METHOD TO PRODUCE RETINYL ACETATE

Abstract

The present invention is related to a novel process for production of retinyl acetate in a host cell, particularly oleaginous yeast such as e.g. Yarrowia, wherein the product purity could be increased with reduction of unwanted side-products. Particularly, the novel process comprises fermentation in the presence of ethanol, such as e.g. in a fed-batch fermentation process. Such process is especially useful in a biotechnological process for production of vitamin A.

Claims

1. A process for production of retinyl acetate in a fungal host cell, particularly oleaginous yeast cell, preferably Yarrowia, said process comprising fermentation of the host cell in the presence of ethanol.

2. The process according to claim 1 wherein the fermentation is a fed-batch fermentation.

3. The process according to claim 2 comprising a concentration of about 5% or less (v/v) ethanol added to the cultivation medium during the batch phase.

4. The process according to claim 1, said process comprising fermentation of the host cell in the absence of triglycerides, preferably vegetable oil.

5. The process according to claim 1, wherein the formation of by-products including fatty acid retinyl esters (FAREs) is reduced or abolished, preferably reduced by at least about 50% based on total retinoids and compared to a process comprising fermentation of the host cell in the absence of ethanol.

6. The process according to claim 1, wherein the percentage of retinyl acetate based on total retinoids is increased, preferably by at least about 25%, compared to a process comprising fermentation of the host cell in the absence of ethanol.

7. The process according to claim 1, wherein the production of total retinoids is increased, preferably by at least about 30%, compared to a process comprising fermentation of the host cell in the absence of ethanol.

8. The process according to claim 1, wherein the host cell is transformed with and is expressing heterologous genes, preferably genes encoding acetylating enzymes catalyzing the conversion of retinol to retinyl acetate, more preferably fungal enzymes, most preferably originated from Lachancea mirantina.

9. The process according to claim 8, wherein the acetylating enzyme comprises one or more amino acid substitution(s) in a sequence with at least about 20%, such as e.g. 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:18, wherein the one or more amino acid substitution(s) are located at position(s) corresponding to amino acid residue(s) selected from the group consisting of position 68, 69, 72, 73, 171, 174, 176, 178, 291, 292, 294, 301, 307, 308, 311, 312, 320, 322, 334, 362, 405, 407, 409, 480, 483, 484, 490, 492, 520, 521, 522, 524, 525, 526 and combinations thereof in a polypeptide according to SEQ ID NO:18.

10. A process for reduction of enzymatic conversion of retinol into FAREs, preferably with a percentage of FARE based on total retinoids being less than 25%, said process comprising fermentation of a retinyl acetate producing host cell in the presence of ethanol.

Description

EXAMPLES

Example 1: General Methods and Plasmids

[0042] 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).

[0043] DNA transformation. Strains were transformed by overnight growth on YPD plate media; 50 ?l of cells was scraped from a plate and transformed by incubation in 500 ?l with 1 ?g transforming DNA, typically linear DNA for integrative transformation, 40% PEG 3550MW, 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 were out grown on YPD liquid media for 4 hours at 30? C. before plating on the selective media. URA3 marker recycling was performed using 5-fluoroorotic acid (FOA). Episomal hygromycin resistance marker plasmids were cured by passage on non-selective media, with identification of Hyg-sensitive colonies by replica plating colonies from non-selective media to hygromycin containing media (100 ?g/mL).

[0044] DNA molecular biology. Plasmids MB9523 containing expression systems for DrBCO, LmATF-S480Q_G409A_V407I_H69A_I484 L, and FfRDH (SEQ ID NO:17) was synthesized at Genscript (Piscataway, NJ, USA). Plasmid MB9523 contains the URA3 for marker selection in Yarrowia lipolytica transformations. For gene insertion by random nonhomologous end joining of the gene and marker, Sfil-digested MB9523 plasmid fragment of interest was purified by gel electrophoresis and Qiagen gel purification column. Clones were verified by sequencing. Typically, genes are synthesized by a synthetic biology at GenScript (Piscataway, NJ).

[0045] Plasmid list. Plasmid, strains, nucleotide and amino acid sequences that were used are listed in Table 1, 2 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 TABLE1 listofplasmidsusedforconstructionofthe strainsforoverexpressionordeletionofthe respectivegenesindicatedasinsert.LmATF1- mutreferstoLachanceamirantina(LmATF1;SEQ IDNO:13inWO2019058001)carryingaasub- stitutionsS480Q_G409A_V407I_H69A_1484L.DrBCO referstoBCOoriginatedfromDaniorerio(see SEQIDNO:16inWO2020141168);FfRDHrefers toRDHoriginatedfromFusariumfujikuroi(see SEQIDNO:22inWO2020141168).Formore explanation,seetext. SEQ Plasmid Insert Marker IDNO MB9523 DrBCO;LmATF1-mut;FfRDH URA3 17

TABLE-US-00002 TABLE 2 list of Yarrowia lipolytica strains used. Construction of ML17544 is described in Table 2 of WO2020141168. For more details, see text. Strain Description ML18812 ML17544 transformed with MB9523

[0046] Fermentation conditions. Fed-batch fermentations were identical to the previously described conditions except using Drakeol 5 (Penreco, Karns City, PA, USA) or another overlay and stirred tank that was corn oil, glucose or ethanol fed in a bench top reactor with 0.5 L to 5 L total volume (see WO2016172282). The batch medium carbon source composition and feed medium are listed in Table 3. Feeding was initiated after the initial batch carbon had been consumed, with feed added in a controlled manner to maintain a dissolved oxygen level (DO) setpoint.

[0047] Briefly, the fermentations were run in 3.0 L flood volume in glass New Brunswick or Eppendorf fermentation systems. The fermentor was batched with the following components: 2228 mL of deionized water, MgSO.sub.4.Math.7H.sub.2O is 1.96 g/kg and NaCl is 0.20 g/kg, 10.46 mL, 1.04 g CaCl.sub.2)-2H.sub.2O, 26.18 g (NH.sub.4)2SO.sub.4, 27.10 g KH.sub.2PO.sub.4, 19.62 g Tastone yeast extract (Marcor, Leominster, MA), 26 mL DF204 antifoam, 0.654 ?L, thiamine HCl at 4 mg/ml, trace elements stock solution 3.27 ml/L 20) containing: 200 g/kg citric acid, 27.3 g/kg FeSO.sub.4.Math.7H.sub.2O, 19.6 g/kg Na.sub.2MoO.sub.4.Math.2H.sub.2O, 18.7 CuSO.sub.4.Math.5H.sub.2O, 4.9 H.sub.3BO.sub.3, 21.9 MnSO.sub.4.Math.H.sub.2O, 30.2 ZnSO.sub.4.Math.7H.sub.2O, and autoclaved. After cooling a carbon source was added along with 800 mL Drakeol 5 or other second phase. The fermentation was inoculated with 200 ml overnight shake flask cultures of YP media grown with 250 RPM agitation at 30? C. and the specific carbon sources shown in Table 3. Fermentation parameters were agitation at 1000 RPM, airflow at 4.6 LPM for ethanol and 2.3 LPM for oil and mixed fatty acids, pH controlled at 5.5 control with NH.sub.4OH, and the temperature set to 30? C. At feed start, feed was added to maintain the DO setpoint at 40%. The DO setpoint was ramped down to 20% in a linear fashion over the following 24 hours by increased feed rate. The DO was then maintained at 20% via feed addition for the remainder of the fermentation.

TABLE-US-00003 TABLE 3 fermentation feeding protocol. Fermentation 2.sup.nd phase was always ~20% by weight Drakeol 5. For more explanation, see text. Run: Batch: Feed: ethanol 5% glucose (w/v) 100% ethanol (w/v) 1% ethanol (w/v) oil 6.25% oleic acid (v/v) 100% corn oil (w/v) 1.4% corn oil (v/v)

[0048] Retinoid quantification. Analysis of retinoids were carried out with a C4 reverse phase retinoid method (see below) and C18 as described elsewhere (WO2020141168). The addition of all added intermediates gives the total amount of retinoids.

[0049] C4 reverse phase chromatography. For exact determination of discrete retinoids the long run reverse phase system was used. We separated analytes at 230 nm and 325 nm through the Agilent 1290 instrument with YMC Pro C4, 150?3.0 mm 3 ?m column (YMC America, Devens, MA) stationary phase, and a 5 ?l injection loop volume and column and sample tray controlled at 23? C. with gradients described in Table 4B. Analytes were detected at 230 nm and 325 nm and the peaks identity verified with LCMS. The analytes separated as discrete peaks that were assigned according to Table 4A.

TABLE-US-00004 TABLE 4A list of analytes using C4-reverse phase method. The addition of all added intermediates gives the total amount retinoids. RT means retention time. For more details, see text. Intermediates RT [min] ? max [nm] trans-retinol 20.21 325 cis-retinol 20.32 325 dihydro-retinol 20.75 290 trans-retinal 20.89 380 cis-retinal 21.02 380 trans-retinyl-acetate 22.15 325 cis-retinyl-acetate 22.35 325 dihydro-retinyl acetate 22.60 290 retinyl esters 26.30 325

TABLE-US-00005 TABLE 4B UPLC Method Gradient with solvent A: acetonitrile; solvent B: water; solvent C: water/acetonitrile/methanesulfonic acid 1000:25:1. For more details, see text. Time Flow [min] % A % B % C [ml/min] 0 5 85 10 0.5 20 98 0 2 0.5 35 98 0 2 0.5 35.1 5 85 10 0.5 40 5 85 10 0.5

[0050] Method Calibration. Method is calibrated using high purity retinyl acetate received from DSM Nutritional Products, Kaiseraugst, CH. Retinols and retinal are quantitated against retinyl acetate. Dilutions were prepared as follows. 40 mg of retinyl acetate was weighed into a 100 mL volumetric flask, and dissolved in ethanol, yielding a 400 ?g/mL solution. This solution was sonicated as required to ensure dissolution. 5 mL of this 400 ?g/mL solution was diluted into 50 mL (1/10 dilution, final concentration 40 ?g/mL), 5 mL into 100 ml (1/20 dilution, final concentration 20 ?g/mL), 5 ml of 40 ?g/mL into 50 mL (1/10 dilution, final concentration 4 ?g/mL), 5 mL of 20 ?g/mL into 50 ml (1/10 dilution, 2 ?g/mL), using 50/50 methanol/methyl tert-butyl ether (MTBE) as the dilutent. All dilutions were done in volumetric flasks. Purity of retinyl acetate was determined by further diluting the 400 ?g/mL stock solution 100-fold (using a 2 mL volumetric pipet and a 200 mL volumetric flask) in ethanol. Absorbance of this solution at 325 nm using ethanol was taken as the blank, with adjustment of the initial concentration using the equation (Abs*dilution (100)*molecular weight (328.5)/51180=concentration in mg/ml). Because of quick out-maximization of UV absorbance of retinyl acetate, lower concentrations are better.

[0051] Sample preparation. Top second-phase layer samples from each strain were diluted at a 25-fold dilution or higher, if needed, into tetrahydrofuran (THF). Fermentation whole broth was prepared using a 2 mL Precellys (Bertin Corp, Rockville, MD) tube, adding 25 ?l of well mixed broth and 975 ?l of THF. Precellys (3?15?7500 rpm) for two cycles with a freeze at ?80? C. for 10 minutes between cycles. Cell debris was spun down via centrifugation for 1 minute at 13000 rpm. These samples were diluted 10-fold in THF for analysis.

Example 2: Impact of Carbon Source on Retinoid Production in Fed-Batch Fermentations Using Yarrowia

[0052] To evaluate the impact of carbon source on the production of retinoids in Yarrowia lipolytica, fermentations in the presence of ethanol or oil (see Table 3) were run, and the purity as well as percentage of retinyl acetate assessed via measuring percentage of FARE from strain ML18812.

[0053] Fermentation in the presence of ethanol resulted in a strong reduction of FARE formation compared to fermentations in the presence of corn oil. Percentage of total retinoids as well as retinyl acetate could also be increased in comparison to corn oil fermentations. The results are shown in Table 5.

TABLE-US-00006 TABLE 5 effect of ethanol on production of FARE, retinoids and retinyl acetate (retAc) using strain ML18812. +++ is in the top quartile of the maximum value, ++ is in the mid quartile and +/? indicates trace amounts observed, while ??? indicates not observed, retAc means retinyl acetate. For more explanation, see text. Carbon source Total retinoids retAc FAREs Ethanol +++ +++ ??? corn oil ++ +/? +++