PRODUCTION OF RETINYL ESTERS

Abstract

The present invention is related to a novel enzymatic process for production of retinyl esters, such as in particular retinyl long chain esters, via conversion of retinol, which process includes the use of enzymes having acyltransferase activity. Said process might be used for biotechnological production of vitamin A.

Claims

1. A carotenoid-producing host cell comprising an enzyme with retinol acylating activity, preferably acyltransferase [EC 2.3.1] activity, more preferably acyltransferase [EC 2.3.1.20] activity, said host cell producing long chain retinyl esters, with a percentage of at least about 20% based on the total amount of retinoids produced by said host cell.

2. The carotenoid-producing host cell according to claim 1 comprising a heterologous acyltransferase.

3. The carotenoid-producing host cell according to claim 1, wherein the host cell is selected from plants, fungi, algae or microorganisms, preferably selected from the group consisting of Escherichia, Streptomyces, Pantoea, Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, Mixococcus, Brevibacterium, Bradyrhizobium, Gordonia, Dietzia, Muricauda, Sphingomonas, Synochocystis, Paracoccus, Saccharomyces, Aspergillus, Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea and Yarrowia, more preferably from Yarrowia lipolytica or Saccharomyces cerevisiae.

4. The carotenoid-producing host cell according to claim 1, wherein the acyltransferase is selected from plants, animals, including humans, algae, fungi, including yeast, or bacteria, preferably selected from the group consisting of Drosophila, Fusarium, Mucor, human, rat and Yarrowia.

5. The carotenoid-producing host cell according to claim 4, wherein the acyltransferase is acyltransferase selected from Yarrowia or Drosophila, preferably selected from Y. lipolytica or D. melanogaster.

6. The carotenoid-producing host cell according to claim 5, wherein the acyltransferase is selected from a polypeptide with at least about 60% identity to acyltransferase according to sequence XM_502557 or sequence NM_135969.

7. The carotenoid-producing host cell according to claim 1, wherein the long chain retinyl esters are further converted into vitamin A.

8. A process for production of retinoids comprising long chain retinyl esters via enzymatic activity of acyltransferase [EC 2.3.1], comprising contacting retinol with said acyltransferase, wherein the ratio of trans- to cis-isoforms in the long chain retinyl ester mix is at least about 4.

9. A process using the carotenoid-producing host cell according to claim 1.

10. A process for production of vitamin A comprising the steps of: (a) introducing a nucleic acid molecule encoding acyltransferase [EC 2.3.1] into a suitable carotene-producing host cell, (b) enzymatic conversion of retinol into a long chain retinyl ester mix comprising trans- and cis-retinyl esters in a ratio of at least about 4, (c) conversion of long chain retinyl esters into vitamin A under suitable culture conditions.

11. Use of acyltransferase [EC 2.3.1] for production of a long chain retinyl ester mix comprising trans- and cis-retinyl esters in a ratio of at least about 4, wherein the acyltransferase is heterologous expressed in a suitable carotenoid-producing host cell.

Description

EXAMPLES

Example 1: General Methods, Strains, and Plasmids

[0060] 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).

[0061] Shake plate assay. Typically, 800 l of 0.075% Yeast extract, 0.25% peptone (0.25 YP) is inoculated with 10 l of freshly grown Yarrowia and overlaid with 200 l of Drakeol 5 mineral oil carbon source 5% corn oil in mineral oil and/or 5% in glucose in aqueous phase. Transformants were grown in 24 well plates (Multitron, 30 C., 800 RPM) in YPD media with 20% dodecane 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.

[0062] DNA transformation. Strains are transformed by overnight growth on YPD plate media 50 l of cells is 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 are out grown on YPD liquid media for 4 hours at 30 C. before plating on the selective media.

[0063] DNA molecular biology. Genes were synthesized with NheI and MluI ends in pUC57 vector. Typically, the genes were subcloned to the MB5082 URA3, MB6157 HygR, and MB8327 NatR vectors for marker selection in Yarrowia lipolytica transformations, as in WO2016172282. For clean gene insertion by random nonhomologous end joining of the gene and marker HindIII/XbaI (MB5082) or Pvull (MB6157 and MB8327), respectively purified by gel electrophoresis and Qiagen gel purification column.

[0064] Plasmid list. Plasmid, strains, and codon-optimized sequences to be used are listed in Table 1, 2 and the sequence listing. ALL sequences were the same as the accession sequence in the database as nucleotides (nt), except for the numbered sequence ID NOs: 1, 2 that were codon optimized for expression in Yarrowia.

TABLE-US-00001 TABLE 1 list of plasmids used for construction of the strains carrying the heterologous acyltransferase-genes of Yarrowia lipolytica (YlDGA1), sequence XM_502557 and Drosophila melanogaster (Dm Dgat1), sequence NM_135969. For more details, see text. SEQ ID NO: MB plasmid Backbone MB Insert (codon-optimized) 8201 5082 DmDGA1 1 8299 5082 YlDGA1 2

TABLE-US-00002 TABLE 2 list of Yarrowia strains used for production of retinoids carrying the heterologous acyltransferase genes. For more details, see text. ML First described strain Description in 7788 Carotene strain W02016172282 15710 ML7788 transformed with W02016172282 MB7311 -Mucor CarG 17544 ML15710 cured of URA3 by FOA here and HygR by Cre/lox 17767 ML17544 transformed with MB6072 here DmBCO-URA3 and MB6732 SbATF1- HygR and cured of markers 17978 ML17968 transformed with MB8200 here FfRDH-URA3 and cured of markers

[0065] Normal phase retinol method. A Waters 1525 binary pump attached to a Waters 717 auto sampler were used to inject samples. A Phenomenex Luna 3 Silica (2), 1504.6 mm with a security silica guard column kit was used to resolve retinoids. The mobile phase consists of either, 1000 mL hexane, 30 mL isopropanol, and 0.1 mL acetic acid for astaxanthin related compounds, or 1000 mL hexane, 60 mL isopropanol, and 0.1 mL acetic acid for zeaxanthin related compounds. The flow rate for each is 0.6 mL per minute. Column temperature is ambient. The injection volume is 20 L. The detector is a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 3.

TABLE-US-00003 TABLE 3 list of analytes using normal phase retinol method. For more details, see text. Retention time Lambda max Intermediates [min] [nm] 11-cis-dihydro-retinol 7.1 293 11-cis-retinal 4 364 11-cis-retinol 8.6 318 13-cis-retinal 4.1 364 dihydro-retinol 9.2 292 retinyl-acetate 3.5 326 retinyl-ester 3 325 trans-retinal 4.7 376 trans-retinol 10.5 325

[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, the samples were processed in a Precellys homogenizer (Bertin Corp, Rockville, Md., USA) on the highest setting 3 according to the manufactures directions. In the washed broth 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 HPLC analysis.

[0067] Fermentation conditions. Fermentations were identical to the previously described conditions using mineral oil overlay and stirred tank that was corn oil fed in a bench top reactor with 0.5 L to 5 L total volume (see WO2016172282). Generally, the same results were observed with a fed batch stirred tank reactor with an increased productivity demonstrating the utility of the system for the production of retinoids.

Example 2: Production of Retinoids in Yarrowia lipolytica

[0068] Retinol producing strain ML17767 was transformed with purified HinDIII/XbaI gene fragments containing codon optimized acyltransferase genes linked to URA3 nutritional marker and selected on minimal media without uracil. Multiple isolates were screened in a shake plate assay for increased acylation of retinol and decreased residual retinol. Successful isolates were run in fed batch stirred tank reactors to show utility of the method for increased production of retinol esters. The result of the experiment is shown below indicating that we have isolated genes with increased retinol acylation activity in a fungal production system.

TABLE-US-00004 TABLE 4 Retinoid production in Yarrowia as enhanced by action of heterologous acyltransferase enzymes. % ester means percentage of long chain retinyl esters in the mix of retinoids. For more details, see text. % SEQ ID Accession increased ML MB NO: Organism no. ester- strain plasmid (aa/nt) D. melanogaster XM_502557 14% 17767 8299 3/4 Y. lipolytica NM_135969 20% 17767 8201 5/6 Y. lipolytica XM_504038 0 17767 8195 7/8 F. oxysporum EXK27040 10% 17767 8200 9/10

Example 3: Production of Retinoids in Saccharomyces cerevisiae

[0069] Typically, a beta carotene strain is transformed with heterologous genes encoding for enzymes such as geranylgeranyl synthase, phytoene synthase, lycopene synthase, lycopene cyclase constructed that is producing beta carotene according to standard methods as known in the art (such as e.g. as described in US20160130628 or WO2009126890). By introducing and/or overexpressing the acyltransferase enzymes as defined herein, similar results regarding production of Long chain retinyl esters, are obtained. Further, when transformed with beta carotene oxidase genes retinal can be produced. Further, when transformed with retinol dehydrogenase, then retinol can be produced. With this approach, similar results regarding productivity towards long chain retinyl esters are obtained.