PRODUCTION OF RETINOL

Abstract

The present invention is related to a novel enzymatic process for production of vitamin A alcohol (retinol) via conversion of retinal, which process includes the use of heterologous enzymes having activity as retinal reductase, particularly wherein the reaction leads to at least about 90% conversion of retinal into retinol. Said process is particularly useful for biotechnological production of vitamin A.

Claims

1. A carotenoid-producing host cell comprising a retinol dehydrogenase [EC 1.1.1.105], preferably a heterologous retinol dehydrogenase, said host cell producing a retinoid mix comprising retinal and retinol, wherein the percentage of retinol is at least about 90%, preferably 92, 95, 97, 98, 99 or even 100% compared to the amount of retinal present in said retinoid mix.

2. The carotenoid-producing host cell of claim 1, wherein the retinal to be reduced via action of the retinol dehydrogenase comprises a mix of trans-retinal and cis-retinal, wherein the percentage of trans-retinal in said retinal mix is in the range of at least about 61 to 98%, preferably at least about 61 to 95%, more preferably at least about 61 to 90%.

3. The carotenoid-producing host cell according to claim 1, wherein the retinol dehydrogenase is selected from fungi, preferably Fusarium, more preferably retinol dehydrogenase is a Fusarium fujikuroi retinol dehydrogenase (FtRDH).

4. The carotenoid-producing host cell according to claim 3, wherein the FtRDH is selected from a polypeptide with at least about 60% identity to a polypeptide according to SEQ ID NO:1.

5. The carotenoid-producing host cell according to claim 1, wherein the host cell is selected from plants, fungi, algae or microorganisms, such as 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, and Blakeslea, preferably selected from fungi including yeast, more preferably selected from the group consisting of Saccharomyces, Aspergillus, Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea and Yarrowia, most preferably from Yarrowia lipolytica or Saccharomyces cerevisiae.

6. The carotenoid-producing host cell according to claim 1, wherein the retinol is further converted into vitamin A.

7. The carotenoid-producing host cell according to claim 1, further comprising a stereoselective beta-carotene oxidizing enzyme selected from Drosophila catalyzing the conversion of beta-carotene to a retinal mix, wherein the mix comprises at least about 61%, preferably 68, 70, 75, 80, 85, 90, 95, 98 or up to 100% retinal in trans-isoform based on the total amount of retinal in the mix, more preferably selected from a sequence with at least 60% identity to a polypeptide according to SEQ ID NO:3.

8. A process for production of a retinoid mix comprising retinol and retinal via enzymatic activity of a retinol dehydrogenase [EC 1.1.1.105], comprising contacting retinal with said retinol dehydrogenase, wherein the ratio of retinol to retinal in the retinoid mix is at least about 9:1.

9. A process for decreasing the amount of retinal in a retinoid mix produced from enzymatic action of retinol dehydrogenase, said process comprising contacting retinal with a retinol dehydrogenase, wherein the amount of retinal in the retinoid mix resulting from said enzymatic action is in the range of about 10% or less compared to the amount of retinol.

10. A process for increasing the amount of retinol in a retinoid mix produced from enzymatic action of retinol dehydrogenase, said process comprising contacting retinal with a retinol dehydrogenase, wherein the amount of retinol in the retinoid mix resulting from said enzymatic action is in the range of at least about 90% compared to the amount of retinol.

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

12. A process for production of vitamin A comprising the steps of: (a) introducing a nucleic acid molecule encoding a retinol dehydrogenase [EC 1.1.1.105] into a suitable carotene-producing host cell, (b) enzymatic conversion of retinal into a retinoid mix comprising retinol and retinal in a ratio of at least about 9:1, (c) conversion of retinol into vitamin A under suitable culture conditions.

13. Use of a carotenoid-producing host cell according to claim 1 for production of a retinoid mix comprising retinol and retinal in a ratio of 9:1.

Description

EXAMPLES

Example 1: General Methods, Strains, and Plasmids

[0069] 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).

[0070] 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.

[0071] 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 3550 MW, 100 mM lithium acetate, 50 mM Dithiothreitol, 5 mM Tris-Cl pH 8.0, 0.5mM 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.

[0072] DNA molecular biology. Genes were synthesized with Nhel and Mlul 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 PvuII (MB6157 and MB8327), respectively purified by gel electrophoresis and Qiagen gel purification column.

[0073] Plasmid list. Plasmid, strains, nucleotide and amino acid sequences to be used are listed are listed in Table 1, 2 and the sequence listing. Nucleotide sequence ID NOs:2, 4, 5, 6, and 7 are codon optimized for expression in Yarrowia.

TABLE-US-00001 TABLE 1 list of plasmids used for construction of the strains carrying the heterologous RDH-genes. The sequence ID NOs refer to the inserts. For more details, see text. SEQ ID NO: MB plasmid Backbone MB Insert (aa/nt) 8200 5082 FfRDH12 1/2 8203 5082 HsRDH12 5 8196 5082 RnRDH12 6 8197 5082 McRDH12 7

TABLE-US-00002 TABLE 2 list of Yarrowia strains used for production of retinoids carrying the heterologous RDH genes. For more details, see text. ML strain Description First described in 7788 Carotene strain WO2016172282 15710 ML7788 transformed with WO2016172282 MB7311-Mucor CarG 17544 ML15710 cured of URA3 by here FOA and HygR by Cre/lox 17767 ML17544 transformed with here MB6072 DmBCO-URA3 and MB6732 SbATF1-HygR and cured of markers 17978 ML17968 transformed with here MB8200 FfRDH-URA3 and cured of markers

[0074] 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 Lambda time 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

[0075] 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.

[0076] 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

[0077] Typically, the beta carotene strain ML17767 was transformed with purified HinDIII/XbaI fragments derived from plasmids containing retinol dehydrogenase (RDH) gene fragments linker to a URA3 promoter. Six to eight isolates were screened for a decrease in the retinol:retinal ratio in a shake plate assay and successful isolates were run in a fed batch stirred tank reactor for eight days which showed an order of magnitude increase in the productivity of the process which indicates a utility in large scale production. The best results were obtained with the Fusarium RDH12 homolog with only 2% or residual retinal maintained after 8 days of shake-flask incubation as described above. The isolate derived from the Fusarium sequence demonstrated an increased reduction of retinol as indicated in the following table.

Example 3: Production of Retinoids in Saccharomyces cerevisiae

[0078] 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). 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 specificity for productivity towards retinol are obtained.

Example 4: Production of Retinol from Beta-Carotene

[0079] In addition to the single modifications described in Examples 2, 3 and 4 a strain was constructed carrying the heterologous together with the heterologous FtRDH12. Fermentation and analysis of the retinoids was done as described before.

[0080] For expression of heterologous BCO from Drosophila melanogaster DmNinaB (DmBC01; SEQ ID NO:3), a beta carotene strain ML17544 was transformed with purified linear DNA fragment by HindII and XbaI mediated restriction endonucleotide cleavage of beta carotene oxidase (BCO) containing codon optimized fragments linked to a URA3 nutritional marker. Transforming DNA were derived from MB6702 Drosophila NinaB BCO gene, whereby the codon-optimized sequences (SEQ ID NO:4) had been used. The gene were then grown screening 6-8 isolates in a shake plate analysis, isolates that performed well were run in a fed batch stirred tank reaction for 8-10 days. Detection of cis-and trans-retinal was made by HPLC using standard parameters as described in WO2014096992, but calibrated with purified standards for the retinoid analytes. The amount of trans-retinal in the retinal mix heterologous expressing the BCO from Drosophila melanogaster (SEQ ID NO:3) resulted in 61% of trans-retinal based on the total amount of retinal (not shown).

[0081] The presence of heterologous FtRDH12 reduced the amount of retinal detected in the analyte from 20% to 4%, which is a good indication for specific retinal-reducing activity of the Fusarium RDH12 (see Ex. 2), with still a percentage of trans-retinol in the range of at least 61%.