Methods and systems for preparing retinol

Abstract

Methods and systems for cell-free bioproduction of retinal and retinol are provided, which may be performed using a combination of a beta-carotene 15,15'-dioxygenase and an alcohol dehydrogenase. Cell-free retinoid production enables reaction rates and productivity not possible / not obtained in cell-based systems.

Description

[0001] Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0002] METHODS AND SYSTEMS FOR PREPARING RETINOL

[0003] SEQUENCE LISTING

[0004] This application incorporates by reference a sequence listing submitted as a text file entitled “DEBU-035-01WO.xml” created on October 17, 2025 and having a size of 38 kilobytes.

[0005] BACKGROUND

[0006] Vitamin A encompasses a group of fat-soluble compounds referred to as retinoids, including retinol, retinal, and retinyl esters. Vitamin A is derived from provitamin (precursor) carotenoids such as beta-carotene, alpha-carotene, and beta-cryptoxanthin. Vitamin A is important for a number of functions in the human body, including development of cells and tissues, normal immune functions, and vision.

[0007] Current commercial retinol production is via chemical synthesis. Certain recent studies have explored microbial production of retinol in organisms such as Saccharomyces cerevisiae. However, improved methods of retinol production are needed in the art.

[0008] SUMMARY

[0009] The present invention provides, in various embodiments, methods and systems for cell- free bioproduction of retinal and retinol.

[0010] In some embodiments, the invention provides a composition for preparing retinol or an isomer thereof from beta-carotene, comprising: beta-carotene; a beta-carotene 15,15’- dioxygenase; and an alcohol dehydrogenase, wherein the composition is cell-free.

[0011] In some embodiments, the beta-carotene concentration is about 0.1 to about 50 mM.

[0012] In some embodiments, the beta-carotene 15,15’-dioxygenase concentration is about 1 to about 500 pM, or 1 to 90 volume% lysate.

[0013] In some embodiments, the alcohol dehydrogenase concentration is about 1 to about 500 pM, or 1 to 30 volume% lysate.

[0014] In some embodiments, the composition includes a buffer.

[0015] In some embodiments, the buffer concentration is about 10 to about 500 mM.

[0016] In some embodiments, the buffer comprises Tris, HEPES, or sodium phosphate.

[0017] In some embodiments, the composition has a pH of about 5.5 to about 9.0. Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0018] In some embodiments, the composition includes a solvent.

[0019] In some embodiments, the solvent concentration is about 1 to about 30 volume%.

[0020] In some embodiments, the solvent comprises tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether, or hexanes.

[0021] In some embodiments, the composition includes a detergent.

[0022] In some embodiments, the detergent concentration is about 0.01 to about 15 volume%.

[0023] In some embodiments, the detergent comprises Tween-80, Tween-60, Tween-40, Tween- 20, Triton-X, or PTS (polyoxyethanyl-oc-tocopheryl sebacate).

[0024] In some embodiments, the composition includes one or more additives.

[0025] In some embodiments, the additive concentration is about 0.01 to about 1 mM.

[0026] In some embodiments, the additives comprise ascorbate or tocopherols.

[0027] In some embodiments, the composition includes an iron source.

[0028] In some embodiments, the iron source concentration is about 1 to about 500 pM.

[0029] In some embodiments, the iron source comprises iron(II) sulfate or Mohr's salt.

[0030] In some embodiments, the composition includes a zinc source.

[0031] In some embodiments, the zinc source concentration is about 0.01 to about 1 mM.

[0032] In some embodiments, the zinc source comprises zinc(II) chloride.

[0033] In some embodiments, the invention provides a method of preparing retinol or an isomer thereof from beta-carotene, comprising: providing a reaction mixture comprising beta-carotene, a beta-carotene 15,15’-dioxygenase, and an alcohol dehydrogenase, wherein the reaction mixture is cell-free; and incubating the reaction mixture containing the beta-carotene, the beta-carotene 15,15’-dioxygenase, and the alcohol dehydrogenase at a temperature of about 20 to about 40 degrees Celsius for at least about 0.5 to about 24 hours.

[0034] In some embodiments, the method is performed in a single reaction vessel.

[0035] In some embodiments, the beta-carotene 15,15’-dioxygenase and the alcohol dehydrogenase are added at the same time.

[0036] In some embodiments, the beta-carotene 15, 15’-dioxygenase and the alcohol dehydrogenase are added sequentially.

[0037] In some embodiments, the method is performed in two or more reaction vessels operating in series, wherein the beta-carotene 15, 15’-dioxygenase and the alcohol dehydrogenase are provided in separate reaction vessels. Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0038] In some embodiments, the beta-carotene 15, 15 ’-dioxygenase comprises an amino acid sequence having at least 90%identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQIDNO:11, SEQIDNO:12, SEQIDNO:13, SEQIDNO:14, SEQIDNO:15, SEQ ID NO: 16, SEQIDNO:17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0039] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQIDNO:11, SEQ IDNO:12, SEQ ID NO:13, SEQIDNO:14, SEQIDNO:15, SEQ IDNO:16, SEQIDNO:17, SEQIDNO:18, or SEQIDNO:19.

[0040] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQIDNO:17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0041] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence having at least 99% identical to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQIDNO ll, SEQIDNO:12, SEQIDNO:13, SEQIDNO:14, SEQIDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQIDNO 18, or SEQ ID NO:19.

[0042] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence provided in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQIDNO 12, SEQIDNO:13, SEQIDNO:14, SEQIDNO:15, SEQIDNO:16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0043] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0044] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0045] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0046] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0047] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence provided in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0048] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 90%, identity to SEQ ID NO:25.

[0049] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 95%identity to SEQ ID NO:25. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:25. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:25. In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence provided in SEQ ID NO: 25.

[0050] Additional features and advantages of embodiments of the present invention are described further below. This summary section is meant merely to illustrate certain features of embodiments of the invention, and is not meant to limit the scope of the invention in any way. The failure to discuss a specific feature or embodiment of the invention, or the inclusion of one or more features in this summary section, should not be construed to limit the invention as claimed.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The foregoing summary, as well as the following detailed description of certain embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purposes of illustrating the systems and methods of the present application, there are shown in the drawings preferred embodiments. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown. In the drawings: Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0053] FIG. 1 shows an overview of cell-free retinol production according to embodiments of the present invention;

[0054] FIG. 2 shows chromatograms of a retinal analytical standard and the results of a first cell- free reaction (beta-carotene to retinal) demonstrating the activity of beta-carotene 15,15’- dioxygenase alone (beta-carotene is not detected by this analytical method);

[0055] FIG. 3 shows chromatograms of a retinol analytical standard and the results of a second cell-free reaction (retinal to retinol) demonstrating the activity of alcohol dehydrogenase alone;

[0056] FIG. 4 shows chromatograms of a retinal analytical standard, a retinol analytical standard, and the results of production of retinol from beta-carotene in a single reaction vessel with both enzymes (beta-carotene 15, 15’ -di oxygenase and alcohol dehydrogenase), according to embodiments of the present invention (beta-carotene is not detected by this analytical method);

[0057] FIG. 5 shows the results of beta-carotene 15, 15 ’ -di oxygenase screening in lysates for retinal production from beta-carotene (reactions were performed as described in Example 1);

[0058] FIG. 6 shows the results of native E. coll retinol dehydrogenase (Ybbo) transformations with and without antioxidant / stabilizer;

[0059] FIG. 7 shows the results of a “one-pot” reaction producing retinol from beta-carotene in a single reaction vessel in vitro, using purified hippo beta-carotene 15,15’-dioxygenase and native E. coli retinol dehydrogenase (Ybbo) lysate;

[0060] FIG. 8 shows a time course of a cell-free reaction converting beta-carotene to retinol (reaction was performed as described in Example 2); and

[0061] FIG. 9 shows results of cell-free reactions converting beta-carotene to retinol, performed with and without an accessory ketoreductase enzyme (reactions were performed as described in Example 3).

[0062] DETAILED DESCRIPTION

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0064] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0065] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0066] Manufacturing of retinoids via chemical synthesis or in cells suffers from problems that limit the efficiency and economy of retinoid production. Chemical synthesis requires extensive, elaborate, expensive, toxic, and inefficient multi-step chemical reactions. Manufacturing processes based on retinoid-producing strains (use of the whole cell) suffer from product toxicity, carbon flux redirection, diffusion problems through cell membranes, toxic byproduct generation, and challenges related to retinoid purification and isolation from complex cellcontaining mixtures.

[0067] To avoid the aforementioned problems, the present inventors have developed methods and systems for cell-free manufacturing of retinoids as a viable alternative to synthetic and cellbased manufacturing methods.

[0068] The present invention provides, in various embodiments, methods and systems for cell- free bioproduction of retinal and retinol.

[0069] In some embodiments, the invention provides a “one-pot” cell-free conversion of betacarotene to retinol. As used herein, the term “retinol” encompasses isomers of retinol, including but not limited to all-trans, 9-cis, 11 -cis, and 13 -cis. FIG. 1 shows an overview of cell-free retinol production according to embodiments of the present invention. Step A (transformation of beta-carotene to retinal) is performed using a beta-carotene 15,15’-dioxygenase (also referred to as beta-carotene 15,15'-monooxygenase or beta-carotene 15, 15 '-oxygenase). Step B (transformation of retinal to retinol) is performed using an alcohol dehydrogenase.

[0070] According to embodiments of the present invention, the overall conversion of betacarotene to retinol may be performed in a single, cell-free reaction vessel. The beta-carotene 15, 15 ’ -di oxygenase and the alcohol dehydrogenase may be added to the reaction vessel at substantially the same time (e.g., both enzymes and the starting material added to the reaction vessel together at the beginning), or may be added in sequence (e.g., the beta-carotene 15,15’- Attorney Docket No. DEBU-035 / 01WO 37396 / 183 dioxygenase is added initially, and the alcohol dehydrogenase is added once the conversion of beta-carotene to retinal is determined to be complete).

[0071] Alternatively, the overall conversion of beta-carotene to retinol may be performed in two or more cell-free reaction vessels operating in series. Step A and Step B may be performed in separate reaction vessels each containing an individual enzyme. For example, in some embodiments, the beta-carotene and the beta-carotene 15, 15 ’-dioxygenase (without the alcohol dehydrogenase) may be provided in a first reaction vessel, and the contents of the first reaction vessel may be transferred to a second reaction vessel containing the alcohol dehydrogenase after Step A is determined to be complete.

[0072] The reaction mixture according to embodiments of the present invention includes betacarotene as a starting material. In some embodiments, the beta-carotene is included in the reaction mixture at a concentration of about 0.1 to about 50 mM.

[0073] For the first cell-free reaction (Step A), the reaction mixture includes a beta-carotene 15, 15 ’-dioxygenase. In some embodiments, the beta-carotene dioxygenase is included in the reaction mixture at a concentration of about 1 to about 500 pM, or of about 1 to 90 volume% cell lysate. In various embodiments, the beta-carotene dioxygenase may be purified, in lysate, or immobilized.

[0074] For the second cell-free reaction (Step B), the reaction mixture includes an alcohol dehydrogenase. In some embodiments, the alcohol dehydrogenase is included in the reaction mixture at a concentration of about 1 to about 500 pM, or of about 1 to 30 volume% lysate. In some embodiments, the alcohol dehydrogenase is retinol dehydrogenase. In various embodiments, the alcohol dehydrogenase may be purified, in lysate, or immobilized.

[0075] In some embodiments, the reaction mixture may include a detergent. The detergent may be included in the reaction mixture at a concentration of about 0.01 to about 15 volume%. A single detergent, or a combination of two or more detergents, may be used. Examples of suitable detergents include, but are not limited to, Tween-80, Tween-60, Tween-40, Tween-20, Triton-X, and PTS (polyoxyethanyl-a-tocopheryl sebacate).

[0076] In some embodiments, the reaction mixture may include a solvent. The solvent may be added to the reaction at a concentration of about 1 to about 30 volume%. A single solvent, or a combination of two or more solvents, may be used. Examples of suitable solvents include, but Attorney Docket No. DEBU-035 / 01WO 37396 / 183 are not limited to, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentyl methyl ether (CPME), and hexanes.

[0077] In some embodiments, the reaction mixture may include a buffer. The buffer may be included in the reaction mixture at a concentration of about 10 to about 500 mM. A single buffer, or a combination of two or more buffers, may be used. Any buffer suitable for maintaining a pH of about 5.5 to about 9.0 may be used. Examples of suitable buffers include Tris (tris(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)piperazine-l -ethane-sulfonic acid), sodium phosphate, and many others.

[0078] In some embodiments, the reaction mixture may include one or more additives. The additive(s) may be included in the reaction mixture at a concentration of about 0.01 to about 1 mM. A single additive, or a combination of two or more additives, may be used. Examples of suitable additives include, but are not limited to, ascorbate and tocopherols.

[0079] In some embodiments, the reaction mixture may include an iron source. The iron source may be included in the reaction mixture at a concentration of about 1 to about 500 pM. Examples of suitable iron sources include iron(II) sulfate, Mohr's salt, and many others.

[0080] In some embodiments, the reaction mixture may include a zinc source. The zinc source may be included in the reaction mixture at a concentration of about 0.01 to about 1 mM. Examples of suitable iron sources include zinc(II) chloride and many others.

[0081] The cell-free conversion of beta-carotene to retinol according to embodiments of the present invention may be performed at a pH of about 5.5 to about 9.0, at a temperature of about 20 to about 40 degrees Celsius, and / or for a time of about 0.5 to about 24 hours. Reaction conditions may be varied according to the exact components and concentrations used.

[0082] Sequences for various homologs of the enzyme beta-carotene 15,15’-dioxygenase, which are suitable to perform Step A described above, are provided below, with the sequence identifier and the NCBI or UniProt accession number followed by the sequence. Sequences with at least 90%, 95%, 98%, 99% or 100% identity to the sequences identified below (SEQ ID NOs: l-19) may also be used to perform Step A.

[0083] In some embodiments, the beta-carotene 15, 15’-dioxygenase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0084] NO: 10, SEQ ID NO : 11, SEQTDNO:12, SEQ TD NO: 13, SEQ ID NO : 14, SEQIDNO:15, SEQ ID NO: 16, SEQIDNON7, SEQ ID NO: 18, or SEQ ID NO: 19.

[0085] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NONO, SEQIDNON1, SEQIDNO:12, SEQ ID NO:13, SEQIDNO:14, SEQIDNO:15, SEQ ID NO: 16, SEQIDNON7, SEQ ID NO: 18, or SEQ ID NO: 19.

[0086] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NON, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NONO, SEQIDNON1, SEQIDNO:12, SEQIDNO:13, SEQIDNO:14, SEQIDNO:15, SEQ ID NO:16, SEQIDNON7, SEQIDNO 18, or SEQ ID NO:19.

[0087] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NONO, SEQIDNO ll, SEQIDNON2, SEQIDNON3, SEQIDNON4, SEQIDNON5, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0088] In some embodiments, the beta-carotene 15,15’-dioxygenase comprises an amino acid sequence identical to SEQ ID NO: 1, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO: 10, SEQ ID NON1, SEQIDNO 12, SEQIDNON3, SEQIDNON4, SEQIDNON5, SEQIDNON6, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0089] SEQ ID NON, A0A6P5KRQ9

[0090] MDMIFGKNTKEQPEPLKAKIRGELPTWLHGTLLRNGPGMHTIGETTYNHWFDGL ALLHSFTIQNGEVHYRSKYLRSDTYNSNIEANRIVVSEFGTMAYPDPCKNIFSKAFSYLS HTIPDFTDNCLINIMKCGEDIYATTETNYIRKINPETLETLEKVDYRNYAAINVATSHPHY DAQGNVLNLGTSIVDKGKTKYLVFKIPPTMPENKKKKNNLKHLEVICSIPSRSLLNPSYY HSFGVTENYIIFLEQPFKMDILKMATAYVRGTSWASCITFHEEDKTYIHIIDRRTKKTLLT KFYADPMVVFHHVNAYEEDGYIVFDVISYKDHSLYQLFYLANLNQHFEHNSKLASIPSL KRF VIPLQ VNKD AEMGENL VKLE STT AT ALKEKDDQ VYCQ SEDLCEGIELPRINYAHNG Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0091] KKYRYIYAAEVQWNPIPTKITKFDILTKSSLKWGEEHCWPAEPVFVPAPNAQQEDDGIIL SVIVSSDPKKLPFLLILDAKQFTELARASIDTEIHLDLHGLFIPSVGSPEEPEDNVLKS

[0092] SEQ ID NO:2, U3JWF5

[0093] MDALYGRNKEEHPDPIKAEVQGQLPTWLQGILLRNGPGMHTIGDSKYNHWFDG MALLHSFTFKNGEVYYRSKYLRSDTYNCNIEANRIVVSEFGTMAYPDPCKNIFAKAFSY LSHTIPEFTDNCLINIMKAGDEFYATGETNFIRKINAQTLETLEKVDYNKYVAVNLATSH PHYD S AGNVLNMGT SIVDKGKTK YLLFKIP S S VPEQEKKKSCFKHLE VVC SIP SRSLLHP SYYHSFGITENYIIFIEQPFRLDIVKMATAYIRGVSWASCLAYNKDDKTWFHFIDRKTKK EVSTKFYTDAMVFFHHINAYEEDGHIVFDIIAYTDNSLYDMFYLKNLDKDFEENVKLTSI PTCKRF VVPLQHDKD AVVD SNLVTLP ST AT AVKEKDGSIYC QPEILCEGIELPRINYD YN GKKYKYVF ATEVQW SPVPTKI AKFNTQTKEMLHWGQDNC WP SEP VF VP SPD AKEEDD GVILTCVVKTDPKDPPFLLVLDAKTFTELGRATVNVEMHMDLHGMFIPQQDVKTGTE

[0094] SEQ ID NO:3, A0A6J2KRQ1

[0095] MDVIFGRNKKEQVEPVRAKVRGKIPTWLQGILLRNGPGMHTVGETSYNHWFDG LALLHSFTIRDGEVYYRSKYLRSDTYNANIEANRIVVSEFGTMAYPDPCKNIF SKAF S YL SHTIPDFTDNCLINIIKCGRDFYATTETNYIRKIDPQTLETLEKVDYRKYVAINLATAHPH YDSAGNVLNMGTSIVDKGKTKYVIFKIPAAVPDRTKEKSPLKHTEVLCSIHSRSLLSPSY YHSFGITENYIVFLEQPFKLDILKLATAYIRGVNWASCLTFHKEEKTHIHVVDRRTGKPV STKFYTDPMVVFHHVNAYEEDGCLLFDAIAYEDSSLYQCFYLAHLKNNSEENSVVTCVS ALKRFAVPLCVDKNAEVGSDLVKLASTTARALKEKDDQVYCQPELLCEGLELPQINYA HNGKRYRYVFAAEIQRSPIPNKMIKYDILTKSSLKWEEEHYWPAEPLFVPTPGAKDEDD GIILSAIISSDPQKPPFLLVLDAKSFTELARAYVDVDMHLDLHGLFIPDADLDLSKRAPPQ DAQDGASEPCVASQT

[0096] SEQ ID NO: 4, A0A2Y9DNM4

[0097] MDIIFGRNKKEQQEPVRTQVTGKIPTWLQGTLLRNGPGMHTVGETKYNHWFDG LALIHSFTIRDSEVYYRSKYLRSDTYTSNIEANRIVVSEFGTMAYPDPCKNIFSKAFSYLS HTIPDFTDNCLINIIKCGEDFYATTETNYIRRINPQTLETLEKVDYRKYVAVNLATAHPHY DAAGNVLNMGTSIVDKGKTKYVIFKIPATVPEDKKKENSCLKHTEVFCSIPSRSLLSPSY YHSFGITENYIVFIEQPFRLDILKMATAYIRGVSWASCLAFHREDKTHIHIIDQRTRKPVVT KYYTDPMVVFHHVNAYEEDGCILFDVIAYKDNSLYQLFYLANLNQDFEENSRLTSIPTL KRFAVPLHVDENAEVGSNLIKLASTTATALKEKDDQVYCQPESLYEGLELPRINYTHNG Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0098] KP YRYVF A AEVQW SPIPTQILK YDILTK S SLKWREEHCWP AEPLF VPTPGAKDEDDGVIL SAIVSTDPQKLPFLLILDAKNFTELARASVDVQMHLDLHGLFIPEMDWEEMSTIVA

[0099] SEQ ID NO:5, A0A663E1X1

[0100] MDTIFGRNKEEHPEPIKAEVQGQLPPWLQGILLRNGPGMHTIGDTKYNHWFDGL

[0101] ALLHSFTFKNGEVYYRSKYLRSDAYNCNIEANRIVVSEFGTMAYPDPCKNIFAKAFSYLS HTIPEFTDNCLINIMKTGDDFYATSETNFIRKINPQTLETLEKADYSKYVAVNVATSHPHY D S AGNILNMGTSI VDKGKTKYVLFKIP AS VP S VPEKEKKKSCLKHLEVVC SIP SRSLLHP S YYHSFGITENYIVFVEQPFKLDIVKMATAYIRGVNWASCLTYHKEDKTWFHFVDKKTK KEVSTKFYTDAMVFFHHVNAYEEDGHIIFDIIAYTDNSLYDLFYLKNLNKNFEENSKLTS IPICKRFVVPLQYDKDAEVGSNLVTLPSTATAVKEKDGSIYCQPEILCEGIELPRINYDYN GKKYKYIFATEVQWSPVPMKIVKFNTQTKEMLHWGEDHSWPSEPIFVPSPDAREEDDG

[0102] VVLTCIVTSDPKKAPFLLVLDAKTFKELGRATVDVEIHLDLHGMFVPEKDLKTETE

[0103] SEQ ID NO:6, XP_004280157.1

[0104] MDIIFGRNNKEQLEPLRARVTGRIPAWLQGTLLRNGPGMHTVGETRYNHWFDGL

[0105] ALLHSFTIRDGEVYYRSKYLRSDTYNANIEANRIVVSEFGTMAYPDPCKNIFSKAFSYLS HTIPDFTDNCLINIMKCGEDFYATTETNYIRKINPQTLETLEKVDYRKHVAVNLATSHPH

[0106] YDDAGNVLNIGTSIVDKGKTKYVIFKIPATVSGGGKKGLSPLKHMEVFCSIASRSLLSPS

[0107] YYHSFGVTKSYIVFLEQPFKMDILKMATAYLRGVSWASCLAFHREDKTYIHIIDQRTRKP VLTKFYTDPMVVFHHVNAYEEDGCLLFDVIAYEDSSLYQLFYLANLNKNFEENSRLTSV PVLKRFSVPLHMDKNAEADSNLIKLSSTTARALKEKEDQVYCQPELLYEGLELPRINYA HNGKR YRYVF AAEVQWSPIPTKIIKYDILTKSSLKWGEVHCWPAEPVFVPTPGAEDEDH GIILSAIVSTDPQKLPFLLVLDAKTFTELARASVDVEMHLDLHGLFIPGADWDAGKQAPS REERDRAAERRVAPRT

[0108] SEQ ID NO: 7, G3GRP4

[0109] MEIIFGRNKKEQLEPVSATVTGKIPTWLQGTLLRNGPGMHTVGDSKYNHWFDGL

[0110] ALLHSFSIKDGEVFYRSKYLQSDTYNANIEANKIVVSEFGTMAYPDPCKNIFSKAFSYLS HTIPDFTDNCLINIMKCGDDFYATTETNYIRKINPQTLETLEKVDYRKYVAVNLATSHPH

[0111] YDEAGNVLNMGTSIVDKGRTKYVIFKIPATAPGTKKKGKSLLKHAEVFCSIPSRSLLSPS YYHSFGVTENYVIFLEQPFKLDILKMATAYVRGVSWASCMSFHREDKTYIHIIDQRTKKP

[0112] VPTKFYTDPMVVFHHVNAYEEDGCVLFDVIAYEDSSLYQLFYLANLNQNFEENSRMTS VPTLKRFAVPLHVDKNAEVGSNLIKLASTRATALKEKDSHVYCQPEVLYEGLELPRINY Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0113] AHNGKPYRYIFAAEVQWSPIPTKILKYDILTKSSLKWGEECCWPAEPLFVPTPGAKDEDD

[0114] GVILSAIVSTHPQKLPFLLVLDAKNFTELARASVDVDMHLDLHGLFIPAADWSAEKQIPD

[0115] KTQEDENSDAPITPQA

[0116] SEQ ID NO:8, A0A8C7SCJ9

[0117] MFQSIIGKNGTESPEPVKAEVTGCVPEWLQGTLLRNGPGLFKVGDTEYNHWFDG MALIHSFTFKDGDVYYRSKFLRSDTFKKNTQANKIVVSEFGTMIYPDPCKNIFSKAFSYL LAAZPDFTDNNLINIIRYGEDYYASSEVNYINQIDPMTLDVIGKMNYRNHIALNMATSHP HYDDEGNTYNMGTALMRFGMPNYVIFKVPVNASDKEHKKPALRKVKQVCNIPFRSTLF PSYFHSFGMTENYIIFVEQPFKLDIVRLATAIFRSVNWASCLRYDKEDITLIHLVDKKTGK AVSTKFYTDALVVFHHINAYEDDGHVVFDMVTYKDSNLYEMFYLANLRKETHEFIESN NVNFSPPVCQRFVLPLTVDKDTPKGTNLVRLKDTTAKTVMQRDGSLYCLPDTIFEGLEL PGMNYKFNGKKYRYFYGSRVEWTPHPNKIGKGDIVTRKYIEWTEEDCYPSEPVFVATP GAVEEDDGVVLTTVINSNPGESGFILVLDAKSFKEVARAYVNAELHMDMHGYFIPKEN

[0118] SEQ ID NO:9, XP_036765240.2

[0119] MDIIFGRNKKEQPEPVRAKVTGKIPAWLQGTLLRNGPGMHTVGETRYNHWFDG LALLHSFTIRDGEVYYRSKYLRSDTYKANIEANRIVVSEFGTMAYPDPCKNIF SKAF S YL SHTLPDFTDNCLINIMKCGEDFFATTETNYIRRINPQTLETLEKVDYHKYVAVNLATSHP HYD AAGNVLNMGT SIMDKRKTRYVMFKIP AT VPEGRSPLKHAEVFC S VASRSLL SP S YY HSFGVTENCIIFLEQPFKLDILKMLTAYFRGVSWASCMTFHKEDKTYIHIVDQRTRKPLPI KFYTDPMVVFHHVNAYEEDGCLLFDVIAYEDSSLYQLFYLANLNQDFEENSRLTSIPTL KRFAVPLHVDKNADVGSNLIKVASTTARALKEKDDQVYCQPEVLYEGLELPRINYAHN GRPYRYVFAAEVQWSPIPTKILKYDVLTKASLKWGEEHCWPAEPLFVPMPGAKDEDDG IILSAIVSTDPQKPPFLLILDAKTFTELARASVDVEMHLDLHGLFIPDTDWDARKQVPSRE ERDQASNGPGAPQT

[0120] SEQ ID NO: 10, A0A663M0Y0

[0121] MDTIFGRNKEEHPEPIKAEVQGQLPPWLQGILLRNGPGMHTIGDTKYNHWFDGL ALLHSFTFKNGEVYYRSKYLRSDAYNCNIEANRIVVSEFGTMAYPDPCKNIFAKAFSYLS HTIPEFTDNCLINIMKTGDDFYATSETNFIRKINPQTLETLEKADYSKYVAVNVATSHPHY D S AGNILNMGTSI VDKGKTKYVLFKIP AS VP S VPEKEKKKSCLKHLEVVC SIP SRSLLHP S YYHSFGITENYIVFVEQPFKLDIVKMATAYIRGVNWASCLTYHKEDKTWFHFVDKKTK KEVSTKFYTDAMVFFHHVNAYEEDGHIIFDIIAYTDNSLYDLFYLKNLNKNFEENSKLTS Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0122] IPICKRFVVPLQYDKDAEVGSNLVTLPSTATAVKEKDGSIYCQPETLCEGIELPRINYDYN GKKYKYIFATEVQWSPVPMKIVKFNTQTKEMLHWGEDHSWPSEPIFVPSPDAREEDDG VVLTCIVTSDPKKAPFLLVLDAKTFKELGRATVDVEIHLDLHGMFVPEKDLKTETE

[0123] SEQ ID NO: 11, C7C6F6

[0124] MEIIFGRNKKEQLEPVRARVTGKIPAWLQGTLLRNGPGMHTVGETRYNHWFDG LALLHSFTIRDGEVYYRSKYLRSDTYTANIEANRIVVSEFGTMAYPDPCKNIF SKAF S YLS HTIPDFTDNCLINIMRCGEDFYATTETNYIRKINPQTLETLEKVDYRKYVAVNLATSHPH YDAAGNVLNVGTSIVDKGKTKYVIFKIPATVPGGRKEGRSPLKDAEVFCSIAARSLLSPS YYHSFGVTENYVVFLEQPFKLDILKMATAYIRGVSWASCLAFHGEDKTHIHIIDRRTRKP VLAKYHTDPMVVFHHVNAYEEDGCLLFDVIAYEDGSLYQLFYLANLNEDFKENSRLTS MPTLKRFVLPLHVDKNAEVGSNLINLSSTTARALKEKDGQVYCQPELLYEGLELPRINY AHNGKPYRYVFAAGVQWSPIPTQIIKYDILTKSSLKWGEEHCWPAEPLFVPTPGAKDED DGIILSAIVSTDPQKSPFLLVLDARTFTELARASIDVEMHLDIHGLFIPDAGWDLGKQAPS REAPARAAAGRAAPQT

[0125] SEQ ID NO: 12, A0A8V5GS96

[0126] MDYGISFLFIGQLPTWLQGILLRNGPGMHTIGDTKYNHWFDGMALLHSFTFKNG EVYYRSKYLRSDTYNSNIEANRIVVSEFGTMAYPDPCKNIFAKAFSYLSHTIPEFTDNCLI NIMKTGDDFYATGETNFIRKINPQTLETLEKVDYTKYTAVNLATSHPHYDSAGNILNMG TSIVDKGKTKYMIFKIPSSVPEREKKKSRFKHLEVLYSIHSRSLLHPSYYHSFGITENYVVF VEQPFKLDMVKLATAYMRGVSWASCLVFNKEEKTWFHFVDRRTKKEVSTKFYTDALV LFHHVNAYEEDGHIVFDVIAYTDNSLYDMFYLKNLTTDFEENTKLTSMPSCKRFVVPLQ YDKDAAVGSNLVKLPSTATAVKQKDGSIYCQPETLCEGIELPRINYDYNGKKYKYVYA TEVKWSPVPTKIAKFNVQTKEMLHWEEDHCWPSEPVFVPNPDAKEEDDGIVLTCIVVSD PKKAPFLLVLDAKTFKELGRAIIDVELHLDLHGMFIPEEDSKTETQ

[0127] SEQ ID NO:13, HOXEX9

[0128] MDIIFGRNKKEQVEPVKAKVTGTIPAWLQGMLLRNGPGMHTVGETRYNHWFDG LALLHSFTIRDGDVYYRSRYLRSDTYNANVEANRIVVSEFGTMAYPDPCKNIFSKAFSYL SHTIPDFTDNCLINIMKCGEDFYATTETNYIRKINPQTLETLEKVDYRKYVAINLATAHPH YDEAGNILNMGTSVVDKGKTKYVMFKIPATVPEDQNKGKSPWKHTEVFCSIPARSFLSP SYYHSFGVTENYIIFLEQPFKLDILKMATAYIRGVSWASCLTFHKEEKTYIHIIDQRTRKPL RTKFYTDPLVVFHHVNAFEEDGCILFDVVAYEDSSLYQLFYLANLNQDFEENSRLTSAP Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0129] TLRRFALPLHVDESAEVGSNEIKVASTTATALKDRDDQVYCQPELLYEGLELPRINYAH NGKQYRYIFAAEVQWSPIPTKIIKYDTLTKSSLKWGEENCWPAEPLFVPTPGATDEDDG VILSAIVSTDPQKLPFLLILDAKNFTELARASVDVEMHLDLHGIFIPDADWGSRKQPPSEE RRDRAPNCDDVAPQT

[0130] SEQ ID NO: 14, A0A8B9NLT2

[0131] MDTIFGRNKEEHPQPIKAEVQGQLPTWLKGILLRNGPGMHTIGDTKYNHWFDGL

[0132] ALLHSFTFKNGEVYYRSKYLRSDTYNCNVEANRIVVSEFGTMAYPDPCKNIFAKAFSYL SHTIPEFTDNCLINIMKTGDDFYATSETNFIRKINPQTLETLEKVDYSKYVAVNVATSHPH

[0133] YDSAGNILNMGTSIVDKGKTKYILFKIPPSVPEKEKKKSHFKHLEVVCSIPSRSLLHPSYY HSFGITENYIVFIEQPFKLDIVKMATAYIRGVNWASCLAYHKEDKTWFHFVDKKTKKEV STKFYTDAMVLFHHINAYEEDGHIIFDIIAYTDNSLYDMFYLKNLTKDFEEKSKLTSIPTC

[0134] KRFVVPLQHDKDAEIGSNLVTLPSTATAVKEKDGSIYCHPEILCEGIELPRINYDYNGKK YNYVFATEVQWSPVPTKIVKFNIQAKEMLYWGEDHSWPSEPVFVPSPDAREEDDGIVLT CVVKSDPKKAPFLLVLDAKTFKELGRAIVDVEMPLDLHGMFIPEKDLKTETE

[0135] SEQ ID NO: 15, V9KPL5

[0136] MQSFFGNNRRESPKPVKAQIRGEIPKWLQGTLIRNGPGMHKIGDTVYNHWFDG

[0137] MALLHSFTFKDGEIFYRSKYLRSDTYRSNMEANRIVVSEFGTMAYPDPCKNIFAKAFSYL SHTIPDFTDNCLINIIRCGEDLYASTETNYIRKIDLETLETNEKVDYRKYVALNLATSHPH YDTNGNT YNMGT SIGDKGKTKYQIIKIPQ SKPGDKEHACFTNCE VIC SIP SRSLLRPS YFH SFGMTENYIVFIEQPFKLDILKLATAYFRGVNWASCLGWYPNDKTYIHLIDRETKKILST KYYTDALVVYHHVNAYEEDGHVIFDIVAYNDNSLYDMFYLKILKLDDAAFEKQGKTFS SPSCRRFVIPLQHDKNVELGTNIVTLENTTASALKEKDGYVYCKPEILFEGIELPRINYDY NGKKYRYIYASKVQWRPVPTKIIKCDILTKTCYEWREDHCWPAEPVFVRAPEAKEEDD

[0138] GVLLSSIVSSDPKTSSFLLVLDAKTFKELGRASVTADIHLDLHGLFIPEK

[0139] SEQ ID NO: 16, M3W391

[0140] MDVIFGRNKKEQLEPVRAQVTGRIPSWLQGTLLRNGPGMHTVGETRYNHWFDG

[0141] LALLHSFTIRDGEVYYRSKYLRSDTYNANTKANRIVVSEFGTMAHPDPCKNIFSKAFSYL SHTIPDFTDNCLINIMKCGEDFYATTETNYIRKINPQTLETLEKVDYRNYVAVNLATSHP HYD AAGNVLNMGT SIMDKGKTKYVIFKIP AT VPED AKGKNPLKHTE VFGSIT SRSLL SP S

[0142] YYHSFGVTENYIVFLEQPFKLDILKMSTAYIRGVNWASCLAFHREDQTYIHIVDQRTGKP LPTKFYTDPMVVFHHVNAYEEDGCLVFDVIAYEDGSLYQLFYLANLNQDFEENSRLTSI Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0143] PTLRRFAVPLDVDKNAEVGSNVVKLASTTARALKEKDDRVYCQPELLYEGLELPRINYA HNGKPYRYVFAAEVHWSPIPTKILKYDILTKSSLKWGQEHCWPAEPLFVPMPGAKDED DGIILSAIVSTDPQKLPFLLVLDAKSFTELARASIDAEVHLDLHGLFIPDADGDTREQAPS QEQPDRVPDCHVAPWT

[0144] SEQ ID NO: 17, A0A7K5WAF4

[0145] MDTLYGRNKEEHPEPIKAEVQGQLPGWLQGILLRNGPGMHTIGDTKYNHWFDG LALLHSFTFKNGEVYYRSKYLRSDTYNCNVEANRIVVSEFGTMAYPDPCKNIFAKAFSY LSHTIPEFTDNCLINIMKVGEDFYATGETNFIRKINPQTLETLEKVDYNKYVAVNVATSH PHYDSAGNVLNMGTSIADKGKTKYLLFKIPASVPEHEKKKSCFKHMEVVCSVPSRSLLH PSYYHSFGITENYIIFIEQPFRLDIVKMATAYIRGVSWASCLAFSKDDKTWFHFIDRKTKK EVATKFYTDAMVFFHHVNAYEEDGHIIFDVIAYTDNSLYDMFYLKNLGKDFKERVRLT

[0146] CIPACKRFVVPLQYDKDAVVDSNLVTLPSTATAVKEKDGNIYCQPEILCEGIELPRINYD YNGKKYKYAYVTEVQWSPIPTKIAKFNTQTKEMLHWGEDDCWPSEPVFVPRPDAKEED DGVVLTCVVKTDPMDPPFLLVLDAKTFTELGRAVVNVEMHLDLHGIFIPQQAMKTETE

[0147] SEQ ID NO: 18, A0A218V6L8

[0148] MDTLYGRNKEEHPEPIKAEVQGQLPTWLQGMLLRNGPGMHTIGDSKYNHWFDG LALLHSFTFKNGEVYYRSKFLRSDTYNCNIEANRIVVSEFGTMAYPDPCKNIFAKAFSYL SHTIPEFTDNCLINIMKAGDDFYATGETNFIRKINPQTLETLEKVDYSKYVSVNLATSHPH YDSAGNVLNMGTSIVDKGKTKYLLFKIPSSVPEQGKKKSCFKQLEVVCSIPSHSLLHPSY YHSFGITENYIVFIEQPFKLDILKMATAYMRGVTWASCLAFNKDDKTWFHFIDRRTKKE VPTKFYTDALVFFHHVNAYEEDGHIVFDIIAYTDNSLYDMFYLKNLNRDFEKNAKLTSIP

[0149] TCRRFVVPLQYDKDALVDSNLVTLPSTATAVKEKDGSIYCQPEILCEGIELPRINYDYNG KKYKYIFATQVQWSPVPTEIAKFNTQTKEMVQWREDDCWPSEPVFVPNPDGKEEDDGV VLTCVVKSDPKDPPFLLILDAKTFTELGRAIVNVDMHMDLHGIFIPQQDMKTETE

[0150] SEQ ID NO: 19, XP_057568740.1

[0151] MDIIFGRNKKEQLEPVKATVTGKIPAWLQGTLLRNGPGMHTVGETRYNHWFDG LALLHSFTIRDGEVYYRSKYLRSDTYNANIEANRIVVSEFGTMAYPDPCKNIF SKAF S YL SHTIPDFTDNCLINIMRCGEDFYATTETNYIRKINPQSLETLEKVDYRKYVAVNLATSHPH YDAAGNVLNIGTSIVDKGKTKYVIFKIPATVPEGGKKGQRPLKHTEVFCSIASRSLLSPSY YHSFGVTENYIVFLEQPFKMDILKMATAYIRGVSWASCLAFHREDKTYIHIIDRRTRKPV LTKFYADPMVVFHHVNAYEEDGCLLFDVIAYEDSSLYQLFYLANLNKDFEENSRLTSVP Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0152] ALKRF A VPLH VDKN AEVGSNLIKL S STT AR ALKEKDDQ VYCQPELL YEGLELPRIN Y AH NGKWYRYVFAAEVQWSPIPTKIIKYDILTRSFLKWEEAHCWPAEPLFVPTPGAEAEDDGI ILSAVVSTDPQKLPFLLVLDAKTFTELARASVDVEMHLDLHGLFIPGVDWDAGKQAPSQ EERDRAVDRHVAPRT

[0153] Sequences for various homologs of the enzyme alcohol dehydrogenase, which are suitable to perform Step B described above, are provided below, with the sequence identifier and the NCBI or UniProt accession number followed by the sequence. Sequences with at least 90%, 95%, 98%, 99% or 100% identity to the sequences identified below (SEQ ID NOs:20-25) may also be used to perform Step B.

[0154] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0155] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25

[0156] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0157] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0158] In some embodiments, the alcohol dehydrogenase comprises an amino acid sequence identical to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0159] SEQ ID NO:20, WP_000148959.1

[0160] MTHKATEILTGKVMQKSVLITGCSSGIGLESALELKRQGFHVLAGCRKPDDVER MNSMGFTGVLIDLDSPESVDRAADEVIALTDNCLYGIFNNAGFGMYGPLSTISRAQMEQ QFSANFFGAHQLTMRLLPAMLPHGEGRIVMTSSVMGLISTPGRGAYAASKYALEAWSD Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0161] ALRMELRHSGIKVSLIEPGPIRTRFTDNVNQTQSDKPVENPGIAARFTLGPEAVVDKVRH

[0162] AFISEKPKMRYPVTLVTWAVMVLKRLLPGRVMDKILQG

[0163] SEQ ID NO:21, KAG1176698.1

[0164] MSKRFEFLFKTSVTPYIHGRFIKSHLGRERVLINPVNEERLCSYHETSIDQLEAALV SSRSVTEEWTSNHRYRRDCLLKLADRIAQHDHDLAVVESRQTGKPIQDALGEMSDVIDC FRHFAGYSDKLFGTTHDILTFTVREPLGTVGLITSFNYPLLLTGWKLAPALAAGNRALVR PAPQTPLSTLALADLATDVLPPGVLSVLPGGDVHVSQAITRRTDKTSFTGSTQVGQEIMR QVSETMTPITLECGGKNAVIICEDADLSRAASEVAAGAFSNAGQNCCAISRVLVHASVH DQFVEELRGHVQKWKPAVCDDYQHDELLYGPLIDKRQYTRVRKYIQEYPQDPIMVGEL NEEKGYFVPPTVYANVQDDAALAQEEIFGPVLSILKPFETLDEAIERVNRSPYGLAFGIFS

[0165] RDYEKTNRAARKVKAGMVWINTYNLTLPSLPFGGTKLSGFGKDLGKTSLDEFTFEKTV MMHAK

[0166] SEQ ID NO:22, A0A2I7G3B0.1

[0167] MSSGANGNSKSLAYDIKFTKLFINGEFVDSISGSTFETIDPATEEVLATVAEGREE DVDLAVKAAREAFDNGPWPRLSGEARRKILLKFADLIEENADEIATLEVIDTGKPFQIAR YVENSWTSETFRYFAGAADKIRGATLKMSSDFQAYTLREPIGVVGHIIPWNAPAYLFAM KVAPALAAGCTVVIKPAENTPLVGLFMAYLSKLAGVPDGVINVVNGFGSTAGAAVSSH MDIDAVTFTGSTKVGRTIMQAAAASNLKPVSLELGGKSPFIVFDDADIEKAAEIAVLGVL SNKGELCVAGSRVFVHEGIYDAFVKKLEATVKNWATGDRFDAATRHGPQNNKQQYEK VLSYIELGKKEGATLVTGGKPFGNKGYYIEPTLFTNVTDEMTIAKEEIFGPVIMVLKFKTI

[0168] EEVIRRANATTYGLAAGIMTKNIDIANTVTRSIRAGSVWVNCYLALDRDTPFGGYKMSG FGREQGLEALEHYLQVKTVTTPIYNSPWL

[0169] SEQ ID NO:23, WP_013034365.1

[0170] MSPSASLPLTHKEQVEVRQTRLLIDGEFRDSLSGKTFATIDPVTEEVIAQVAEGDA EDIDLAVKAARKAFDTGPWQQMDARERGRRMLKWADLIETHMEELAKLEVLDNGKPI NEALGYDIPSAAATIRYFAGWADKIHGKTIPVSGPFFTYTRREPVGVCGLIIPWNFPLAM AAWKLGPALAAGCTTILKPAEQTPLTALRAGELALEAGIPPGVLNIVPGFGPTAGAALV QHPLVEKIAFTGEYKTAQIIKQATVNSMKRLSFELGGKSPNIIFNDANLEDAITGSFGAIFL NQGQNCCAGSRAFVQDNIYNEFVERFADKAEKRRLGDPFDSNTEHGAQIDKAQFDKIM HYIALGKEQGAACVTGGNQAFDRGYFIQPTVFSEVNENMAIATDEIFGPVVSVLRFKDIN Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0171] EVIAKANNTMFGLAAAVWTQDIDKANAVAEGVKAGTVWVNCYNIVDPAAPFGGFKM

[0172] SGVGRELSEQALDAYTETKTVTVLRK

[0173] SEQ ID NO:24, NP 057110.3

[0174] MFPLLLLLLPFLLYMAAPQIRKMLSSGVCTSTVQLPGKVVVVTGANTGIGKETA KELAQRGARVYLACRDVEKGELVAKEIQTTTGNQQVLVRKLDLSDTKSIRAFAKGFLA EEKHLHVLINNAGVMMCPYSKTADGFEMHIGVNHLGHFLLTHLLLEKLKESAPSRIVNV SSLAHHLGRIHFHNLQGEKFYNAGLAYCHSKLANILFTQELARRLKGSGVTTYSVHPGT VQSELVRHSSFMRWMWWLFSFFIKTPQQGAQTSLHCALTEGLEILSGNHFSDCHVAWV SAQARNETIARRLWDVSCDLLGLPID

[0175] SEQ ID NO:25

[0176] MTHKATEILTGKVMQKSVLITGCSSGIGLESALELKRQGFHVLAGCRKPDDVER MNSMGFTGVLIDLDSPESVDRAADEVIALTDNCLYGIFNNAGFGMYGPLSTISRAQMEQ QF S ANFFG AHQLTM RLLP AM LPHGEGRI VMTS SVMGLISTPGRGAYAASKYALEAWSD ALRMELRHSGIKVSLIEPGPIRTRFTDNVNQTQSDKPVENPAIAARFTLGPEAVVDKVRH AFISEKPKMRYPVTLVTWAVMVLKRLLPGRVMDKILQG

[0177] FIG. 2 shows chromatograms of a retinal analytical standard and the results of a first cell- free reaction (beta-carotene to retinal) demonstrating the activity of beta-carotene 15,15’- dioxygenase alone. Here the first cell-free reaction (enzymatic Step A) was isolated from the second cell-free reaction (the reaction mixture included beta-carotene 15, 15 ’ -di oxygenase only, no alcohol dehydrogenase). Beta-carotene is not detected by this analytical method.

[0178] FIG. 3 shows chromatograms of a retinol analytical standard and the results of a second cell-free reaction (retinal to retinol) demonstrating the activity of alcohol dehydrogenase alone. Here the second cell-free reaction (enzymatic Step B) was isolated (the reaction mixture included alcohol dehydrogenase only, no beta-carotene 15,15’-dioxygenase, and used retinal as a starting material).

[0179] FIG. 4 shows chromatograms of a retinal analytical standard, a retinol analytical standard, and the results of production of retinol from beta-carotene in a single reaction vessel with both enzymes (beta-carotene 15, 15 ’ -di oxygenase and alcohol dehydrogenase) added at the same time, according to embodiments of the present invention. Beta-carotene is not detected by this analytical method. Attorney Docket No. DEBU-035 / 01WO 37396 / 183

[0180] Systems and methods according to embodiments of the present invention advantageously provide cell-free bioproduction of retinal and retinol, using a specific combination of two enzymes (beta-carotene 15,15’-dioxygenase and alcohol dehydrogenase) in a single reaction pot. The cell-free systems and methods according to embodiments of the present invention can outperform microbial production, and can provide, for example, higher titers of product, higher conversion of carbon inputs to product, higher selectivity for desired product formation, no toxicity issues, and high purity of the isolated product retinoid. The cell-free systems and methods according to embodiments of the present invention enable users to use / engineer enzymes tolerant of high solvent and / or detergent levels that cannot be tolerated by cells, thereby allowing higher retinoid solubility. This ability to use substrates at high concentrations in turn enables reaction rates and productivity not possible / not obtained in cell-based systems.

[0181] EXAMPLES Example 1 Cell-free transformations of beta-carotene to retinal were conducted using lysates prepared from cells expressing various beta-carotene 15,15’-dioxygenases. BL21(DE3) cells were transformed with pT5 plasmid (IPTG-inducible, KAN resistance) containing the dioxygenase gene. In deep-well 96-well plates, 500 pL LB media with 50 pg / mL kanamycin were inoculated from single colonies or with 5 pL of a glycerol stock (15% glycerol). Cultures were grown for 16-20 hours at 30 °C, 750 rpm (70% humidity). These seed cultures (5 pL) were then used to inoculate expression cultures (500 pL TB, 50 pg / mL kanamycin in a second deepwell 96-well plate). Expression cultures were incubated at 30 °C, 750 rpm for 4 hours, then induced with 0.1-0.5 mM IPTG and incubated for an additional 20 hours at 30 °C, 750 rpm (70% humidity). Culture plates were then centrifuged at 4000 xg (4 °C) for 15 minutes. The media supernatant was decanted, and the cell pellets were resuspended with 100-200 pL B-PER complete reagent. The plates were incubated at 30 °C, 400 rpm (70% humidity) for 45 minutes and then centrifuged at 4000 xg (4 °C) for 20 minutes; the supernatant was then employed in cell-free reactions.

[0182] To perform cell-free reactions, beta-carotene (12 mg) was combined with THF (800 pL) and Tween-80 (200 pL). In 1.5 mL Eppendorf tubes, lysate containing the dioxygenase (90 pL) was combined with sodium ascorbate (5 pL of a 50 mM stock), Fe2SC>4 (2 pL of a 2.5 mM Attorney Docket No. DEBU-035 / 01WO 37396 / 183 stock), and 3 pL of the beta-carotene / Tween-80 solution. Reactions were then incubated at 32.5 °C, 750 rpm, shielded from light. After 20h, reactions were quenched with 100% EtOH (3x dilution), filtered through 0.45 pm filters, and analyzed by HPLC.

[0183] FIG. 5 shows the results of beta-carotene 15, 15 ’ -di oxygenase screening in lysates for retinal production. The Y-axis shows the retinal produced (pM), and the X-axis shows the sequence ID of the dioxygenase.

[0184] FIG. 6 shows the results of native E. coli retinol dehydrogenase (Ybbo) transformations with and without antioxidant / stabilizer. The Y-axis shows the retinoid concentration (pM), and the X-axis shows the antioxidant / stabilizer tested: no antioxidant, 1 mM BHT (butylated hydroxytoluene), 0.15% PTS (polyoxyethanyl-a-tocopheryl sebacate).

[0185] FIG. 7 shows the results of a “one-pot” reaction producing retinol from beta-carotene in a single reaction vessel in vitro, using purified hippo beta-carotene 15, 15 ’-dioxygenase and native E. coli retinol dehydrogenase (Ybbo) lysate. The Y-axis shows the retinoid concentration (pM), and the X-axis shows the antioxidant / stabilizer used: none, 1 mM BHT, 0.15% PTS. In certain preferred embodiments, one or more detergents were added to organic solvents such as tetrahydrofuran / methyltetrohydrofuran (THF / mTHF) to solubilize the beta-carotene prior to reaction loading. Antioxidants / stabilizers are preferred to help minimize retinol degradation.

[0186] Example 2

[0187] Cell-free transformations of beta-carotene to retinol were performed using lysates prepared from cells expressing beta-carotene 15, 15 ’ -di oxygenases or alcohol dehydrogenases. BL21(DE3) cells were transformed with pT5 plasmid (IPTG-inducible, KAN resistance) containing either the dioxygenase or the alcohol dehydrogenase genes. Cells containing the dioxygenase plasmid were additionally transformed with the GroES / EL plasmid pGro7 from Takeda. In 250 mL flasks, 50 mL LB media with 50 pg / mL kanamycin was inoculated from single colonies or with 5 pL of a glycerol stock (15% glycerol). These cultures were grown for 16-20 hours at 30 °C, 750 rpm (70% humidity). These seed cultures (10 mL) were then used to inoculate expression cultures (1 L TB, 50 ug / mL kanamycin in 3 L shake flasks). Dioxygenase expression cultures additionally contained Ig / L arabinose. Expression cultures were incubated at 30 °C, 750 rpm for 4 hours, then induced with 0.1-0.5 mM IPTG and incubated for an additional 20 hours at 30 °C, 750 rpm (70% humidity). Cells were then centrifuged at 5000 xg (4 °C) for 30 Attorney Docket No. DEBU-035 / 01WO 37396 / 183 minutes. The media supernatant was decanted, and the cell pellets were resuspended with 60 mL lysis buffer (50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 10% glycerol). The cells were mechanically homogenized, then centrifuged at 30,000 xg (4 °C) for 60 minutes; the supernatant was then employed in cell-free reactions.

[0188] To perform cell-free transformations of beta-carotene to retinol, beta-carotene (435 mg) was combined with CPME (38 mL) and heated with stirring to 55 °C for 15 min, at which point the beta-carotene is fully dissolved. Tween-60 (20 mL) was then added, followed by 50 mM sodium phosphate buffer (pH 7.5, 87 mL). This mixture was concentrated to a volume of 93 mL and then added to a 0.75 L Infers HT Multifors Bioreactor equipped with overhead stirring. To this solution, heated to 30 °C, were added KRED-P1-A12 (commercially available from Codexis, 7.5 mL of a 10 g / L stock), NADP+(1.5 mL of a 50 mM stock), dioxygenase lysate (37.5 mL), alcohol hydrogenase lysate (7.5 mL), isopropanol (3 mL), and 1 mL of antifoam (AF- 204). The reaction was then incubated under air at 700 rpm, 30 °C, shielded from light. At each timepoint, two reaction samples were removed and quenched with either 100% EtOH (3x dilution) or CPME (3x dilution), filtered through a 0.45 pm filter, and analyzed by HPLC.

[0189] FIG. 8 shows the time course of a cell-free reaction in which beta-carotene is converted to retinol, producing 3.68 mM retinol (1.06 g / L retinol) after 23 hours.

[0190] Example 3

[0191] Cell-free transformations testing the role of the ketoreductase were carried out using lysates prepared from cells expressing the dioxygenase (SEQ ID NO:3) and the alcohol dehydrogenase (SEQ ID NO:25). BL21(DE3) cells were transformed with pT5 plasmid (1PTG- inducible, KAN resistance) containing the dioxygenase, as well as the GroES / EL plasmid pGro7 from Takeda. The dioxygenase was produced according to the general expression protocol of Example 1) with the addition of 1 g / L arabinose during the expression step. Alcohol dehydrogenase (SEQ ID NO:25) was expressed from a pT5 plasmid (IPTG-inducible, KAN resistance) in BL21(DE3) cells according to the general expression protocol of Example 1. To perform cell-free transformations of beta-carotene to retinol, beta-carotene (30 mg) was combined with CPME (2.62 mL) and heated with stirring to 55 °C for 15 min, at which point the beta-carotene is fully dissolved. Tween-40 (1.38 mL) was then added, followed by 50 mM sodium phosphate buffer (pH 7.5, 6.0 mL). This mixture was concentrated to a volume of 6.4 Attorney Docket No. DEBU-035 / 01WO 37396 / 183 mL. In a 1.5 mL Eppendorf tube, 62 pL of the beta-carotene / Tween-40 solution is added, followed by KRED-P1-A12 (commercially available from Codexis, 0 pL or 5 pL of a 10 g / L stock), NADP+(1 pL of a 50 mM stock), dioxygenase lysate (37.5 pL), alcohol dehydrogenase lysate (2.5-10 pL), isopropanol (2 pL). The reactions were then incubated at 750 rpm, 30 °C, shielded from light for 24 hours. The reactions were then quenched with 100% EtOH (3x dilution) filtered through a 0.45 pm filter, and analyzed by HPLC.

[0192] FIG. 9 shows transformations of beta-carotene to retinol carried out in the presence and absence of ketoreductase (KRED) P1-A12. The data demonstrates that a two-enzyme system (dioxygenase and alcohol dehydrogenase) achieves in excess of 1 g / L retinol production in the absence of ketoreductase P1-A12.

[0193] While there have been shown and described fundamental novel features of the invention as applied to the preferred and illustrative embodiments thereof, it will be understood that omissions and substitutions and changes in the form and details of the disclosed invention may be made by those skilled in the art without departing from the spirit of the invention. Moreover, as is readily apparent, numerous modifications and changes may readily occur to those skilled in the art. For example, various features and structures of the different embodiments discussed herein may be combined and interchanged. Hence, it is not desired to limit the invention to the exact construction and operation shown and described and, accordingly, all suitable modification equivalents may be resorted to falling within the scope of the invention as claimed. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.

Claims

Attorney Docket No. DEBU-035 / 01WO 37396 / 183CLAIMS1. A composition for preparing retinol or an isomer thereof from beta-carotene, comprising: beta-carotene; a beta-carotene 15,15’-dioxygenase; and an alcohol dehydrogenase, wherein the composition is cell-free.

2. The composition of claim 1, wherein the beta-carotene concentration is about 0.1 to about 50 mM.

3. The composition of claim 1, wherein the beta-carotene 15,15’-dioxygenase concentration is about 1 to about 500 pM, or 1 to 90 volume% lysate.

4. The composition of claim 1, wherein the alcohol dehydrogenase concentration is about 1 to about 500 pM, or 1 to 30 volume% lysate.

5. The composition of claim 1, wherein the composition includes a buffer.

6. The composition of claim 5, wherein the buffer concentration is about 10 to about 500 mM.

7. The composition of claim 5, wherein the buffer comprises Tris, HEPES, or sodium phosphate.

8. The composition of claim 1, wherein the composition has a pH of about 5.5 to about 9.0.

9. The composition of claim 1, wherein the composition includes a solvent.

10. The composition of claim 9, wherein the solvent concentration is about 1 to about 30 volume%.Attorney Docket No. DEBU-035 / 01WO 37396 / 18311 . The composition of claim 9, wherein the solvent comprises tetrahydrofuran, 2- methyltetrahydrofuran, cyclopentyl methyl ether, or hexanes.

12. The composition of claim 1, wherein the composition includes a detergent.

13. The composition of claim 12, wherein the detergent concentration is about 0.01 to about 15 volume%.

14. The composition of claim 12, wherein the detergent comprises Tween-80, Tween-60, Tween- 40, Tween-20, Triton-X, or PTS (polyoxyethanyl-a-tocopheryl sebacate).

15. The composition of claim 1, wherein the composition includes one or more additives.

16. The composition of claim 15, wherein the additive concentration is about 0.01 to about 1 mM.

17. The composition of claim 15, wherein the additives comprise ascorbate or tocopherols.

18. The composition of claim 1, wherein the composition includes an iron source.

19. The composition of claim 18, wherein the iron source concentration is about 1 to about 500 pM.

20. The composition of claim 18, wherein the iron source comprises iron(II) sulfate or Mohr's salt.

21. The composition of claim 1, wherein the composition includes a zinc source.

22. The composition of claim 21, wherein the zinc source concentration is about 0.01 to about 1 mM.Attorney Docket No. DEBU-035 / 01WO 37396 / 18323. The composition of claim 21, wherein the zinc source comprises zinc(TI) chloride.

24. A method of preparing retinol or an isomer thereof from beta-carotene, comprising: providing a reaction mixture comprising beta-carotene, a beta-carotene 15,15’- dioxygenase, and an alcohol dehydrogenase, wherein the reaction mixture is cell-free; and incubating the reaction mixture containing the beta-carotene, the beta-carotene 15,15’- dioxygenase, and the alcohol dehydrogenase at a temperature of about 20 to about 40 degrees Celsius for at least about 0.5 to about 24 hours.

25. The method of claim 24, wherein the method is performed in a single reaction vessel.

26. The method of claim 25, wherein the beta-carotene 15, 15 ’ -di oxygenase and the alcohol dehydrogenase are added at the same time.

27. The method of claim 25, wherein the beta-carotene 15, 15 ’ -di oxygenase and the alcohol dehydrogenase are added sequentially.

28. The method of claim 24, wherein the method is performed in two or more reaction vessels operating in series, wherein the beta-carotene 15,15’-dioxygenase and the alcohol dehydrogenase are provided in separate reaction vessels.

29. The composition of claim 1, wherein the beta-carotene 15, 15 ’ -di oxygenase comprises an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID N0:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID N0: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, or SEQ ID NO:19.

30. The composition of claim 1 or claim 29, wherein the alcohol dehydrogenase comprises an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.Attorney Docket No. DEBU-035 / 01WO 37396 / 18331. The composition of claim 1 or claim 29, wherein the alcohol dehydrogenase comprises an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO:25.

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