LIPASE-MODIFIED STRAIN

Abstract

The present invention is related to a retinoid-producing host cell, particularly oleaginous yeast, modified such that the percentage of retinyl acetate based on the total retinoids produced by such host cell is increased during fermentation using triglyceride oils, like for example vegetable oil, as carbon source, wherein the activity of certain endogenous hydrolases or transferases involved in undesired conversions of retinol or retinol acetate is reduced or abolished. Particularly, such modified host cell might be useful in a biotechnological process for production of vitamin A.

Claims

1. A retinoid-producing host cell capable of retinyl acetate formation, particularly retinyl acetate-producing host cell, such as fungal host cells, preferably oleaginous yeast cell such as e.g. Yarrowia, comprising one or more genetic modification(s), such as reduction or abolishment, preferably abolishment, of endogenous enzymes involved in pre-digestion of vegetable oil into glycerol and fatty acids, preferably endogenous enzymes belonging to EC class 3.1.1.-, more preferably enzymes with esterase or lipase activity.

2. The host cell according to claim 1, wherein the expression of endogenous genes is reduced or abolished, preferably abolished, said genes encoding enzymes with activities corresponding to enzyme activities selected from the group consisting of Yarrowia LIP2, LIP3, LIP4, LIP8, TGL1, LIP16, LIP17, LIP18, and combinations thereof.

3. The host cell according to claim 1, wherein the modification leads to an increase in the percentage of retinyl acetate to at least about 70%, such as at least about 70-90%, based on total retinoids compared to a host cell, wherein the respective genes are still expressed and active.

4. The host cell according to claim 1, comprising a modification in a polypeptide obtainable from Yarrowia lipolytica with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to a polypeptide selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15 and combinations thereof.

5. The host cell according to claim 1, wherein the endogenous enzyme corresponding to Yarrowia LIP8 is reduced or abolished, preferably abolished.

6. The host cell according to claim 1, wherein formation of retinal acetate is increased during fermentation compared to the formation of retinyl acetate using the respective non-modified host cell, and wherein a percentage of at least about 70%, such as e.g. about 75, 80, 85, 90, 95, 98% or more, including 100%, retinyl acetate based on total retinoids present in/produced by said modified host cell is obtained.

7. The host cell according to claim 1 used in a fermentation process for production of retinoids with vegetable oil as carbon source, wherein the percentage of retinyl acetate present in said retinoid mix is about 70% or more, preferably about 75, 80, 85, 90, 95, 98% or more, including 100%, retinyl acetate based on total retinoids present in or produced by said host cell.

8. The host cell according to claim 1, wherein the host cell is selected from Yarrowia, preferably Yarrowia lipolytica, comprising inactivation, preferably deletion, of the LIP8 gene, optionally combined with inactivation, preferably deletion, of a gene selected from the group consisting of LIP2, LIP3, LIP4, TGL, LIP16, LIP17, LIP18, and combinations thereof.

9. Use of a host cell according to claim 1 in a process for production of retinoids selected from the group consisting of retinol, retinyl acetate, retinyl fatty esters, vitamin A or mixtures thereof.

10. Use according to claim 9, wherein the percentage of retinyl acetate is in the range of about 70% or more based on the total amounts of retinoids.

11. Use according to claim 9, wherein the host cell is grown on vegetable oil as carbon source, preferably corn oil.

12. A process for reducing or abolishing the percentage of retinoids other than retinyl acetate in a retinoid mix generated in a fermentation process, comprising the steps of: (1) introducing into a retinoid-producing host cell heterologous genes encoding enzymes involved in retinol to retinyl acetate conversion and optionally enzymes involved in retinal to retinol conversion and/or beta-carotene to retinal conversion, (2) introducing one or more modification(s) in endogenous enzyme activities involved in pre-digestion of vegetable oil into glycerol and fatty acids, preferably enzymes, belonging to EC class 3.1.1.-, more preferably enzymes having lipase or esterase activity, most preferably with activities corresponding to Yarrowia LIP8, LIP2, LIP3, LIP4, TGL, LIP16, LIP17, LIP18, and combinations thereof, wherein the modification is a reduction or abolishment of such enzyme activity, preferably abolishment of said enzyme activity.

13. A process for production of a product selected from the group consisting of retinol, retinyl acetate, vitamin A, and a mix comprising retinol, retinyl acetate and vitamin A, said process comprising the steps of: (a) providing a retinoid-producing host cell capable of formation of retinyl acetate, (b) introduction of one or more modification(s) into the genome of said host cell, such as modification(s) into enzyme(s) belonging to the EC class 3.1.1.- having lipase activity, such as e.g. reducing/abolishing the enzyme activity including but not limited to deletion of the respective genes, particularly abolishment of lipase activity corresponding to Yarrowia LIP8 and optionally further abolishing enzyme activity corresponding to Yarrowia LIP2 and/or LIP3 and/or LIP4 and/or TGL and/or LIP16 and/or LIP17 and/or LIP18, wherein the modified host cell is still able to grow on vegetable oil as carbon source; (c) optionally introduction of further modification(s) comprising expression of one or more copies of (heterologous) enzymes involved in retinol, retinyl acetate and/or vitamin A production as known to a person skilled in the art, (d) cultivation of such modified host cell under suitable conditions resulting in formation of retinol, retinyl acetate and/or vitamin A, wherein the modified host cell is grown on vegetable oil as carbon source; and (e) optionally isolation and/or further purification of retinol, retinyl acetate and/or vitamin A from the cultivation (fermentation) medium.

14. A process for the identification of suitable endogenous hydrolases to be modified in order to increase the percentage of retinyl acetate in a fermentation of a retinyl acetate producing host cell grown on vegetable oil as carbon source, comprising the steps of: pre-digestion of vegetable oil into glycerol and fatty acids, (2) selection of endogenous lipase or esterase enzymes based on sequence homology of at least about 50%, such as e.g. 60, 70, 80, 90, 95, 98 or 100% to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, (3) overexpression of selected genes and comparison of retinyl acetate percentage based on total retinoids, (4) selection of genes, wherein overexpression had a negative impact on retinyl acetate percentage in the retinoid mix, and (5) reduction or abolishment, e.g. inactivation, such as e.g. via deletion, of selected genes for enhancement of retinyl acid formation in a retinoid mix.

Description

EXAMPLES

Example 1: General Methods and Strains

[0058] All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998). All genetic manipulations exemplified were performed in Yarrowia lipolytica.

[0059] Shake plate assay. Typically, 800 μl of 0.075% Yeast extract, 0.25% peptone (0.25×YP) is inoculated with 10 μl of freshly grown Yarrowia and overlaid with 200 μl of Drakeol 5 (Penreco, Karns City, Pa., USA) mineral oil with either 2% oleic acid as a carbon source in mineral. Clonal isolates of transformants were grown in 24 well plates (Multitron, 30° C., 800 RPM) in YPD media with 20% mineral oil for 4 days. The mineral oil fraction was removed from the shake plate wells and analyzed by HPLC on a normal phase column, with a photo-diode array detector.

[0060] DNA transformation. Strains are transformed by overnight growth on YPD plate media 50 μl of cells is scraped from a plate and transformed by incubation in 500 μl with 1 μg transforming DNA, typically linear DNA for integrative transformation, 40% PEG 3550 MW, 100 mM lithium acetate, 50 mM Dithiothreitol, 5 mM Tris-Cl pH 8.0, 0.5 mM EDTA for 60 minutes at 40° C. and plated directly to selective media or in the case of dominant antibiotic marker selection the cells are out grown on YPD liquid media for 4 hours at 30° C. before plating on the selective media.

[0061] DNA molecular biology. Genes were synthesized with NheI and MluI ends in pUC57 vector. Typically, the genes were subcloned to the MB5082 ‘URA3’ vector (SEQ ID NO:35) for marker selection in Yarrowia lipolytica transformations. For clean gene insertion by random nonhomologous end joining of the gene and marker HindIII/XbaI (MB5082) the restriction fragment was purified by gel electrophoresis and Qiagen gel purification column. To generate a retinyl acetate producing strain from a beta-carotene producing strain, the strain was transformed with plasmid MB9232, see Table 2, cut with SfiI and double selected for HOM3 and URA3 autotrophy. Plasmids MB9287 and MB9953, containing a Cas9, and guide RNA expression systems to target LIP2, LIP3, and LIP8 in the case of MB9287, and LIP4 in the case of MB9953, were synthesized at Genscript (Piscataway, N.J., USA).

[0062] Plasmid list. Plasmid, strains, nucleotide and amino acid sequences to be used are listed in Table 1, 2, 3 and the sequence listing. In general, all non-modified sequences referred to herein are the same as the accession sequence in the database for reference strain CLIB122 (Dujon B, et al, Nature. 2004 Jul. 1; 430(6995):35-44).

TABLE-US-00001 TABLE 1 list of plasmids used for construction of the strains for overexpression or deletion of the respective genes indicated as “Insert” or for construction used for CRISPR/Cas9 method using the insert as gRNA driver together with the marker as indicated. “LmATF1-mut” refers to Lachancea mirantina (LmATF1; SEQ ID NO: 13 in W02019058001) carrying aa substitutions S480Q_G409A_V407I_H69A_I484L. For more details, see text. Plasmid Insert Marker MB8388 Hh/hdv, snr52 Hyg MB7452 None (pre-Cas9) Nat MB8845 Cas9; lip2 targeting guide RNA Hyg MB8699 Cas9; lip3 targeting guide RNA Hyg MB9953 Cas9; lip4 targeting guide RNA Hyg MB9373 Cas9; lip8 targeting guide RNA Hyg MB9148 Cas9; lip16 targeting guide RNA Hyg MB9149 Cas9; lip17 targeting guide RNA Hyg MB9276 Cas9; lip18 targeting guide RNA Hyg MB9702 Cas9; tgl1 targeting guide RNA Hyg MB9282 Cas9; Ku targeting guide RNA Hyg MB9150 Cas9; ura3 targeting guide RNA Hyg MB9232 LmATF1-mut HOM3 URA3

TABLE-US-00002 TABLE 2 list of Yarrowia strains used. Construction of ML7788 and ML15710 is described in WO2016172282 (Table 2 and Ex. 5). For more details, see text or Table 1. Strain Description ML17544 ML15710 cured of URA3 by FOA and HygR by Cre/lox ML17968 ML17544 transformed with MB8457 UmCCO1 ML18183 ML17968 transformed with MB7452 [Cas9 NatR CEN] ML18210 ML18183 transformed with MB8549 Cas9 hom3 ML18210-1 ML18210 transformed with MB9232 HOM3::LmATF1-mut::URA3 ML18210-2 ML18210-1 transformed with MB7452 [precas9] MB9282 ku70 MB9373 lip8 ML18210-3 ML18210-2 transformed with MB7452 [precas9] MB9282 ku70 MB9373 lip8 MB8845 lip2 ML18210-4 ML18210-3 transformed with MB7452 [precas9] MB9282 ku70 MB9373 lip8 MB8845 lip2 MB8699 lip3 ML18210-5 ML18210-3 transformed with MB7452 [precas9] MB9282 ku70 MB9373 lip8 MB8845 lip2 MB8699 lip3 MB9953 lip4

TABLE-US-00003 TABLE 3A list of sequences used for construction of the plasmids/strains. For details of the sequences, see sequence listings. SEQ ID NO: Name (aa/nt) lip2 1/2 lip3 3/4 lip8 5/6 tgl-1 7/8 lip16  9/10 lip17 11/12 lip18 13/14 lip4 15/16 est1 17/18 lip11 19/20 lip12 21/22 lip20 23/24 lip1 25/26 lip15 27/28 lipR 29/30 ipf3594 31/32

TABLE-US-00004 TABLE 3B list of primers for CRISPR Cas9 method, PCR, sequencing as described in Ex. 3. For more details on the sequences, see sequence listings. Primer Description SEQ ID NO: 13304 Ku70-d-Top-66 36 13305 Ku70-d-Bot-66 37 13308 Ku70-c-Top-24 38 13309 Ku70-c-Bot-24 39 12491 ura3-Cas9-Top-66 40 12492 ura3-Cas9-Bot-66 41 12493 ura3-2-Top-24 42 12494 ura3-2-Bot-24 43 14054 lip16_pcr_rev_full 44 14053 lip16_pcr_for_full 45 14052 Lip16Dbtm 46 14051 Lip16Dtop 47 13418 LIP17-24-Bot 48 13417 LIP17-24-Top 49 13324 LIP18 rev seq 50 13323 LIP18 for seq 51 13322 LIP18 rev pcr 52 13321 LIP18 for pcr 53 13315 LIP3-Cas9-24-b-Bot 54 13314 LIP3-Cas9-24-b-Top 55 13313 LIP8-Cas9-34-Bot 56 13312 LIP8-Cas9-34-Top 57 13259 LIP18-Cas9-24-Bot 58 13258 LIP18-Cas9-24-Top 59 13257 LIP18-Cas9-66-Bot 60 13256 LIP18-Cas9-66-Top 61 13147 LIP17 rev seq 62 13146 LIP17 for seq 63 13145 LIP17 rev pcr 64 13144 LIP17 for pcr 65 13143 LIP16 rev seq 66 13142 LIP16 for seq 67 13141 LIP16 rev pcr 68 13140 LIP16 for pcr 69 13111 LIP17-Cas9-24-Bot 70 13110 LIP17-Cas9-24-Top 71 13109 LIP17-Cas9-66-Bot 72 13108 LIP17-Cas9-66-Top 73 13107 LIP16-Cas9-24-Bot 74 13106 LIP16-Cas9-24-Top 75 13105 LIP16-Cas9-66-Bot 76 13104 LIP16-Cas9-66-Top 77 12850 Lip8 rev seq 78 12849 Lip8 for seq 79 12848 Lip8 rev pcr 80 12847 Lip8 for pcr 81 12840 LIP2ioRevXba 82 12839 LIP2ioFwdMlu 83 12838 LIP2iorevMlu 84 12837 LIP2ioFwdkpn 85 12821 LIP8-Cas9-24-Bot 86 12820 LIP8-Cas9-24-Top 87 12819 LIP8-Cas9-66-Bot 88 12818 LIP8-Cas9-66-Top 89 12707 LIP2 for seq 90 12706 LIP2 rev pcr 91 12705 LIP2 for pcr 92 12602 LIP2-Cas9-24-Bot 93 12601 LIP2-Cas9-24-Top 94 12600 LIP2-Cas9-66-Bot 95 12599 LIP2-Cas9-66-Top 96 12564 LIP3 rev seq 97 12563 LIP3 for seq - (really reverse) 98 12562 LIP3 rev pcr 99 12561 LIP3 for pcr 100 12464 LIP3-Cas9-24-Bot 101 12463 LIP3-Cas9-24-Top 102 12462 LIP3-Cas9-66-Bot 103 12461 LIP3-Cas9-66-Top 104 14025 tglseq-rev 105 14024 tglseq-fwd 106 14023 TglDelta-rev 107 14022 TglDelta-fwd 108 13307 Ku70-e-Bot-66 109 13306 Ku70-e-Top-66 110 12074 ku70RightseqFwd 111 12073 ku70LeftseqFwd 112 14152-2 Lip4-5′-top-24 113 14152-3 Lip4-5′-bot-24 114 14152-4 Lip4-3′-top-66 115 14152-5 Lip4-3′-bot-66 116 14151 LIP4-5′seq-fwd 117 14152 LIP4-3′seq-rev 118

[0063] Fermentation conditions. Fermentations were identical to the previously described conditions using mineral oil overlay and stirred tank in a bench top reactor with 0.5 L to 5 L total volume (see WO2016/172282, Ex. 5 and 6 but with a different oil), however, they were oleic acid fed. Generally, the same results were observed with a fed batch stirred tank reactor with an increased productivity, which demonstrated the utility of the system for the production of retinoids. Preferably, fermentations were batched with 6% glucose and 20% mineral oil was added after dissolved oxygen dropped below about 20% and feed was resumed to achieve 20% dissolved oxygen throughout the feeding program. Fermenters were harvested and compared at 138 hrs.

[0064] UPLC reverse phase retinol method. For rapid screening this method does not separate cis-isomers, only major functional groups. A Waters Acquity UPLC with PDA detection (or similar) with auto sampler was used to inject samples. An Acquity UPLC HSS T3 1.8 um P/N 186003539 was used to resolve retinoids. The mobile phase consisted of either, 1000 mL hexane, 30 mL isopropanol, and 0.1 mL acetic acid for retinoid related compounds. Column temperature was 20° C. The injection volume was 5 μL. The detector was a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 4.

TABLE-US-00005 TABLE 4A list of analytes using reverse phase retinol method. The addition of all added intermediates gives the total amount retinoids. Beta- carotene* can be detected in 325 nm and will interfere with retinyl ester quantitation, therefore care must be taken to observe the carotene peak and not include them in the retinoid quantification. “N/A” means “not available”. For more details, see text. Retention time Lambda max Response Intermediates [min] [nm] factor retinyl-acetate 2.93 325 1.00 retinyl-esters 3.2-3.8 325 1.68 retinal 2.77 325 0.87 retinol 2.73 325 0.87 Beta-carotene* 3.56 450 N/A

TABLE-US-00006 TABLE 4B UPLC Method Gradient with solvent A: water; solvent B: acetonitrile; solvent C: methanol; solvent D: tert-butyl methyl ether. Time Flow Pressure [min] % A % B % C % D [ml/min] [psi/bar] 0 50 50 0 0 0.5 9500-14000max 0.5 50 50 0 0 0.5 1.0 0 50 50 0 0.5 1.25 0 0 100 0 0.5 3.25 0 0 5 95 0.5 3.5 0 0 5 95 0.5 4.0 0 0 100 0 0.5 4.25 0 50 50 0 0.5 4.5 50 50 0 0 0.5

[0065] Method Calibration. Method is calibrated on retinyl acetate, retinols and retinals are quantitated against retinyl-acetate using the indicated response factor. Retinyl Acetate is dissolved in THF at −200 μg/ml for stock solution using a volumetric flask. Using volumetric flasks, ×20, ×50 and ×100 dilutions of stock solution in 50/50 methanol/MTBE were made. UV absorbance of retinyl acetate becomes nonlinear fairly quickly, so care must be taken to stay within the linear range. Consequently, lower concentrations might be better. Retinyl palmitate can also be used as retinyl ester calibration.

[0066] Sample preparation. Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples the broth was placed in a Precellys® tube, weighed, and mobile phase was added. Briefly in a 2 ml Precellys® tube, add 25 μl of well mixed broth and 975 μl of THF. The samples were then processed in a Precellys® homogenizer (Bertin Corp, Rockville, Md., USA) on the highest setting 3× according to the manufacturer's directions, typically 3 repetitions×15 minutes×7500 rpms. For the washed pellet the samples were spun in a 1.7 ml tube in a microfuge at 10000 rpm for 1 minute, the broth decanted, 1 ml water added, mixed, pelleted and decanted, and brought up to the original volume. The mixture was pelleted again and brought up in appropriate amount of mobile phase and processed by Precellys® bead beating. For analysis of mineral oil fraction, the sample was spun at 4000 RPM for 10 minutes and the oil was decanted off the top by positive displacement pipet (Eppendorf, Hauppauge, N.Y., USA) and diluted into mobile phase mixed by vortexing and measured for retinoid concentration by UPLC analysis.

Example 2: Lipase/Esterase Overexpression in Yarrowia lipolytica

[0067] To test the influence of endogenous lipases and/or esterases on production of retinoids in a suitable Yarrowia host, overexpression experiments were carried out, wherein only 1 gene at the time was overexpressed (no combination of 2 or more genes).

[0068] Lipases were overexpressed as described above (Example 1). Native Yarrowia lipase genes were synthesized and sequence verified by GenScript then cloned into the NheI and MluI sites of MB5082. The genes are TEF1 promoter driven that allows selection for by complementation of an uracil auxotroph strain (ura3).

[0069] Plasmids containing the respective lipase/esterase genes cleaved by XbaI/HindIII were transformed into retinoid producing strain ML18210-9 carrying the wild-type lip8 gene (see Example 1, Table 2) and selected for uracil prototrophy. Clonal isolates of transformations were grown for four days in 0.25× Yeast/Peptone (YP) with 2% corn oil as a carbon source and a 20% mineral oil overlay in the standard shake plate assay and assayed by the previously described UPLC analytical method. At least two individual clonal isolates of transformed Yarrowia strains were tested by shake plate and measured by UPLC assay % retinyl esters and % retinol per mass of total retinoids. The result is depicted in Table 5, showing production of retinyl fatty esters and retinol. Best performance on accumulation of retinyl fatty esters and conversion of retinol is achieved with overexpression of LIP8, some minor effect was visible with LI P3 overexpression.

TABLE-US-00007 TABLE 5 performance of Yarrowia strains overexpressing single endogenous lipases or esterases as indicated. The percentage of retinyl esters (“% esters”) and retinol (“% retinol”) based on the total amount of retinoids is given. Empty vector is the plasmid without an ORF inserted, that can be interpreted as a negative control. For more details, see text. Insert % esters % retinol empty 8 26 LIP3 24 18 LIP8 95 3 TGL1 8 43 LIP16 7 61 LIP17 8 65 LIP18 9 45 EST1 7 25 LIP11 6 26 LIP12 6 25 LIP20 7 25 LIP1 6 26 LIP15 7 25 LIPR 7 24 IPF3594 5 25

Example 3: Deletion of Lipase Genes in Yarrowia lipolytica

[0070] Lipase genes were deleted using modern CRISPR Cas9 methods. The strains were pre-transformed with MB7452 expressing Cas9 (SEQ ID NO:34) under nourseothricin selection, that increased the deletion frequency when a subsequent guide RNA was transformed. Cas9 guide RNAs were selected using the Geneious® 10.1.3 software (Biomatters Ltd). Sites were selected that are as close to the beginning of the open reading frame (ORF) for single cuts or at 5′ and 3′ to remove most of gene. Guides were inserted into SapI cloning sites of the vector MB8388 (SEQ ID NO:33) and were synthesized and sequence verified by GenScript (see Table 3 for sequences). Strains were transformed and selected on YPD Hygromycin at 200 μg/ml then replica plated to YPD. Plasmids are passed by outgrowth on YPD plates containing Nourseothricin 100 μg/ml and replica plating to YPD Hygromycin at 200 μg/ml to identify colonies that have lost the guide RNA fragment, but still contain the PreCas9 plasmid, MB7452. Then these clones were screened for deletion by PCR over the gene using primers 100 bp upstream and 100 bp downstream, identifying the deletion by gel mobility, and sequencing the deletion. To precisely remove the ORF for the Cas9 deletions template DNA (100 bp with 50 bp 5′ of the ORF, and 50 bp 3′ of the ORF as in strain CLIB122) was used in strains where the ku70 gene (YALI0008701g) was previously deleted using MB9282. Sequences of the guide RNA expressing region are referenced in Table 3. Nucleotides that code for guides in the sequence anneal and ligate to the SapI sites and result in removal of the SapI site that was present in the oligonucleotide. The annealing of the guides is directed by the specific overhangs in the guide sequence (5′ to 3′ on the top strand: ATG, GTT, CGT, TTT). The first three nucleotides of the guide containing the SapI site is included in the insert sequence for clarity in alignment and the annealed overhangs can be assembled into the vector MB8388 (SEQ ID NO:33) by matching the overhangs. The 24 base pair inserts are inserted into a guide RNA that is driven and processed by a hammerhead ribozyme system (hh, hdv), and the 66 base pair insert is driven by the Yarrowia SNR52 promoter. Single stranded oligonucleotides can assemble the guide sequences by annealing top and bottom sets and using these for ligation into appropriate the SapI sites. Plasmids containing these inserts in MB8388 have been routinely synthesized at the DNA provider GenScript, (Piscataway, N.J., USA). Examples of the oligonucleotides used in these assemblies are included in Table 3B.

Example 4: Effect of Lipase Knockouts on Formation of Retinyl Acetate

[0071] To explore the effects-on retinyl acetate production, we constructed lipase deletions in retinyl acetate producing strain ML18210-1 expressing a highly active acetyl transferase derived from Lachancea mirantina, i.e. LmATF1 (see WO2019058001: SEQ ID NO:13), carrying amino acid substitutions S480Q_G409A V407I_H69A_I484L. The lineage of said strain is known from Table 2. Removal of the open reading frames of lipase genes was carried out using CRISPR Cas9 methods. This scheme was performed by primary introduction of a ku70 mutation, using MB9282 and subsequently co-transforming lipase deletion plasmids with template DNA (100 nucleotide base pairs 5′ and 3′ of the ORF ordered as FragmentGENE from Genewiz.com, Cambridge, Mass., USA) that directs a precise deletion of the ORF, since homologous recombination is required to repair the double strand break in a ku70 mutant. Deletion of only one or several lipase genes, i.e. serial deletion, was performed with this technique. Said modified strains were tested for formation of retinoids, in particular formation of retinyl acetate, as shown in Table 6, with focus on purity, i.e. the percentage of retinyl acetate based on the total amount of retinoids, and abundance, i.e. comparison between retinyl acetate formation with a lipase-deleted strain to retinyl acetate formation with strain ML18210-1 (wild-type strain for all endogenous lipase genes). Strains were grown in 2% oleic acid in 0.25× yeast peptone fed shake plate and fermentations with a 20% mineral oil overlay for four days at 30° C. in shake plates as described in Example 1. The results are shown in Table 6 for deletion of LIP8 alone, leading to a percentage of 70% retinyl acetate based on total retinoids, or in combination with LIP2 and/or LIP8 and/or LIP4, with some further increase of the percentage. Addition of further deletions selected from TGL1 and/or LIP16 and/or LIP17 and/or LIP18 might result in at least the same retinyl acetyl percentages, i.e. in the range of at least about 70-90% retinyl acetate based on total retinoids, with further increase of at least about 10% compared to retinyl acetate formation with deletion of LIP8 only.

TABLE-US-00008 TABLE 6 Effect of lipase deletions on purity and abundance of retinyl acetate formation in a retinyl acetate-producing Yarrowia host. “% retAc” means purity of retinyl acetate, “increase [%]” means abundance of retinyl acetate with the value for strain ML18210-2 being zero, “deletion” refers to the deleted genes. For further details, see text. ML strain deletion % retAc increase [%] 18210-1 N/A N/A N/A 18210-2 lip8 70%  0 18210-3 lip8 lip2 78% 12 18210-4 lip8 lip2 lip3 92% 31 18210-5 lip8 lip2 lip3 lip4 94% 32