Fermenative production of retinyl esters and other retinoids

Abstract

A fermentative process for producing a desired retinyl ester of a fatty acid (desired retinyl fatty acid ester) is described herein. The process generally comprises: (i) providing retinol-producing cells that express an enzyme that catalyzes the conversion of retinol to a retinyl ester; and (ii) culturing the retinol-producing cells in a production medium comprising the fatty acid and / or an alkyl ester thereof, under conditions enabling the production of the desired retinyl fatty acid ester. The production medium may further comprise a liquid alkane and / or alkene mixture, resulting in improved production and / or purity of desired retinyl fatty acid ester. Also described herein are microorganisms engineered for the production of retinoids, such as retinyl fatty acid esters.

Description

[0001] FERMENATIVE PRODUCTION OF RETINYE ESTERS AND OTHER RETINOIDS

[0002] The present technology relates to a fermentative process for the production of retinyl esters and other retinoids, as well as microorganisms engineered for same.

[0003] The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Vitamin A belongs to a group of compounds known as retinoids and is largely known as a fat soluble vitamin which plays an essential role in human health, including eyesight, bone growth, immunity and reproductive health. For this reason, retinoids are commercially valuable molecules which are globally sold as supplements, added to food products, or used in cosmetic products. Currently, retinoids are synthesized by complex chemical processes which frequently have significant constraints, including harsh reaction conditions, and excess byproduct generation. These conventional chemical processes necessitate energy-intensive purification steps, and leave a large environmental impact. As such, environmentally friendly and high-specificity alternative manufacturing routes of retinoid production are needed to overcome at least some of these drawbacks.

[0006] SUMMARY

[0007] In a first aspect, described herein is a process for producing a retinyl ester of a fatty acid. The process generally comprises: (i) providing retinol-producing cells that express an enzyme that catalyzes the conversion of retinol to a retinyl ester; and (ii) culturing the retinol-producing cells in a production medium comprising the fatty acid and / or an alkyl ester thereof, under conditions enabling the production of the retinyl ester of the fatty acid. In some embodiments, the production medium further comprises a liquid alkane and / or alkene mixture (e.g., mineral oil, paraffin oil, or other non-toxic liquid hydrocarbon mixture).

[0008] In a further aspect, described herein is a microorganism engineered for the production of retinoids, which overexpresses: (a) a geranylgeranyl diphosphate synthase (EC 2.5.1.29) that catalyzes the synthesis of geranylgeranyl diphosphate; (b) a phytoene synthase (EC 2.5. 1.32) that catalyzes the conversion of geranylgeranyl diphosphate to phytoene; (c) a phytoene desaturase (EC 1.3.99.31) that catalyzes the conversion of phytoene to lycopene; (d) a lycopene cyclase (EC 5.5.1.19) that catalyzes the conversion of lycopene to a beta-carotenoid; (e) a beta-carotene oxidizing enzyme (EC 1.13.11.63) that catalyzes the conversion of beta-carotene to retinal; (f) a retinol dehydrogenase (EC 1.1.1.105) that catalyzes the conversion of retinal to retinol; (g) a retinol acyltransferase that catalyzes the conversion retinol to the retinyl ester of the fatty acid, or (h) any combination of (a) to (g).

[0009] General Definitions

[0010] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0011] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0012] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0013] The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0014] Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0015] SEQUENCE LISTING

[0016] This application contains a Sequence Listing in computer readable form created May 27, 2025. The computer readable form is incorporated herein by reference.

[0017] DETAILED DESCRIPTION

[0018] Described herein are processes and microorganisms for producing retinoids, and more specifically retinyl esters.

[0019] In a first aspect, described herein is a process for producing a desired retinyl ester of a fatty acid (desired retinyl fatty acid ester) or a desired profile thereof. The process comprises: (i) providing retinol- producing cells that express an enzyme that catalyzes the conversion of retinol to a retinyl ester, and (ii) culturing the retinol-producing cells in a production medium comprising the fatty acid and / or an alkyl ester thereof, under conditions enabling the production of the retinyl fatty acid ester. As used herein, the expression “fatty acid and / or an alkyl ester thereof’ refers to the fact that the fatty acid(s) may be free and / or in their corresponding alkyl ester form(s).

[0020] In some embodiments, the production medium may further comprise a liquid alkane and / or alkene mixture (e.g., mineral oil, paraffin oil, or other non-toxic liquid hydrocarbon mixture). In some embodiments, the liquid alkane and / or alkene mixture is substantially free of fatty acids and / or alkyl fatty acid esters, for example, so as not to compete with or otherwise influence the species of retinyl fatty acid ester produced from the process. In some embodiments, inclusion of the liquid alkane and / or alkene mixture in the production medium increases the proportion of the desired retinyl fatty acid ester produced (e.g., with respect to all carotenoids), as compared to culturing the retinol-producing cells in a corresponding production medium lacking the liquid alkane and / or alkene mixture. In some embodiments, the proportion or ratio of the fatty acid and / or an alkyl ester thereof to the liquid alkane and / or alkene mixture in the production medium may be between about 5, 10, 15, 20, 25, 30, 35, or 40% w / w and about 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% w / w. For example, a 20% w / w proportion or ratio of the fatty acid and / or an alkyl ester thereof corresponds to 80% w / w of the liquid alkane and / or alkene mixture. In some embodiments, the fatty acid and / or an alkyl ester thereof may be premixed with the liquid alkane and / or alkene (e.g., at the aforementioned proportions / ratios) prior to addition to the production medium. In some embodiments, the production medium may comprise a medium- or long-chain fatty acid, and / or an alkyl ester thereof. In some embodiments, the production medium may comprise a fatty acid which is: palmitic acid, oleic acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, stearic acid, arachidic acid, or any combination thereof; and / or alkyl ester(s) thereof. In some embodiments, the retinyl fatty acid ester produced from the process may comprise: retinyl palmitate, retinyl oleate, retinyl caproate, retinyl caprylate, retinyl caprate, retinyl laurate, retinyl myristate, retinyl stearate, retinyl arachidate, or any combination thereof. In some embodiments, the production medium may comprise an alkyl fatty acid ester, wherein the alkyl group is: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl. For greater clarity, these alkyl groups also include suitable isomers thereof, such as isopropyl, isobutyl, sec-butyl, tert-butyl, etc.). In some embodiments, the production medium may comprise a defined profile of a mixture of fatty acids and / or an alkyl esters thereof, resulting the production of corresponding profile of a mixture of retinyl fatty acid esters.

[0021] In some embodiments, the retinol-producing cells may be cultured until the formation of an emulsion layer containing biosurfactants is generated by the cells into which the retinyl ester of the target fatty acid may accumulate. In some embodiments, the process further comprises (iii) isolating the retinyl ester of the fatty acid from the emulsion layer. In some embodiments, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% w / w of total carotenoids or retinoids produced from the process are the retinyl fatty acid ester.

[0022] In some embodiments, the retinol-producing cells may express (e.g., endogenously express) or overexpress (e.g., engineered to overexpress): (a) a geranylgeranyl diphosphate synthase (EC 2.5.1.29) that catalyzes the synthesis of geranylgeranyl diphosphate; (b) a phytoene synthase (EC 2.5.1.32) that catalyzes the conversion of geranylgeranyl diphosphate to phytoene; (c) a phytoene desaturase (EC 1.3.99.31) that catalyzes the conversion of phytoene to lycopene; (d) a lycopene cyclase (EC 5.5.1.19) that catalyzes the conversion of lycopene to a beta-carotenoid; (e) a beta-carotene oxidizing enzyme (EC 1.13.11.63) that catalyzes the conversion of beta-carotene to retinal; (f) a retinol dehydrogenase (EC 1.1.1.105) that catalyzes the conversion of retinal to retinol; (g) a retinol acyltransferase that catalyzes the conversion retinol to the retinyl ester of the fatty acid, or (h) any combination of (a) to (g).

[0023] In some embodiments, the geranylgeranyl diphosphate synthase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 14; or a fragment thereof having geranylgeranyl diphosphate synthase activity. In some embodiments, the phytoene desaturase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or a fragment thereof having phytoene synthase activity. In some embodiments, the phytoene synthase and the lycopene cyclase are comprised in a single bifunctional enzyme comprising an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 16; or a fragment thereof having phytoene desaturase and lycopene cyclase bifunctional activity. In some embodiments, the beta-carotene oxidizing enzyme comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; or a fragment thereof having beta-carotene oxidizing activity. In some embodiments, the retinol dehydrogenase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 18; or a fragment thereof having retinol dehydrogenase activity. In some embodiments, the retinol acyltransferase comprises or consists of an acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1), a diacylglycerol O-acyltransferase (DGAT; EC 2.3.1.20) (e.g., DGAT1, DGAT2), an acyl-CoA: retinol acyltransferase (ARAT), or an O-acyltransferase (e.g., encoded by mdy). In some embodiments, the retinol-producing cells described herein may express or overexpress any combination of the above-mentioned enzymes.

[0024] In some embodiments, the geranylgeranyl diphosphate synthase may be encoded by a polynucleotide sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 2. In some embodiments, the phytoene desaturase may be encoded by a polynucleotide sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 6. In some embodiments, the phytoene synthase and the lycopene cyclase may be comprised in a single bifunctional enzyme encoded by a polynucleotide sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 7. In some embodiments, the beta-carotene oxidizing enzyme may be encoded by a polynucleotide sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11. In some embodiments, the retinol dehydrogenase may be encoded by a polynucleotide sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13. In some embodiments, the retinol acyltransferase comprises or consists of an acyl- CoA:diacylglycerol acyltransferase (EC 2.3.1), a diacylglycerol O-acyltransferase (DGAT; EC 2.3.1.20) (e.g., DGAT1, DGAT2), an acyl-CoA:retinol acyltransferase (ARAT), or an O-acyltransferase (e.g., encoded by mdy). In some embodiments, the retinol-producing cells described herein may express or overexpress any combination of the above-mentioned enzymes.

[0025] In some embodiments, the retinol-producing cells may further express (e.g., endogenously express) or overexpress (e.g., engineered to overexpress): (a) an acetyl-CoA C-acetyltransferase (EC 2.3.1.9) that catalyzes the synthesis of acetoacetyl-CoA; (b) a hydroxymethylglutaryl-CoA synthase (EC

[0026] 2.3.3.10) that catalyzes the conversion of acetoacetyl-CoA to hydroxymethylglutaryl-CoA; (c) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) that catalyzes the conversion of hydroxymethylglutaryl-CoA to mevalonate; (d) a mevalonate kinase (EC 2.7.1.36) that catalyzes the conversion of mevalonate to a phosphomevalonate; (e) a phosphomevalonate kinase (EC 2.7.4.2) that catalyzes the conversion of phosphomevalonate to diphosphomevalonate; (f) a diphosphomevalonate decarboxylase (EC 4.1.1.33) that catalyzes the conversion of diphosphomevalonate to isopentenyl diphosphate; (g) a isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2) that catalyzes the conversion of isopentenyl diphosphate to dimethylallyl diphosphate; (h) a famesyl diphosphate synthase (EC 2.5.1.1; EC:2.5. 1. 10) that catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to geranyl diphosphate, and / or catalyzes the conversion of geranyl diphosphate and isopentenyl diphosphate to famesyl diphosphate; or (i) any combination of (a) to (h). In some embodiments, expression or overexpression of one or more of the aforementioned enzymes is beneficial (e.g., in terms of carbon flux) for the production of retinoids and retinyl esters through the pathways described herein.

[0027] In some embodiments, at least one, two, three, four, five, or six of the enzymes (a) to (g) under the control of an inducible promoter. In some embodiments, the fatty acid inducible promoter, such as a promoter of a fatty acid inducible gene, such as: HDE1, POX, PEX11 (peroxin), SPS19 (Peroxisomal 2-4- dienoyl CoA reductase), ICL1 (Isocitrate lyase), or FOX3 (3 -ketoacyl CoA thiolase). In some embodiments, the retinol-producing cells, prior to contact with the production medium, were subjected to an initial growth phase in a growth medium comprising a main carbon source (e.g., glucose) that may contribute to repression of fatty acid metabolism of the cells.

[0028] In some embodiments, at least one of enzymes (a) to (g) is / are: heterologous or exogenous with respect to the retinol-producing cells; encoded by polynucleotide(s) comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the microorganism in one or more copies); encoded by polynucleotide(s) comprised in an expression vector (e.g., plasmid); encoded by polynucleotide(s) operably linked to a heterologous promoter; or any combination thereof.

[0029] In some embodiments, the retinol-producing cells may be from an oleaginous microorganism. In some embodiments, the retinol-producing cells may be bacteria, yeast, fungi, algae, or other microorganisms. In some embodiments, the retinol-producing cells may be from the Blastobotrys, Saccharomyces, Aspergillus, Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, or Yarrowia genus. In some embodiments, the retinol-producing cells may be from Blastobotrys adeninivorans , Yarrowia lipolytica, or Saccharomyces cerevisiae. In some embodiments, the retinol-producing cells may be ones in which an endogenous activity of at least one of sfkl, yer!34C, dos2, xbpl, roxl, vba5, ynr063w, and ygr259c is inactivated or decreased (Trikka et al., 2015).

[0030] In some aspects, described herein a microorganism engineered to overexpress one or more of the enzymes described herein. In some embodiments, the microorganism corresponds to retinol-producing cells described herein. In some embodiments, the microorganism described herein may be for use in the production of: (i) phytoene, as a product or intermediate, from geranylgeranyl diphosphate; (ii) lycopene, as a product or intermediate, from phytoene; (iii) a beta-carotenoid, as a product or intermediate, from lycopene; (iv) retinal, as a product or intermediate, from beta-carotene; (v) retinol, as a product or intermediate, from retinal; or (vi) a retinyl ester, as a product or intermediate, from retinol.

[0031] In some aspects, described herein is a fermentation broth comprising a microorganism as described herein, and a production medium as described herein.

[0032] ITEMS

[0033] In some aspects, described here is one or more of the following items:

[0034] 1. A process for producing a desired retinyl ester of a fatty acid (desired retinyl fatty acid ester), the process comprising: (i) providing retinol-producing cells that express an enzyme that catalyzes the conversion of retinol to a retinyl ester; and (ii) culturing the retinol-producing cells in a production medium comprising the fatty acid and / or an alkyl ester thereof, under conditions enabling the production of the desired retinyl fatty acid ester.

[0035] 2. The process of item 1, wherein the production medium further comprises a liquid alkane and / or alkene mixture (e.g., mineral oil, paraffin oil, or other non-toxic liquid hydrocarbon mixture).

[0036] 3. The process of item 2, wherein the proportion of the fatty acid and / or an alkyl ester thereof to the liquid alkane and / or alkene mixture in the production medium is between about 5, 10, 15, 20, 25, 30, 35, or 40% w / w and about 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% w / w.

[0037] 4. The process of any one of items 1 to 3, wherein the fatty acid and desired retinyl fatty acid ester produced are: palmitic acid and retinyl palmitate; oleic acid and retinyl oleate; caproic acid and retinyl caproate; caprylic acid and retinyl caprylate; capric acid and retinyl caprate; lauric acid and retinyl laurate; myristic acid and retinyl myristate; stearic acid and retinyl stearate; arachidic acid and retinyl arachidate; or any combination thereof.

[0038] 5. The process of any one of items 1 to 4, wherein the alkyl group of the fatty acid ester is: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or any combination thereof. The process of any one of items 1 to 5, wherein the retinol-producing cells are cultured until the formation of an emulsion layer containing biosurfactants is generated by the cells into which the desired retinyl fatty acid ester accumulates. The process of item 6, further comprising (iii) isolating the desired retinyl fatty acid ester from the emulsion layer. The process of any one of items 1 to 7, wherein at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% w / w of total carotenoids or retinoids produced from the process are the desired retinyl fatty acid ester. The process of any one of items 1 to 8, wherein the retinol-producing cells express or overexpress: (a) a geranylgeranyl diphosphate synthase (EC 2.5.1.29) that catalyzes the synthesis of geranylgeranyl diphosphate; (b) a phytoene synthase (EC 2.5.1.32) that catalyzes the conversion of geranylgeranyl diphosphate to phytoene; (c) a phytoene desaturase (EC 1.3.99.31) that catalyzes the conversion of phytoene to lycopene; (d) a lycopene cyclase (EC 5.5.1.19) that catalyzes the conversion of lycopene to a beta-carotenoid; (e) a beta-carotene oxidizing enzyme (EC 1.13.11.63) that catalyzes the conversion of beta-carotene to retinal; (f) a retinol dehydrogenase (EC 1.1.1.105) that catalyzes the conversion of retinal to retinol; (g) a retinol acyltransferase that catalyzes the conversion retinol to the desired retinyl fatty acid ester, or (h) any combination of (a) to (g). The process of item 9, wherein: the geranylgeranyl diphosphate synthase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 14; or a fragment thereof having geranylgeranyl diphosphate synthase activity; the phytoene desaturase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or a fragment thereof having phytoene synthase activity; the phytoene synthase and the lycopene cyclase are comprised in a single bifunctional enzyme comprising an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 16; or a fragment thereof having phytoene desaturase and lycopene cyclase bifunctional activity; the beta-carotene oxidizing enzyme comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; or a fragment thereof having beta-carotene oxidizing activity; the retinol dehydrogenase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 18; or a fragment thereof having retinol dehydrogenase activity; the retinol acyltransferase comprises or consists of an acyl-CoA: diacylglycerol acyltransferase (EC 2.3.1), a diacylglycerol O- acyltransferase (DGAT; EC 2.3.1.20) (e.g., DGAT1, DGAT2), an acyl-CoA:retinol acyltransferase (ARAT), or an O-acyltransferase (e.g., encoded by mdy , or any combination thereof. The process of any one of items 1 to 10, wherein the retinol-producing cells further express or overexpress: (a) an acetyl-CoA C-acetyltransferase (EC 2.3. 1.9) that catalyzes the synthesis of acetoacetyl-CoA; (b) a hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) that catalyzes the conversion of acetoacetyl-CoA to hydroxymethylglutaryl-CoA; (c) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) that catalyzes the conversion of hydroxymethylglutaryl-CoA to mevalonate; (d) a mevalonate kinase (EC 2.7.1.36) that catalyzes the conversion of mevalonate to a phosphomevalonate; (e) a phosphomevalonate kinase (EC 2.7.4.2) that catalyzes the conversion of phosphomevalonate to diphosphomevalonate; (f) a diphosphomevalonate decarboxylase (EC 4.1.1.33) that catalyzes the conversion of diphosphomevalonate to isopentenyl diphosphate; (g) a isopentenyl -diphosphate Delta-isomerase (EC 5.3.3.2) that catalyzes the conversion of isopentenyl diphosphate to dimethylallyl diphosphate; (h) a famesyl diphosphate synthase (EC 2.5.1.1; EC:2.5.1.10) that catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to geranyl diphosphate, and / or catalyzes the conversion of geranyl diphosphate and isopentenyl diphosphate to famesyl diphosphate; or (i) any combination of (a) to (h). The process of any one of items 9 to 11, wherein at least one of the expressed or overexpressed enzymes is under the control of an inducible promoter. The process of item 12, wherein the inducible promoter is a promoter of a fatty acid inducible gene, such as: HDE1, POX, PEX11 (peroxin), SPS19 (Peroxisomal 2-4-dienoyl CoA reductase), ICL1 (Isocitrate lyase), or FOX3 (3 -ketoacyl CoA thiolase). The process of any one of items 9 to 13, wherein at least one of enzymes (a) to (g) is / are: (1) heterologous or exogenous with respect to the retinol-producing cells; (2) encoded by polynucleotide(s) comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the microorganism in one or more copies); (3) encoded by polynucleotide(s) comprised in an expression vector (e.g., plasmid); (4) encoded by polynucleotide(s) operably linked to a heterologous promoter; or (5) any combination of (1) to (4). The process of any one of items 1 to 14, wherein the retinol-producing cells are from an oleaginous microorganism. The process of any one of items 1 to 15, wherein the cells are bacteria, yeast, fungi, algae, or other microorganisms. The process of any one of items 1 to 16, wherein the cells are from Blastobotrys, Saccharomyces, Aspergillus, , Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, or Yarrowia. 18. The process of any one of items 1 to 17, wherein the cells are from Blastobotrys adeninivorans , Yarrowia lipolytica, or Saccharomyces cerevisiae.

[0039] 19. The process of any one of items 1 to 18, wherein the retinol-producing cells are ones in which an endogenous activity of at least one of sfkl, yerl34C, dos2, xbpl, roxl, vba5, ynr063w, and ygr259c is inactivated or decreased.

[0040] 20. A microorganism engineered to overexpress one or more of the enzymes as defined in any one of items 9 to 11.

[0041] 21. The microorganism of item 20, which is as defined in any one of items 12 to 19.

[0042] 22. The microorganism of item 20 or 21, for use in the production of: (i) phytoene, as a product or intermediate, from geranylgeranyl diphosphate; (ii) lycopene, as a product or intermediate, from phytoene; (iii) a beta-carotenoid, as a product or intermediate, from lycopene; (iv) retinal, as a product or intermediate, from beta-carotene; (v) retinol, as a product or intermediate, from retinal; or (vi) a retinyl ester, as a product or intermediate, from retinol.

[0043] 23. A fermentation broth comprising a microorganism as defined in any one of items 20 to 22, and a production medium as defined in any one of items 1 to 5.

[0044] EXAMPLES

[0045] Example 1: Transformation Procedure

[0046] 5 mL YPD starter cultures were inoculated with a colony of Blastobotrys adeninivorans strain ATCC 76597 (LCYBS1) and incubated overnight at 30 °C, with shaking at about 200 rpm. The following day, fresh 25 mL YPD cultures were inoculated to an initial OD600 nm of 0.4 and the culture incubated at 30 °C, with shaking at about 200 rpm until an OD600 nm of 1.0-2.0 was reached. Cells were pelleted by centrifugation at 1,000 x g, 4 °C for 10 minutes. Cells were washed by resuspending in 10 mL sterile water, pelleted, resuspended in 1 mL sterile water, and transferred to a 1.5 mL microcentrifuge tube. The cells were then washed in 1 mL sterile TE / LiOAC solution (100 mM Tris-EDTA (TE), 100 mM LiOAC, pH 7.5), pelleted, resuspended in 0.25 mL TE / LiOAC solution and incubated with shaking at 30 °C for 30 minutes. The cell solution was divided into 50 pL aliquots in 1 .5 mL tubes to which was added 1-3 pg of both donor DNA, guide RNA, and 5 pL of carrier DNA (boiled and cooled salmon sperm DNA, 10 mg / mL). 300 pL of sterile PEG solution (40% polyethylene glycol (PEG) 3350, 100 mM TE, 100 mM LiOAC) was added, mixed thoroughly, and incubated at 30 °C for 60 minutes with gentle mixing every 15 minutes. 40 pL of DMSO was added, mixed thoroughly and the cell solution was incubated at 42 °C for 15 minutes. Cells were then pelleted by centrifugation at 1,000 x g for 30 seconds, resuspended in 500 pL of YPD media and incubated at 30 °C with shaking at about 200 rpm for 2 hours. Cells were then pelleted by centrifugation and resuspended in 1 mb 100 mM TE, cells were pelleted again, resuspended in 0.2 mb 100 mM TE and plated on selective media. Plates were incubated at 30 °C for growth of transformants.

[0047] Example 2: Construction of Strain LCYBSA1 (SFK1::CRTE)

[0048] An SFK1 deletion cassette was constructed by assembling three DNA fragments using overlap PCRto produce a strain with the endogenous SFK1 gene (SEQ ID NO: 1) interrupted with the geranylgeranyl diphosphate synthase (EC 2.5.1.29) (CRTE) gene (SEQ ID NO: 2). The SFK1 upstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The SFK1 downstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The third fragment contained a synthesized and codon-optimized CRTE gene under the control oiw HDEl promoter (SEQ ID NO: 3) and HDE1 terminator (SEQ ID NO: 4), which was created using overlapping PCR. All three fragments were assembled using Gibson cloning (Gibson et al., 2009) to generate the plasmid pLCYBAl, which contains an SFK1 deletion cassette that overexpresses the CrtE gene. The deletion cassette was then generated via a PCR reaction containing plasmid pLCYBAl and suitable primers. The SFK1 deletion cassette was then purified and used to replace the SFK1 gene in strain LCYBS1 using a CRISPR / Cas9-based system, generating strain LCYBSA1.

[0049] Example 3: Construction of Strain LCYBSA2 (YER134C::CRTI_CRTBY)

[0050] A YER134C deletion cassette was constructed by assembling three DNA fragments using overlap PCRto produce a strain with the endogenous YER134C gene (SEQ ID NO.: 5) interrupted with the phytoene desaturase (EC 1.3.99.31) (CRTI) (SEQ ID NO: 6) and phytoene synthase (EC 2.5.1.32) / lycopene cyclase (EC 5.5.1.19) (CRTBY) genes (SEQ ID NOs: 7 and 8). The YER134C upstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The YER134C downstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The third fragment contained a synthesized and codon-optimized Crtl gene under the control of an HDE1 promoter (SEQ ID NO: 3) and HDE1 terminator (SEQ ID NO: 4), and codon-optimized CrtBY gene under the control of a Pox promoter (SEQ ID NO: 9) and Pox terminator (SEQ ID NO: 10), which was created using overlapping PCR. All three fragments were then assembled using Gibson cloning (Gibson et al., 2009) to generate the plasmid pLCYBA2, which contains a YER134C deletion cassette that overexpresses both Crtl and CrtBY genes. The deletion cassette was then generated via a PCR reaction containing plasmid pLCYBA2 and suitable primers. The YER134C deletion cassette was then purified and used to replace the YER134C gene in strain LCYBSA1 using a CRISPR / Cas9-based system, generating strain LCYBSA2.

[0051] Example 4: Construction of Strain LCYBSA3 (DOS2::BCO)

[0052] A DOS2 deletion cassette was constructed by assembling three DNA fragments using overlap PCRto produce a strain with the endogenous DOS2 gene (SEQ ID NO: 11) interrupted with a betacarotene oxidizing enzyme (EC 1.13.11.63; beta-carotene 15, 15 ’-dioxygenase) (BCO) (SEQ ID NO: 12). The DOS2 upstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The DOS2 downstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The third fragment contained a synthesized and codon-optimized BCO gene under the control oiwHDEl promoter (SEQ ID NO: 3) and HDE1 terminator (SEQ ID NO: 4), which was created using overlapping PCR. All fragments were assembled using Gibson cloning (Gibson et al, 2009) to generate the plasmid pLCYBA3, which contains a. DOS2 deletion cassette that overexpresses the BCO gene. The deletion cassette was then generated via a PCR reaction containing plasmid pLCYBA3 and suitable primers. The DOS2 deletion cassette was purified and used to replace the DOS2 gene in strain LCYBSA2 using a CRISPR / Cas9-based system, generating strain LCYBSA3.

[0053] Example 5: Construction of Strain LCYBSA4 (XBP1::RDH)

[0054] An XBP1 deletion cassette was constructed by assembling three DNA fragments using overlap PCRto produce a strain with the endogenous XBP1 gene (SEQ ID NO: 13) interrupted with a retinol dehydrogenase (RDH) enzyme (EC 1.1.1.105) (SEQ ID NO: 14) capable of converting retinal into retinol. The XBP1 upstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The XBP1 downstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The third fragment contained a synthesized and codon-optimized RDH gene under the control oiw HDEl promoter (SEQ ID NO: 3) and HDE1 terminator (SEQ ID NO: 4), which was created using overlapping PCR. All fragments were assembled using Gibson cloning (Gibson et al., 2009) to generate the plasmid pLCYBA4, which contains an XBP1 deletion cassette that overexpresses the RDH gene. The deletion cassette was then generated via a PCR reaction containing plasmid pLCYBA4 and suitable primers. The XBP1 deletion cassette was purified and used to replace the XBP1 gene in strain LCYBSA3 using a CRISPR / Cas9-based system, generating strain LCYBSA4. Example 6: Carotenoid production by LCYBSA1, LCYBSA2, LCYBSA3, and LCYBSA4 in shake flasks using canola oil

[0055] Starter cultures (5 mL) of LCYBSA1, LCYBSA2, LCYBSA3, and LCYBSA4 in YPD were incubated overnight at 30° C, with shaking at approximately 250 rpm. The overnight cultures were used to inoculate 25 mL of fresh GM Gly media (6.7 g / L yeast nitrogen base, 3.0 g / L yeast extract, 3.0 g / L (NEL SCL, 1.0 g / L K2HPO4, 1.0 g / L KH2PO4, 75 g / L glycerol) to an initial OD600 nm of 0.4 and incubated approximately 24 hours at 30 °C, and 300 rpm shaking. Cells were pelleted by centrifugation for 10 minutes at 3,000 x g at 4 °C, and then resuspended in 12.5 mL Production Media (yeast nitrogen base without amino acids and without ammonium sulfate, 1.7 g / L; yeast extract, 3.0 g / L; potassium phosphate monobasic, 10.0 g / L; potassium phosphate dibasic, 10.0 g / L), and added to 250 mL baffled shake flasks. 1 mL of canola oil was added to the shake flasks, which were shaken at approximately 300 rpm at 30 °C. Incubation of the cultures continued for 48 hours and samples were taken at 24 and 48 hours for analysis of carotenoid production by HPLC. Shake flask results from samples taken at 48 hours are shown in Table 1 and are presented as a percentage of total carotenoids produced. Strain LCYBSA1 (engineered to overexpress only CrtE) produced no detectable levels of carotenoids. Strain LCYBSA2 (engineered to overexpress CrtE, Crtl, and CrtBY) produced 59.6% of carotenoids as beta-carotene. Interestingly, strains LCYBSA3 (engineered to overexpress CrtE, Crtl, CrtBY, and BCO) and LCYBSA4 (engineered to additionally overexpress RDH) each produced comparable percentages of carotenoids as retinol and retinyl esters. These results suggest the presence of endogenous RDH enzymatic activity in the host Blastobotrys microorganism that may catalyze the conversion of retinol to retinyl esters such that overexpression of an exogenous RDH did not result in significantly higher retinol and retinyl esters concentrations. Furthermore, while both LCYBSA3 and LCYBSA4 were not engineered to overexpress an acyl transferase enzyme that catalyzes the conversion of retinol to retinyl ester, the host Blastobotrys microorganism was observed to possess such enzymatic activity endogenously.

[0056] Table 1. Carotenoid production using canola oil as a carbon source in shake flask tests

[0057] Example 7: Carotenoid production by LCYBSA4 in shake flasks using coconut oil

[0058] Starter culture (5 mL) of LCYBSA4 in YPD was incubated overnight at 30° C, with shaking at approximately 250 rpm. The overnight culture was used to inoculate 25 mL of fresh GM Gly mediate an initial OD600 nm of 0.4, and incubated approximately 24 hours at 30 °C and 300 rpm shaking. Cells were pelleted by centrifugation for 10 minutes at 3,000 x g at 4 °C, and then resuspended in 12.5 mL Production Media, and added to 250 mL baffled shake flasks. 1 mL of coconut oil was added to the shake flasks, which were shaken at approximately 300 rpm at 30 °C. Incubation of the culture was continued for 48 hours and samples were taken at 48 hours for analysis of carotenoid production. Shake flask results for these experiments are shown in Table 2 and are presented as a percentage of total carotenoids produced. For ease of comparison, the results in Table 2 using coconut oil are presented side-by-side with the results of Table 1 using canola oil for the LCYBSA4 strain. While the percentage of retinol was nearly 4- fold lower using coconut oil (0.8%) versus canola oil (3.1%), the percentage of retinyl esters produced (*) were observed to be similar whether using coconut oil or canola oil as a carbon source.

[0059] Table 2. Carotenoid production by LCYBSA4 using coconut oil as a carbon source

[0060] Example 8: Analysis of retinyl esters produced by LCYBSA4 in shake flasks using coconut oil or canola oil

[0061] The proportions of different species of retinyl esters produced by LCYBSA4 in the presence of coconut oil or canola oil were analyzed and the results are shown in Table 3. Interestingly, while the percentage of retinyl esters with respect to total carotenoids produced were similar whether using coconut oil (51.3%) or canola oil (50.6%), as shown in Example 7, the results in Table 3 reveal very distinct retinal ester profiles produced for each carbon source. For example, when using coconut oil as a carbon source, the retinyl ester species produced in the highest proportion was retinyl dodecanoate (47.3%). In contrast, when using canola oil as a carbon source, the retinyl ester species produced in the highest proportion was retinyl oleate (62.9%). These results show that different species of retinyl esters are produced depending on the carbon source added to the Production Media. Subsequent analysis of the fatty acid profiles of the coconut oil and canola oil that were used as carbon sources during the production phase (Table 4) revealed a striking correlation with the retinyl ester profiles ultimately produced, as can be seen by comparing the results shown in Tables 3 and 4. These results suggest that the retinyl ester profile produced from fermentation is governed by the fatty acid profile of the carbon source provided to the host microorganism during the production phase. Table 3. Profile of retinyl esters produced by LCYBSA4 using coconut or canola oil

[0062] Table 4. Profile of fatty acids in coconut and canola oil

[0063] Example 9: Carotenoid production by LCYBSA4 in bench-scale fermenter using liquid paraffin and / or ethyl palmitate as carbon sources

[0064] Strain LCYBSA4 was grown using a fed-batch process conducted in a bench-scale fermenter. The fermentation was a two-phase process comprising a first growth phase and a second production phase. The initial growth phase medium contained 60 g / L glucose as the primary carbon source. After the initial batch glucose had been consumed, a rapid rise in the fermenter dissolved oxygen level was observed. Once the glucose had been consumed and paraffin and / or ethyl palmitate started feeding, these yeast strains were observed to develop lipid bodies and created an emulsion layer or phase containing biosurfactants (generated by the cells), which changed the fermentation environment and into which the retinoid products accumulated. Next, during the production phase, the LCYBSA4 strain was fed with 100% liquid paraffin, 100% ethyl palmitate, or a mixed feed containing 80% liquid paraffin and 20% ethyl palmitate. The feed rate of all three fermenters was set at 4 g / L / hour. In order to determine retinyl palmitate and carotenoid production levels, 5 mL samples were taken 48 hours after induction and tested using HPLC. Fermentation results are shown in Table 5 and are presented as a percentage of total carotenoids produced. Advantageously, it was observed that a portion of the desired retinyl esters accumulated in the emulsion layer that accumulated at the top of the medium in a distinct cream -like phase or layer (e.g., upon centrifugation), separate from the layer of liquid paraffin. Table 5. Carotenoid production by LCYBSA4 using different feeds

[0065] Liquid paraffin 0.9% 0.2% 0.2% 18.6% 0.7% 75.8% 1.5% 2.0%

[0066] Ethyl palmitate 0.5% 0.2% 0.1% 29.7% 0.2% 5.8% 62.0% 1.5%

[0067] Liquid paraffin + Ethyl palmitate 1.1% 0.6% 0.5% 11.2% 0.5% 7.1% 77.6% 1.3%

[0068] As shown in Table 5, using a heterogenous mixture of alkane / alkenes (e.g., liquid paraffin, which contains a mixture of C10-C14 alkanes) as the main carbon source during the production phase resulted in a marked accumulation of retinol (75.8%), but only low amounts of the retinyl esters (e.g., retinyl palmitate and retinyl oleate), despite the LCYBSA4 strain having all the necessary enzymatic machinery to produce them. These results suggest that the microorganism metabolizes the mixture of alkane / alkenes, which involves the synthesis of fatty acids, but that these endogenous fatty acids synthesized by the microorganism itself are not significantly converted to retinyl fatty acid esters. Conversely, use of an exogenous alkyl fatty acid ester (i.e., ethyl palmitate) as the main carbon source during the production phase resulted in a marked production of the corresponding retinyl fatty acid ester, namely retinyl palmitate (62.0%), with low retinol levels (5.8%). These results, along with those in Example 8, demonstrate that the retinyl fatty acid ester species produced from fermentation is governed by the exogenous fatty acid species present in the carbon source provided to the host microorganism during the production phase. In summary, these results demonstrate that the microorganism host metabolizes exogenously provided fatty acids quite differently from endogenously synthesized fatty acids in the sense that the former are converted to retinyl fatty acid esters, whereas the latter are not significantly, which goes against what was observed in some prokaryotic systems, such as in E. colt (US patent no. 9,834,794).

[0069] Unexpectedly, use of a mixture of 80% alkanes / alkenes (i.e., liquid paraffin) and 20% alkyl palmitate (i.e., ethyl palmitate) as the main carbon source during the production phase resulted a 1.25-fold increase in the retinyl palmitate produced, as compared to when using ethyl palmitate alone. Furthermore, use of the 80% liquid paraffin / 20% ethyl palmitate mixture resulted in a concomitant reduction of the intermediate phytoene to 11.2%, which was strikingly lower than the level of phytoene present when using either paraffin or ethyl palmitate alone. Example 10: Carotenoid production by LCYBSA2, LCYBSA3, and LCYBSA4 in bench-scale fermenter using liquid paraffin / ethyl palmitate mixture as a carbon source

[0070] Strains LCYBSA1, LCYBSA2, LCYBSA3, and LCYBSA4 were grown using a fed-batch process in a bench-scale fermenter. The two-phase fermentation process comprised a first growth phase and a second production phase. The initial growth phase medium contained 60 g / L glucose as the primary carbon source. After the glucose was consumed, a rapid rise in the fermenter dissolved oxygen level was observed. Next, during the production phase, a feed containing 80% paraffin and 20% ethyl palmitate was started with the feed rate of 4 g / L / hour. In order to determine retinyl palmitate and carotenoid production levels, 5 mL samples were taken 48 hours after induction and tested using HPLC. Fermentation results are shown in Table 6 and are presented as a percent of total carotenoids produced.

[0071] Table 6. Carotenoid production by LCYBSA2, LCYBSA3, and LCYBSA4 in fed-batch cultures

[0072] As seen in Table 6, LCYBSA2 produced the vast majority (86.0%) of carotenoids as betacarotene, while strains LCYBSA3 and LCYBSA4 produced a striking 88.5% and 84.7% of carotenoids, respectively, as retinyl palmitate.

[0073] REFERENCES

[0074] Gibson et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009 May;6(5):343-5. doi: 10.1038 / nmeth.l318. Epub 2009 Apr 12.

[0075] Trikka et al. Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production. Microb Cell Fact. 2015 Apr 24;14:60. doi: 10.1186 / sl2934-015-0246-0.

[0076] US patent no. 9,834,794

Claims

CLAIMS1. A process for producing a desired retinyl ester of a fatty acid (desired retinyl fatty acid ester), the process comprising:(i) providing retinol-producing cells that express an enzyme that catalyzes the conversion of retinol to a retinyl ester; and(ii) culturing the retinol-producing cells in a production medium comprising the fatty acid and / or an alkyl ester thereof, under conditions enabling the production of the desired retinyl fatty acid ester.

2. The process of claim 1, wherein the production medium further comprises a liquid alkane and / or alkene mixture (e.g., mineral oil, paraffin oil, or other non-toxic liquid hydrocarbon mixture).

3. The process of claim 2, wherein the proportion of the fatty acid and / or an alkyl ester thereof to the liquid alkane and / or alkene mixture in the production medium is between about 5, 10, 15, 20, 25, 30, 35, or 40% w / w and about 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% w / w.

4. The process of any one of claims 1 to 3, wherein the fatty acid and desired retinyl fatty acid ester produced are: palmitic acid and retinyl palmitate; oleic acid and retinyl oleate; caproic acid and retinyl caproate; caprylic acid and retinyl caprylate; capric acid and retinyl caprate; lauric acid and retinyl laurate; myristic acid and retinyl myristate; stearic acid and retinyl stearate; arachidic acid and retinyl arachidate; or any combination thereof.

5. The process of any one of claims 1 to 4, wherein the alkyl group of the fatty acid ester is: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or any combination thereof.

6. The process of any one of claims 1 to 5, wherein the retinol-producing cells are cultured until the formation of an emulsion layer containing biosurfactants is generated by the cells into which the desired retinyl fatty acid ester accumulates.

7. The process of claim 6, further comprising (iii) isolating the desired retinyl fatty acid ester from the emulsion layer.

8. The process of any one of claims 1 to 7, wherein at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% w / w of total carotenoids or retinoids produced from the process are the desired retinyl fatty acid ester.

9. The process of any one of claims 1 to 8, wherein the retinol-producing cells express or overexpress:(a) a geranylgeranyl diphosphate synthase (EC 2.5.1.29) that catalyzes the synthesis of geranylgeranyl diphosphate;(b) a phytoene synthase (EC 2.

5. 1.32) that catalyzes the conversion of geranylgeranyl diphosphate to phytoene;(c) a phytoene desaturase (EC 1.3.99.31) that catalyzes the conversion of phytoene to lycopene;(d) a lycopene cyclase (EC 5.5.1.19) that catalyzes the conversion of lycopene to a betacarotenoid;(e) a beta-carotene oxidizing enzyme (EC 1.13.11.63) that catalyzes the conversion of betacarotene to retinal;(f) a retinol dehydrogenase (EC 1.1.1.105) that catalyzes the conversion of retinal to retinol;(g) a retinol acyltransferase that catalyzes the conversion retinol to the desired retinyl fatty acid ester, or(h) any combination of (a) to (g).

10. The process of claim 9, wherein: the geranylgeranyl diphosphate synthase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, or 99% identical to SEQ ID NO: 14; or a fragment thereof having geranylgeranyl diphosphate synthase activity; the phytoene desaturase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 15; or a fragment thereof having phytoene synthase activity; the phytoene synthase and the lycopene cyclase are comprised in a single bifunctional enzyme comprising an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 16; or a fragment thereof having phytoene desaturase and lycopene cyclase bifunctional activity;the beta-carotene oxidizing enzyme comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 17; or a fragment thereof having beta-carotene oxidizing activity; the retinol dehydrogenase comprises an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 18; or a fragment thereof having retinol dehydrogenase activity; the retinol acyltransferase comprises or consists of an acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1), a diacylglycerol O-acyltransferase (DGAT; EC 2.3.1.20) (e.g., DGAT1, DGAT2), an acyl-CoA:retinol acyltransferase (ARAT), or an O-acyltransferase (e.g., encoded by mdy , or any combination thereof.

11. The process of any one of claims 1 to 10, wherein the retinol-producing cells further express or overexpress:(a) an acetyl-CoA C-acetyltransferase (EC 2.

3. 1.9) that catalyzes the synthesis of acetoacetyl- CoA;(b) a hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) that catalyzes the conversion of acetoacetyl-CoA to hydroxymethylglutaryl-CoA;(c) a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) that catalyzes the conversion of hydroxymethylglutaryl-CoA to mevalonate;(d) a mevalonate kinase (EC 2.7.1.36) that catalyzes the conversion of mevalonate to a phosphomevalonate;(e) a phosphomevalonate kinase (EC 2.7.4.2) that catalyzes the conversion of phosphomevalonate to diphosphomevalonate;(f) a diphosphomevalonate decarboxylase (EC 4.1.1.33) that catalyzes the conversion of diphosphomevalonate to isopentenyl diphosphate;(g) a isopentenyl -diphosphate Delta-isomerase (EC 5.3.3.2) that catalyzes the conversion of isopentenyl diphosphate to dimethylallyl diphosphate;(h) a famesyl diphosphate synthase (EC 2.5.1.1; EC:2.5.1.10) that catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to geranyl diphosphate, and / or catalyzes the conversion of geranyl diphosphate and isopentenyl diphosphate to famesyl diphosphate; or(i) any combination of (a) to (h).

12. The process of any one of claims 9 to 11, wherein at least one of the expressed or overexpressed enzymes is under the control of an inducible promoter.

13. The process of claim 12, wherein the inducible promoter is a promoter of a fatty acid inducible gene, such as: HDE1, POX, PEX11 (peroxin), SPS19 (Peroxisomal 2-4-dienoyl CoA reductase), ICL1 (Isocitrate lyase), or F0X3 (3 -ketoacyl CoA thiolase).

14. The process of any one of claims 9 to 13, wherein at least one of enzymes (a) to (g) is / are:(1) heterologous or exogenous with respect to the retinol-producing cells;(2) encoded by polynucleotide(s) comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the microorganism in one or more copies);(3) encoded by polynucleotide(s) comprised in an expression vector (e.g., plasmid);(4) encoded by polynucleotide(s) operably linked to a heterologous promoter; or(5) any combination of (1) to (4).

15. The process of any one of claims 1 to 14, wherein the retinol-producing cells are from an oleaginous microorganism.

16. The process of any one of claims 1 to 15, wherein the cells are bacteria, yeast, fungi, algae, or other microorganisms.

17. The process of any one of claims 1 to 16, wherein the cells are from Blastobotrys, Saccharomyces, Aspergillus, , Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, or Yarrowia.

18. The process of any one of claims 1 to 17, wherein the cells are from Blastobotrys adeninivorans, Yarrowia lipolytica, or Saccharomyces cerevisiae.

19. The process of any one of claims 1 to 18, wherein the retinol-producing cells are ones in which an endogenous activity of at least one of sfkl, yerl34C, dos2, xbpl, roxl, vba5, ynr063w, and ygr259c is inactivated or decreased.

20. A microorganism engineered to overexpress one or more of the enzymes as defined in any one of claims 9 to 11.

21. The microorganism of claim 20, which is as defined in any one of claims 12 to 19.

22. The microorganism of claim 20 or 21, for use in the production of:(i) phytoene, as a product or intermediate, from geranylgeranyl diphosphate;(ii) lycopene, as a product or intermediate, from phytoene;(iii) a beta-carotenoid, as a product or intermediate, from lycopene;(iv) retinal, as a product or intermediate, from beta-carotene;(v) retinol, as a product or intermediate, from retinal; or(vi) a retinyl ester, as a product or intermediate, from retinol.

23. A fermentation broth comprising a microorganism as defined in any one of claims 20 to 22, and a production medium as defined in any one of claims 1 to 5.

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