BB-hygR-rv GATTCAAATAATGCCAAGTTTGCTCTTCATATGAACAAACGACCCAACACCG SEQ ID N° 104: TP3874_pox- BB-fw ATGAAGAGCAAACTTGGCATTATTTG For amplification of Ylelo2 overexpression construct Name Oligonucleotide sequence (5’→3’) SEQ ID N° 105: TP4048_pox- UF-fw CCTTGGTCTGGTAGTCAATTCC SEQ ID N° 106: TP4049_pox- DF-rv CAGCGAGCCTCGACCCATTATGC For Ylelo2 overexpression yeast colony PCR Name Oligonucleotide sequence (5’→3’) SEQ ID N° 24: TP2504_PoxKO_chk_F CGCAGAGTAAAGCGCGTAC SEQ ID N° 119: TP4114_Yelo2ex-new-rv TTATGCTCGTCGAGATCGGGTAGTG SEQ ID N° 108:TP3926_hygR- ATATGCGCGATTGCTGATCC seq_fw SEQ ID N° 27: TP2619_PoxKO_chk_R2 CTTTTGGCGGCTCGAATTCC 4.2.2 Shakeflask experiment and LCDA extraction See Example 2. 4.2.3 LCDA detection and quantification See Example 1. 4.3 Results The fatty acid elongation activities of YlELO1 and YlELO2 were compared with SbELO1 as a read out of longer LCDA production (≥C20). To evaluate the characteristics of the fatty acid elongation gene overexpression strains in Lang medium, three parameters were analysed: cell growth, pH and LCDA titers in shake flasks (Fig. 9). The overexpression of Ylelo2 resulted in significantly reduced cell growth during the first 24h, similar to the Sbelo1 overexpression strain. In contrast, the cell growth of Ylelo1 overexpression strain was comparable to that of the parental strain (E3; Fig.9A). In the longer production experiment where rapeseed oil was added, the dry cell weight (DCW) of the strains overexpressing either SbELO1 or YlELO2 was significantly improved after 8 days of cultivation (Fig.9B). The culture broths remained acidic (Fig.9C). Of the two heterologous elongases, the fatty acid elongation activity of YlELO2 in S. bombicola was the strongest. Although the total LCDA titer was significantly reduced (0.45 g / l), the production of longer LCDAs (≥C20) increased to 0.34 g / l. As a result of the strong fatty acid elongation, trace amounts of C16-C18 LDCA were detected (Fig. 9D-E). In contrast, the production of longer LCDAs in Ylelo1 overexpression strain did not show any improvement compared to the parental strain (E3; Fig.9E). However, the effect of the Ylelo2 overexpression on the longer LCDA production was similar to the result of the overexpression of Sbelo1. Notably, the highest C24:0 LCDA production (0.05 g / l) was achieved with the S. bombicola strain containing Ylelo2 overexpression (Fig.9F). In essence, two strong fatty acid elongases are available for improvement of longer LCDA production in S. bombicola (SbELO1 and YlELO2). Example 5: combining the over-expression of CYP52E3 and “the best elongases from 5 example 3 and 4” with an faa1 knock-out in modified S. bombicola strains for increased production of ≥C20 LCDAs 5.1 Introduction In Example 2, we demonstrated the unexpected beneficial effect of inactivating the 10 fatty acyl-CoA forming route by faa1 deletion, resulting in increased C16-C18 LCDA production. In example 5 we evaluate if faa1 deletion can be beneficial in combination with overexpression of the elongase to obtain more ≥C20 LCDAs. 5.2 Materials and methods 15 5.2.1 Strain construction For the faa1 gene deletion construct, the ura3 marker of the CYP52E3 overexpression strain with Δpox1Δugta1 background was recycled as described in Example 2, Section 2.2.1.1 kb long upstream and downstream regions of the faa1 were amplified via PCR. The gene fragments and a selection marker gene cassette (ura3) were assembled 20 through CPEC. The deletion cassette gene fragment was amplified to be introduced into the genome and the right transformants were selected on SD-ura agar plates. The successful deletion was confirmed by performing colony PCR. Used oligonucleotides in the context of Example 5 are listed in the Table 3. Furthermore, the selected strong fatty acid elongase genes (Sbelo1 and Ylelo2) were overexpressed at the pox1 locus as 25 described in Example 3 and 4. 5.2.2 Shake flask experiment and LCDA extraction See Example 2. In addition, to ensure accurate LCDA extraction from cell culture broths containing high concentrations of longer LCDAs (≥C20), a total extraction was 30 performed. An equal volume of ethyl acetate (1:1 ratio) was added to the shake flasks after 8 days of cultivation. The shake flasks were continuously shaken at 200 rpm for 1 h. The ethyl acetate phase was harvested by centrifugation at 7,690 × g for 20 min and air-dried. 5.2.3 LCDA detection and quantification 5 See Example 1.5.3 Results Blocking the fatty acid activation route by faa1 deletion in S. bombicola resulted in slow cell growth (E3 vs ^faa1; Fig. 10A). The cell growth defect caused by Ylelo2 overexpression was alleviated by faa1 deletion during the growth phase (Fig. 10A). However, after 8 days of cultivation, thefinal dry cell weight (DCW) was significantly 10 lower in strains carrying the faa1 deletion (Fig.10B). In addition, the culture broths from these strains remained less acidic after 8 days. (Fig.10C). The total LCDA titer of the engineered strain carrying Ylelo2 overexpression and faa1 deletion was slightly decreased (3.7 g / l) compared to the titer of the strain without Ylelo2 overexpression (^faa1; 3.9 g / l). The additional faa1 deletion significantly increased amounts of C16- 15 C18 LCDA titer in the Ylelo2 overexpression strain (Fig.10D-E). Despite the production of significant amounts of C16-C18 LCDAs in the strain with both Ylelo2 overexpression and faa1 deletion, the fatty acid elongation activity of YlELO2 was evident. The overexpression of Ylelo2 notably reduced the concentrations of C16-C18 LCDAs (2.4 g / l) in the faa1 deletion strain compared to the strain without Ylelo2 overexpression (3.3 20 g / l; Fig.10E). As a result, the reduction in C16-C18 LCDA production in thefinal strain, which carried both Ylelo2 overexpression and faa1 deletion, led to a significant increase in longer LCDA titers (≥C20; 0.6 g / l), with a 3.5-fold improvement compared to the ^faa1 strain (Fig.10F). Specifically, the titers of C20:0 (0.07 g / l), C22:0 (0.34 g / l), C24:0 (0.2 g / l) were significantly improved, with the production of C24:0 LCDA increasing by 10- 25 fold compared to the faa1 deletion strain without Ylelo2 (Fig.10G). In addition, there was no significant difference in the cell growth between the two S. bombicola strains carrying either Sbelo1 overexpression combined with faa1 deletion or Ylelo2 overexpression combined with faa1 deletion during the growth phase (Fig.11A). The final dry cell weight (DCW) of the strain with Sbelo1 overexpression and faa1 deletion (15 g / l) was higher 30 compared to the faa1 deletion strain (^faa1; 8 g / l) after 8 days of cultivation with supplemented rapeseed oil. This improvement was notable, as the DCW of engineered strain carrying both Ylelo2 overexpression and faa1 deletion reached only 9.9 g / l under the same growth conditions (Fig. 11B). Furthermore, the culture broth of the strain carrying both Sblelo1 overexpression and faa1 deletion was more acidic than that of the strain carrying both Ylelo2 overexpression and faa1 deletion (Fig. 11C). The combination of Sbelo1 overexpression and faa1 deletion improved the total LCDA titer to 6.1 g / l, compared to 3.9 g / l in the parental strain (^faa1; Fig.11D). Unlike the reduction of C16-C18 LCDA production 5 observed in the strain carrying both Ylelo2 overexpression and faa1 deletion, the concentrations of C16-C18 LCDAs did not decrease in the case of the engineered strain with both Sbelo1 overexpression and faa1 deletion (Fig.11E). Notably, the longer LCDA (≥C20) titer in this strain reached 2.3 g / l, representing a 5.8-fold increase compared to the titer in the faa1 deletion strain (0.4 g / l). Furthermore, this improvement corresponds to a 9-fold 10 increase compared to the titer in the triple deletion strain (^pox1^ugta1^faa1), which is referred to as ^3 (Fig. 11F). The best engineered S. bombicola strain for longer LCDAs produced 0.3 g / l of C20:0, 1 g / l of C22:0 and 1 g / l of C24:0 (Fig.11G). In summary, the additional faa1 deletion not only improved C16-C18 LCDA production but also enhanced the longer LCDA production. The combined metabolic engineering strategy, 15 which includes the active hydroxylation of intracellular longer fatty acid, chain-length oriented intracellular fatty acid pool modification and the preservation of high levels of intracellular fatty acids, is essential for achieving high production of longer (≥C20) LCDAs. 20

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Claims

Claims 1. A modified yeast strain wherein said yeast strain is a non-pathogenic yeast strain selected of Starmerella (Candida) bombicola, Starmerella (Candida) apicola, 5 Starmerella (Candida) batistae, Starmerella (Candida) magnolia, Candida gropengiesseri, Starmerella (Candida) floricola, Starmerella (Candida) riodocensis, Starmerella (Candida) stellata, Starmerella (Candida kuoi), Starmerella cerana, Rhodotorula muciliginosa, Candida rugosa, Cyberlindnera samutprakarnensis, Cryptoococcus sp. VITGBN2, Lachancea thermotolerans, Candida sp. NRRL Y- 27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis sp., Wickerharmiella domericqiae, Candida antarctica, Pseudohyphozyma antarctica, Candida lipolytica and a sophorolipid-producing strain or potential sophorolipid- producing strain of the Starmerella clade and beyond, comprising a non-functional or dysfunctional UGTA1 and FAA1 enzyme and / or not containing a functional ugta1and faa1 gene, combined with a non-functional or dysfunctional β-oxidation.

2. A modified yeast strain wherein said yeast strain is a non-pathogenic yeast strain selected of Starmerella (Candida) bombicola, Starmerella (Candida) apicola, Starmerella (Candida) batistae, Starmerella (Candida) magnolia, Candida gropengiesseri, Starmerella (Candida) floricola, Starmerella (Candida) riodocensis, Starmerella (Candida) stellata, Starmerella (Candida kuoi), Starmerella cerana, Rhodotorula muciliginosa, Candida rugosa, Cyberlindnera samutprakarnensis, Cryptoococcus sp. VITGBN2, Lachancea thermotolerans, Candida sp. NRRL Y- 27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis sp., Wickerharmiella domericqiae, Candida antarctica, Pseudohyphozyma antarctica, Candida lipolytica and a sophorolipid-producing strain or potential sophorolipid- producing strain of the Starmerella clade and beyond, comprising a non-functional or dysfunctional UGTA1, POX1 and FAA1 enzyme and / or not containing a functional ugta1, pox1 and faa1 gene.

3. Use of a modified strain according to claims 1-2 to produce long chain dicarboxylic acids.

4. Use of a modified strain according to claim 3 wherein said long chain dicarboxylic 5 acids have a dominant chain length of 16 and / or 18 carbon atoms.

5. A method to produce long chain dicarboxylic acids comprising: ^ providing oil or fats, ^ providing a modified strain according to claims 1-3, ^ bringing said oil or fat and said modified strain in a fermenter, ^ adjusting the pH of said mixture of oil or fat and strain in said fermenter to about 5.8, ^ allowing the production of long chain dicarboxylic acids by said strain for a certain period of time.

6. Use of a modified yeast strain comprising a non-functional or dysfunctional UGTA1 and POX1 enzyme and / or not containing a functional ugta1and pox1 gene, combined with an over-expressed or heterologous expressed elongase gene and / or an over-expressed or heterologous expressed cytochrome P450 monooxygenase gene or system to produce long chain dicarboxylic acids.

7. Use of a modified yeast strain comprising a non-functional or dysfunctional UGTA1 and POX1 enzyme and / or not containing a functional ugta1and pox1 gene, combined with an over-expressed or heterologous expressed elongase gene and / or an over-expressed CYP52E3 gene to produce long chain dicarboxylic acids.

8. Use of a modified yeast strain according to claims 6-7 in addition comprising a non- functional or dysfunctional FAA1 enzyme and / or not containing a functional faa1 gene.

9. Use of a yeast strain according to claims 6-8 wherein said yeast strain is a yeast strain according to claim 3.

10. Use of a modified strain according to claim 6-9 wherein said long chain dicarboxylic acids have a chain length of 20, 22 or 24 carbon atoms.

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