NOVEL BETA-CAROTENE OXIDASES

Abstract

The present invention is related to a method for increasing the trans-specificity of a beta-carotene oxidase (BCO), particularly insect BCO, to be used in the production of vitamin A aldehyde (retinal) from conversion of beta-carotene, with at least about 75 to 100% of retinal in the trans-isoform.

Claims

1. A beta-carotene oxidase enzyme, preferably insect enzyme, more preferably enzyme originated from Drosophila, comprising one or more amino acid substitution(s) in a sequence with at least about 60%, such as 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:1, wherein the one or more amino acid substitution(s) are located at position(s) corresponding to amino acid residue(s) selected from 91 and/or 499 in the polypeptide according to SEQ ID NO:1 and wherein the amino acid of residue 91 being tryptophan or phenylalanine and/or amino acids of residue 499 being selected from methionine, leucine or isoleucine.

2. The enzyme according to claim 1 catalyzing the conversion of beta-carotene into retinal with a ratio of at least about 78% as trans-retinal based on total retinoids.

3. The enzyme according to claim 1, wherein at least about 5% of beta-carotene is converted into retinal.

4. The enzyme according to claim 1, wherein the specificity towards trans-isoforms including the formation of trans-retinal is increased by at least about 7% based on total retinoids compared to the trans-specificity of the respective enzyme without carrying one or more of said amino acid substitution(s).

5. The enzyme according to claim 1, comprising a single amino acid substitution located at a position corresponding to amino acid residues selected from 91 and/or 499, preferably selected from residue 499, in the polypeptide according to SEQ ID NO:1.

6. The enzyme according to claim 1, comprising at least two amino acid substitutions at positions corresponding to amino acid residues selected from 91 and 499 in the polypeptide according to SEQ ID NO:1.

7. The enzyme according to claim 1 which is expressed in a carotenoid-producing host cell, preferably a fungal host cell, more preferably selected from Yarrowia or Saccharomyces.

8. A carotenoid-producing host cell, particularly fungal host cell, comprising an enzyme according to claim 1, wherein said host cell being preferably selected from Yarrowia or Saccharomyces and being transformed with a polynucleotide expressing said enzyme.

9. A process for production of trans-retinal comprising providing a carotenoid-producing host cell according to claim 8, cultivating said host cell in a suitable culture medium under suitable culture conditions, and optionally isolating and/or purifying the trans-retinal from the medium, wherein the ratio of trans-retinal is in the range of at least about 78% based on total retinoids.

10. A process for increasing the conversion of beta-carotene into trans-retinal by at least 7% based on total retinoids in a carotenoid-producing host cell comprising transforming said host cell, preferably fungal host cell, more preferably a host cell selected from Yarrowia or Saccharomyces, with an enzyme according to claim 1.

11. A process for production of vitamin A comprising the steps of: (a) introducing a nucleic acid molecule encoding one of the modified BCO enzymes according to claim 1 into a suitable carotenoid-producing host cell, particularly fungal host cell, (b) enzymatic conversion, i.e. stereo-selective oxidation, of beta-carotene via action of said expressed modified BCO into at least about 78% of trans-retinal based on total retinoids, (c) optionally, enzymatic conversion of retinal with a percentage of at least about 75% trans-retinal into retinol via action of retinol dehydrogenases, (d) optionally, enzymatic conversion, i.e. acetylation, of retinol via action of acetyl transferase enzymes; and (e) optionally, conversion of said retinyl acetate into vitamin A under suitable conditions known to the skilled person.

12. Use of an enzyme according to claim 1 in a process for production of retinyl acetate in a suitable host cell, comprising the step of conversion of beta-carotene into retinal by the action of said enzyme and optionally further enzymatic conversion into retinyl acetate.

13. Use according to claim 12, wherein the percentage of trans retinyl acetate is in the range of at least about 78% based on total retinoids.

14. Method for increasing the trans-specificity of a beta-carotene oxidase enzyme comprising the steps of: (1) alignment of different beta-carotene oxidase enzymes, including but not limited to enzymes originated from insects, preferably from Drosophila, such as e.g. identified via BLAST search against UNIREF/UNIPROT databases, with SEQ ID NO:1, wherein the selected enzymes show high activity towards retinal production, i.e. in the range of at least about 5%, such as e.g. at least 2-fold higher than the BCO of Danio rerio, (2) identification of the positions in the selected enzymes corresponding to amino residue 91 and/or 499 in the polypeptide according to SEQ ID NO:1, (3) introduction of at least one or two amino acid substitution(s) on position(s) corresponding to amino acid residue(s) selected from 91, 499 and combinations thereof identified in SEQ ID NO:1 in the aligned sequences; and (4) screening for trans-retinal activity in a carotenoid-producing host cell, preferably selected from Yarrowia or Saccharomyces, with conversion rates of at least about 78 to 100% towards formation of trans-retinal based on total retinoids, whereby the activity of the enzyme, i.e. conversion of beta-carotene into retinal, is in the range of at least about 5%.

Description

EXAMPLES

Example 1: General Methods, Strains and Plasmids

[0066] 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).

[0067] 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 200 μl 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.

[0068] 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.

[0069] 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 Nat® 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 PvulI (MB6157 and MB8327), respectively purified by gel electrophoresis and Qiagen gel purification column.

[0070] Plasmid list. Plasmid, strains and codon-optimized sequences to be used are listed in Table 1, 2 and the sequence listing. Nucleotide sequence ID NO:2 is codon optimized for expression in Yarrowia.

TABLE-US-00001 TABLE 1 list of plasmids used for construction of the strains carrying the heterologous modified 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 DmBCO ½

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

[0071] Normal phase retinol method. A Waters 1525 binary pump attached to a Waters 717 auto sampler were used to inject samples. A Phenomenex Luna 3μ 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 Intermediates time [min] max [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

[0072] 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.

[0073] Fermentation conditions. Fermentations (especially on larger scale) 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

[0074] 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 (non-modified or modified 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 sequence (SEQ ID NO:2) 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 at least 78.1 up to 96.5% using the modified BCOs. The wild-type, i.e. non-modified, BCO from Drosophila melanogaster (SEQ ID NO:1) resulted in only 73% of trans-retinal based on total retinoids (see Table 4). Further, a native RDH reduces retinal to retinol in Yarrowia lipolytica. These isomers can also be monitored as surrogates for the retinal cis/trans isomers. The enzyme activity indicating the generation of retinal from conversion of beta-carotene was about the same irrespectively whether the wildtype or modified BCOs were used (about 5 to 20% conversion into retinal).

TABLE-US-00004 TABLE 4 Retinal production in Yarrowia as enhanced by action of modified BCOs originated from Drosophila melanogaster (DmNinaB). “% trans” means percentage of trans-retinal in the mix of retinoids; “DCW” means dry cell weight”. For more details, see text. % % retinoids/ ML MB BCO gene trans- DCW strain plasmid DmNinaB wt 73 14 17544 6702 DmNinaB T499L 95.3 9.8 17544 9343 DmNinaB L91W-T499L 96.5 7.3 17544 9357 DmNinaB L91F-T499L 91.7 7.6 17544 9358 DmNinaB T499M 96.4 9.9 17544 9360 DmNinaB T4991 83.2 5.2 17544 9363 DmNinaB L91F 90.8 14 17544 9339 DmNinaB L91W 78.1 14 17544 9338

[0075] Furthermore, various insect BCOs with at least about 60% identity to SEQ ID NO:1 were tested for occurrence of the amino acid residues on positions corresponding to L91, L336, M364, T499, and L611 (see Table 5).

[0076] Surrounding amino acids were identified by modeling the structure of the enzyme encoded by SEQ ID NO:1 using the software program Yasara (https://www.yasara.org/) using the following parameters and PDB code 4RSC (downloadable from http://www.pdb.org) as the template structure: Modeling speed (slow=best): Slow

[0077] Number of PSI-BLAST iterations in template search (PsiBLASTs): 3

[0078] Maximum allowed (PSI-)BLAST E-value to consider template (EValue Max): 0.5

[0079] Maximum number of templates to be used (Templates Total): 1

[0080] Maximum number of templates with same sequence (Templates SameSeq): 1

[0081] Maximum oligomerization state (OligoState): 4 (tetrameric)

[0082] Maximum number of alignment variations per template: (Alignments): 3

[0083] Maximum number of conformations tried per loop (LoopSamples): 50

[0084] Maximum number of residues added to the termini (TermExtension): 10

[0085] The homology model that is produced by Yasara can subsequently be inspected by someone skilled in the art to identify residues surrounding the mutated positions 91 and 499. Subsequently, an alignment was made from the closest homologous sequences from the Uniref/Swissprot database (https://www.uniref.org) that score 58% and up compared to SEQ ID NO:1, and the conservancy of the 5 positions above is checked. This data is shown in Table 5. All residues are strictly conserved of the mutated positions 91 and 499 and their surrounding residues 336, 364 and 611, which are directly in the active site where the beta-carotene substrate binds and close to the metal ion bound by the conserved catalytic His cluster in the enzyme, and therefore it is expected that the effect of the claimed mutations on cis/trans-specificity will also be the same in these homologous sequences.

TABLE-US-00005 TABLE 5 Blast search for insect BCOs with at least 60% identity to SEQ ID NO: 1 showing conserved amino acids corresponding to position 91, 499, 336, 364 and 611. The “reference #” is the UNIREF-SWISSPROT database code (www.uniprot.org), “identity” is the longest identity to DmBCO1 (SEQ ID NO: 1), “L91” means corresponding AA on 91L mutation position, “T499” means corresponding AA on 499T mutation position, “L336” means corresponding AA on 336L surrounding position, “M364” means corresponding AA on 364M surrounding position, and “L611” means corresponding AA on 611L surrounding position. The molecular function annotation for all shown sequences is beta-carotene 15,15′-monooxygenase. For more details, see text. Identity Reference # Organism [%] L91 T499 L336 M364 L611 SEQ ID NO: 1 Drosophila melanogaster 100 L T L M L B3P415 Drosophila erecta 96.0% L T L M L A0A1W4V4X0 Drosophila ficusphila 93.6% L T L M L B3LWV8 Drosophila ananassae 90.2% L T L M L B4G3J1 Drosophila persimilis 84.5% L T L M L Q299Z6 Drosophila pseudoobscura pseudoobscura 84.4% L T L M L B4NH74 Drosophila willistoni 84.0% L T L M L A0A3B0K9S8 Drosophila guanche 83.9% L T L M L B4JYB7 Drosophila grimshawi 83.1% L T L M L B4KBR0 Drosophila mojavensis 83.1% L T L M L A0A3B0K2Y2 Drosophila guanche 82.3% L T L M L A0A0M3QXZ7 Drosophila busckii 82.3% L T L M L B4MBL2 Drosophila virilis 81.4% L T L M L A0A0Q9WZH1 Drosophila mojavensis 81.3% L T L M L W8AFR2 Ceratitis capitata 71.8% L T L M L A0A1A9V8V6 Glossina austeni 65.1% L T L M L A0A1A9XM81 Glossina fuscipes fuscipes 64.7% L T L M L A0A1B0FGT0 Glossina morsitans morsitans 64.4% L T L M L A0A1B0BNS4 Glossina palpalis gambiensis 64.2% L T L M L A0A182NUF8 Anopheles dirus 61.5% L T L M L A0A182Y0A2 Anopheles stephensi 61.3% L T L M L A0A182WU08 Anopheles quadriannulatus 61.2% L T L M L A0A182URE3 Anopheles merus 60.9% L T L M L A0A182PPK6 Anopheles epiroticus 60.7% L T L M L A0A182W715 Anopheles minimus 60.5% L T L M L A0A182LRC9 Anopheles culicifacies 60.4% L T L M L A0A182JU85 Anopheles christyi 60.2% L T L M L A0A182F4X0 Anopheles albimanus 60.2% L T L M L A0A1Y9GLE5 Anopheles arabiensis 60.2% L T L M L A0A182L1F0 Anopheles coluzzii 60.1% L T L M L A0A182Q6V3 Anopheles farauti 60.0% L T L M L A0A182SJU9 Anopheles maculatus 59.5% L T L M L W5J3D8 Anopheles darlingi 59.0% L T L M L A0A182H5Q0 Aedes albopictus 58.7% L T L M L Q17FY3 Aedes aegypti 58.5% L T L M L

Example 3: Production of Trans-Retinal in Saccharomyces cerevisiae

[0086] 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, WO2009126890 or Verwaal et al., Applied and Environmental Microbiology, Vol. 73, No. 13, pp. 4342-4350, 2007). Carotene producing strain MY4378 (CEN.PK113-7D FBA1p-crtE; TEF1p-crtYB; ENO1p-crtI) is transformed with modified BCOs that are codon optimized for expression in Saccharomyces like vector MB8433 (DmNinaB wt HYGR) to make strain MY4382(CEN.PK113-7D FBA1p-crtE; TEF1p-crtYB; ENO1p-crtI TEF1p-DmNinaB wt HYGR) or in an analogous fashion to result in at least 78% trans-retinal based on total retinoids including retinal. Optionally, when transformed with retinol dehydrogenase from vector MB8431, then retinol can be produced. Vector MB8433 is constructed as an integrating Hygromycin selectable vector based on the backbone MB7622 (SEQ ID NO:3) by insertion of the coding sequence into the unique BamHI/EcoRI sites to yield vectors MB8431 (SEQ ID NO:4) and MB8433 (SEQ ID NO:5). Further, optionally the enzymes can be selected to produce and acetylate the trans form of retinol to yield a high amount of all-trans retinyl acetate.

[0087] Using the BCOs according to Table 4 in S. saccharomyces as host cell, conversion of beta-carotene into retinal with percentage of at least about 5% can be obtained, with a selectivity for trans-retinal based on total retinoids in the range of at least about 78%. The % retinoids/DCW is much lower as compared to Yarrowia lipolytica as host cell, such as e.g. in the range of about 2 to 3 (data not shown).