PRODUCTION OF TRANS-RETINAL

Abstract

The present invention is related to a novel enzymatic process for production of vitamin A aldehyde (retinal) via stereoselective conversion of beta-carotene which process includes the use of trans-selective enzymes having activity as beta-carotene oxidases (BCOs), in particular having preference for trans-retinal. 5 Said process is in particular useful for biotechnological production of vitamin A.

Claims

1. A carotenoid-producing 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.

2. The carotenoid-producing host cell of claim 1, wherein the percentage of trans-retinal in the retinal mix comprising trans- and cis-retinal is in the range of about at least 65 to 98%, preferably about at least 65 to 95%, more preferably at least about 65 to 90% based on the total amount of retinal produced by said host cell.

3. The carotenoid-producing host cell according to claim 1 comprising a heterologous stereoselective BCO.

4. 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.

5. The carotenoid-producing host cell according to claim 4, wherein the BCO is selected from a polypeptide with at least about 60% identity to a polypeptide according to sequences known from the database such as EAK81726, AJ854252, Q84K96.1, or with at least 50% identity to a polypeptide according to sequence known from the database as Q90WH4.

6. The carotenoid-producing host cell according to claim 5, expressing a polynucleotide encoding 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.

7. 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.

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

9. A process for production of a retinal mix comprising trans- and cis-retinal via enzymatic activity of a stereoselective BCO, comprising contacting beta-carotene with said BCO, wherein the ratio of trans-retinal to cis-retinal in the retinal mix is at least about 2:1.

10. A process for decreasing the amount of cis-retinal produced from enzymatic cleavage of beta-carotene, said process comprising contacting beta-carotene with a stereoselective BCO, wherein the amount of cis-retinal in the retinal mix resulting from cleavage of beta-carotene is in the range of about 35% or less based on the total amount of retinal.

11. A process for increasing the amount of trans-retinal produced from enzymatic cleavage of beta-carotene, said process comprising contacting beta-carotene with a stereoselective BCO, wherein the amount of trans-retinal in the retinal mix is in the range of at least about 65 to 98% based on the total amount of retinal.

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

13. A process for production of vitamin A comprising the steps of: (a) introducing a nucleic acid molecule encoding a stereoselective BCO, into a suitable carotene-producing host cell, (b) enzymatic conversion of beta-carotene into a retinal mix comprising cis- and trans-retinal, wherein the percentage of trans-retinal is at least about 65% based on the total amount of retinal, (c) conversion of trans-retinal into vitamin A under suitable culture conditions.

14. Use of a carotenoid-producing host cell according to claim 1 for production of a retinal mix comprising trans- and cis-retinal in a ratio of 2:1, wherein said host cell expressing a heterologous BCO with stereoselectivity towards production of trans-isoforms.

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).

Shake Plate Assay.

[0070] Typically, 800 l of 0.075% Yeast extract, 0.25% peptone (0.25YP) 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.

DNA Transformation.

[0071] 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, 0.5 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.

DNA Molecular Biology.

[0072] 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 PvuII (MB6157 and MB8327), respectively purified by gel electrophoresis and Qiagen gel purification column.

Plasmid List.

[0073] Plasmid, strains and codon-optimized 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 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-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

TABLE-US-00002 TABLE 2 list of Yarrowia strains used for production of retinoids carrying the heterologous BCO 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

Normal Phase Retinol Method.

[0074] 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. The addition of all added intermediates gives the amount of total retinoids. 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

Sample Preparation.

[0075] 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.

Fermentation Conditions.

[0076] 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 Trans-Retinal in Yarrowia lipolytica

[0077] Typically, 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, MB6703 Crocus BCO gene, MB8456 Fusarium BCO gene, 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 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 only 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. BCO % % ML MB Organism gene trans- retinoids/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: Production of Trans-Retinal 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 as described herein retinal can be produced. Optionally, when transformed with retinol dehydrogenase, then retinol can be produced. The retinol can optionally be acetylated by transformation with genes encoding alcohol acetyl transferases. Optionally, the endogenous retinol acylating genes can be deleted. Further, optionally the enzymes can be selected to produce and acetylate 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-retinal as described herein with Yarrowia lipolytica as host are obtained.

Example 4: Optimization of Trans-Retinal Production Using Fungal BCOs

[0079] Typically, the Ustilago BCO was codon optimized for Yarrowia lipolytica and subcloned using MluI/NheI into vectors in Table 5 below and examined for activity. These plasmids were then transformed into the carotene producing strain MB17544, a lycopene producing strain, MB14925 (erg9::ura3 car8 HMG-tm GGS carRP(E78G) alk1D alk2D) and a phytoene producing strain, MB7206(erg9::ura3bart car8 HMG GGS ura3 ade1) (see Table 5). Surprisingly, there was an optimal activity and we could show that there was an increased production of retinol from a lower activity promoters ALK1, and ACT1. We also observed decreased attenuation of the precursors in the lycopene and phytoene strains.

TABLE-US-00005 TABLE 5 list of plasmids used for construction of the strains. For more details, see text. MB plasmid gene description 6222 ENO enolase 6224 CWP cell wall protein 6226 TPI triose phospate isomerase 6228 GAPDH glycerol phosphate dehydrogenase 6230 ACT actin 7311 ALK alkane assimilating 6655 HYPO Hypothetical 6674 HSP Heat shock protein