BIOSYNTHESIS OF RETINOIDS

Abstract

The present invention is related to a novel enzymatic process for production of retinoids via a multi-step process, which process includes the use of heterologous enzymes having activity in a carotene-producing host cell, particularly wherein such process results in high percentage of retinoids, in trans-isoform.

Claims

1. A carotenoid-producing host cell, particularly fungal host cell, comprising: (a) stereoselective beta-carotene oxidizing enzyme (BCO), said host cell producing a retinal mix comprising cis- and trans-retinal, wherein the percentage of trans-retinal in the mix is at least about 65%, preferably 68, 70, 75, 80, 85, 90, 95, 98% or up to 100% produced by said host cell; and (b) an acetyl transferase (ATF) [EC 2.3.1.84], preferably an enzyme with acetyl transferase 1 (Atf1) activity, said enzyme catalyzing the conversion of retinol, preferably trans-retinol, to a retinyl acetate mix, with a percentage of at least 10% of acetylated retinol, i.e. retinyl acetate, based on the total amount of retinoids produced by said host cell.

2. The carotenoid-producing host cell according to claim 1, wherein the acetyl transferase [EC 2.3.1.84], preferably an enzyme with acetyl transferase 1 activity, catalyzes the conversion of retinol to a retinyl acetate mix, wherein the mix comprises at least about 65%, preferably 68, 70, 75, 80, 85, 90, 95, 98% or up to 100% retinyl acetate, such as e.g. at least 65-90% retinyl acetate, in trans-isoform.

3. The carotenoid-producing host cell according to claim 1, further comprising a preferably heterologous retinol dehydrogenase (RDH) [EC 1.1.1.105] capable of converting retinal into retinol with a total conversion of at least about 90% towards generation of retinol, preferably selected from fungi, more preferably from Fusarium, such as a polypeptide with at least 60% identity to a polypeptide according to SEQ ID NO:19.

4. The carotenoid-producing host cell according to claim 1, further comprising a modification in the endogenous acyltransferase activity, wherein the endogenous acyltransferase activity, preferably [EC 2.3.1] activity, more preferably acyltransferase [EC 2.3.1.20] activity, has been reduced or abolished.

5. The carotenoid-producing host cell according to claim 1, wherein the BCO is selected from fungi, plants or animal, preferably selected from Fusarium, Ustilago, Crocus, Drosophila, Danio, Ictalurus, Esox, Latimeria, more preferably selected from Fusarium fujikuroi, Ustilago maydis, Crocus sativus, Drosophila melanogaster, Danio rerio, Ictalurus punctatus, Esox Lucius, Latimeria chalumnae, even more preferably selected from a polypeptide with at least about 60% identity to a polypeptide according to SEQ ID NOs:1, 3, 5 or 7 or a polypeptide with at least about 50% identity to a polypeptide sequence according to SEQ ID NOs:9, 11, 13, 15 or 17.

6. The carotenoid-producing host cell according to claim 1, wherein the acetyl transferase is selected from plants, animals, including humans, algae, fungi, including yeast or bacteria, preferably selected from Saccharomyces, Fragaria, Escherichia, Euonymus, Malus, Petunia or Lachancea, more preferably selected from a polypeptide with at least about 60% identity to a polypeptide according to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 36, or 38.

7. The carotenoid-producing host cell according to claim 1, producing a retinyl acetate mix comprising at least about 65%, preferably 68, 70, 75, 80, 85, 90, 95, 98% or up to 100% trans-retinyl acetate isoform, such as at least 65-90% trans-retinyl acetate isoform.

8. 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.

9. The use of the carotenoid-producing host cell according to claim 1 in a process for conversion of beta-carotene into vitamin A.

10. A process for production of trans-retinyl acetate comprising cultivation of the carotenoid-producing host cell according to claim 1 in an aqueous medium under suitable culture conditions and isolating and optionally further purifying said trans-retinyl acetate from the medium and/or host cell.

11. A process for production of vitamin A comprising the steps of: (a) introducing a nucleic acid molecule encoding stereoselective BCO, acetyl transferase [EC 2.3.1.84], optionally retinol dehydrogenase [EC 1.1.1.105], into a suitable host cell; (b) optionally reducing or abolishing the endogenous acyltransferase activity [EC 2.3.1] of the cell of (a), (c) enzymatic conversion of beta-carotene into retinoids comprising at least a percentage of 10% retinyl acetate, said retinyl acetates comprising at least a percentage of 65% in trans isoform based on the total amount of produced retinoids; and (d) conversion of retinyl acetate into vitamin A under suitable culture conditions.

Description

EXAMPLES

Example 1: General Methods, Strains, and Plasmids

[0090] 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).

[0091] 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 800 μl of mineral oil (Drakeol 5, Penreco Personal Care Products, Karns City, Pa., USA) carbon source 5% corn oil in mineral oil and/or 5% in glucose in aqueous phase. Transformants were grown in 24 well plates (Microplate Devices 24 Deep is Well Plates Whatman 7701-5102), covered with mat seal (Analytical Sales and Services Inc. Plate Mats 24010CM), sterile sealed with Qiagen Airpore Tape Sheets (19571) and shaken in Infors multi plate shaker (Multitron), 30° C., 800 RPM in YPD media with 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. This method is used in Examples 2, 3, 4.

[0092] 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 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.

[0093] DNA molecular biology. Genes were synthesized with NheI and MluI ends in pUC57 vector (GenScript, Piscataway, N.J.). 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. MB5082 ‘URA3’ marker could be reused due to gratuitous repeated flanking sequences that enable selection of circular excisants of the URA3 cassette on FOA. The NatR and HygR markers can be removed by transient expression of Cre recombinase that results in excisants due to the flanking Lox sites.

[0094] Plasmid list. Plasmid, strains, nucleotide and amino acid sequences to be used are listed in Table 1, 2 and the sequence listing. Nucleotide sequence ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 35, 37, and 39 are codon optimized for expression in Yarrowia.

TABLE-US-00001 TABLE 1 list of plasmids used for construction of the strains carrying the heterologous BCO, RDH and ATF1-genes. The sequence ID NOs refer to the inserts. For more details, see text. SEQ ID NO: MB plasmid Backbone MB Insert (aa/nt) 8457 5082 UmCCO1 1/2 8456 5082 FfCarX 3/4 6703 5082 CsZCO 5/6 6702 5082 DmNinaB 7/8 9068 5082 DrBCO  9/10 9279 5082 DrBCO-TPI 11/12 9123 5082 IpBCO 13/14 9121 5082 ElBCO 15/16 9126 5082 LcBCO 17/18 8200 5082 FfRDH12 19/20 8064 5082 SbATF1 21/22 8509 6157 FaATF 23/24 8510 6157 EcCAT 25/26 8511 6157 EaCAcT 27/28 8512 6157 MdATF 29/30 8513 6157 PhATF 31/32 8849 5082 LmATF1 33/35 8610 5082 LfATF1 36/37 8806 5082 LffATF1 38/39

TABLE-US-00002 TABLE 2 list of Yarrowia strains used for production of retinoids carrying the heterologous BCO, RDH and ATF1-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 17968 ML17544 transformed with here MB8457 UmCCO1- URA3 and cured of markers 17978 ML17968 transformed with here MB8200 FfRDH-URA3 and cured of markers

[0095] Normal phase retinol method. A Waters 1525 binary pump attached to a Waters 717 auto sampler were used to inject samples. A Phenomenex Luna 3p Silica (2), 150×4.6 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 μL. 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. The addition of all added intermediates gives the amount of total retinoids. 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

[0096] 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.

[0097] Fermentation conditions. Fermentations were identical to the previously described conditions using preferably a silicone oil or a mineral oil overlay and stirred tank that was preferably glucose or 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. Preferably, fermentations were batched with 5% glucose and 20% silicone oil was added after dissolved oxygen plummeted and feed was resumed to achieve 20% dissolved oxygen throughout the feeding program. Alternatively, corn oil was used as a feed and mineral oil was used as a second phase to collect the aliphatic retinoids.

Example 2: Conversion of Beta-Carotene to Retinal in Yarrowia lipolytica

[0098] For expression of heterologous BCOs, a beta carotene strain ML17544 was transformed with purified linear DNA fragment by HindII and XbaI mediated restriction endonucleotide cleavage and gel purification 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, MB6703 Crocus BCO gene, MB8456 Fusarium BCO gene, and MB8457 Ustilago BCO gene and MB6098 Dario BCO gene, whereby the codon-optimized sequences (SEQ ID NOs:2, 4, 6, 8, 10, 12) had been used. The genes were then grown screening 6-8 isolates in a shake plate analysis, and isolates that is 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 could be increased to 90% (using the Crocus BCO), 95% (using the Fusarium BCO), 98% (using the Ustilago BCO) and 98% (using Dario BCO), respectively. A comparison with the BCO from Drosophila melanogaster (SEQ ID NO:7) resulted in 61% of trans-retinal based on the total amount of retinal (see Table 4).

TABLE-US-00004 TABLE 4 Retinal production in Yarrowia as enhanced by action of heterologous BCOs. “% trans” means percentage of trans-retinal in the mix of retinoids. For more details, see text. % retinoids/ ML MB Organism BCO gene % trans- DCW strain plasmid Drosophila DmNinB 61 14 17544 6702 Ustilago UmCCO1 98 8 17544 8457 Fusarium FfCarX 95 5 17544 8456 Crocus ZsZCO 90 0.01 17544 6703 Dario DrBCO 98 6 17544 9068 Dario DrBCO-TPI 98 6 17544 9279 Ictalurus IpBCO 98 5 17544 9123 Esox ElBCO 98 3 17544 9121 Latimeria LcBCO 98 2 17544 9126

Example 3: Conversion of Retinal to Retinol in Yarrowia lipolytica

[0099] For expression of heterologous RDHs, 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.

Example 4: Conversion of Retinol to Retinyl Acetate in Yarrowia lipolytica

[0100] For expression of heterologous ATF1, the trans retinol producing strain ML17968 was transformed with purified PvuII gene fragments containing acetyltransferase gene fragments linked to a Hygromycin resistance marker (HygR) for selection rich media (YPD) containing 100 ug/ml hygromycin. Prior to plating the cultures were outgrown in YPD for four hours to synthesize the antibiotic resistance genes. Isolates were screened for acylation in shake plate assays and successful isolates were screened in fed batch stirred tank reactor which showed an order of magnitude increased productivity indicating utility in the production of retinoids. The data from the analysis are shown in Table 5).

TABLE-US-00005 TABLE 5 Trans retinoid production in Yarrowia as enhanced by action of heterologous ATF1 enzymes. “% acetylation” means percentage of trans-retinyl acetate in the mix of retinoids. For more details, see text. ML MB Organism ATF1 gene % acetylation- strain plasmid S. bayanus SbATF1 10.3 17968 6832 P. hybrida PhATF 2.1 17968 8513 E. alatus EaCAcT 0.45 17968 8511 E. coli EcCAT 0.35 17968 8510 L. fermentata LfATF1 9.6 18523 8610 L. fermentata LffATF1 11.7 18523 8806 L. mirantina LmATF1 40.4 18523 8849

Example 5: ATF1 Activity Assay

[0101] For expression of heterologous ATF1, the trans retinol producing strain ML17968 was transformed with purified PvuII gene fragments containing acetyltransferase gene fragments linked to a Hygromycin resistance marker (HygR) for selection rich media (YPD) containing 100 ug/ml hygromycin. Prior to plating the cultures were outgrown in YPD for four hours to synthesize the antibiotic resistance genes. Isolates were screened for acylation in shake plate assays, specifically using 10% glucose as a carbon source in 0.25×YP with silicone oil as an overlay and successful isolates were further screened in fed batch stirred tank reactor with glucose feed and silicone oil overlay, which showed an order of magnitude increased productivity indicating utility in the production of retinoids. The data from the analysis are shown in Table 5.

Example 6: Conversion of Beta-Carotene to Retinyl Acetate in Saccharomyces cerevisiae

[0102] 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. The retinol can be acetylated by transformation with genes encoding alcohol acetyl transferases. Optionally, the endogenous retinol acylating genes can be deleted. Further, the enzymes can be selected to produce and acylate the trans form of retinol to yield all trans retinyl acetate, and long chain esters of trans retinol, respectively. With this approach, similar results regarding specificity for trans-isoform or productivity towards retinyl acetate are obtained.