WO2023275212A1

WO2023275212A1 - Sustainable production of retinyl fatty esters

Info

Publication number: WO2023275212A1
Application number: PCT/EP2022/068004
Authority: WO
Inventors: Peter Louis HOUSTON; Valmik Kanubhai VYAS
Original assignee: Dsm Ip Assets B.V.
Priority date: 2021-07-01
Filing date: 2022-06-29
Publication date: 2023-01-05

Abstract

The present invention is related to a fermentation process wherein long-chain fatty acid retinyl esters (FAREs) are produced using retinoid-producing host cell, particularly oleaginous yeast, modified such that the activity of certain endogenous lipases is enhanced or overexpressed, said host cells being capable of using triglyceride oils or free fatty acids as carbon source, with the proviso that palm oil or palmitic acid are omitted, to generate those FAREs in a sustainable way.

Description

Sustainable production of retinyl fatty esters

Retinoids, including vitamin A, are one of very important and indispensable nutrient factors for human beings which must be supplied via nutrition. Retinoids promote well-being of humans, inter alia in respect of vision, the immune system and growth.

The most commonly used and stable form of vitamin A is vitamin A palmitate, that is naturally found in eggs, chicken or beef but that is also available as a nutritional supplement. Chemical synthesis as common in the art starts from retinyl acetate and palmitic acid, using harch conditions including acidic and alkaline solvents with negative impact on both the starting material (i.e. retinyl acetate) and the equipment. Due to high energy consumption and pollution said process is not very sustainable and maybe not in line with the UN Sustainable Development Goals.

Alternatively, vitamin A palmitate can be produced in a bio-transesterification process, wherein retinyl acetate and palmitic acid are converted into vitamin A palmitate with the help of immobilized enzymes. However, such process still requires the use of palmitic acid in organic solvents. Palmitic acid (also known under the lUPAC name hexadecenoic acid) is a saturated fatty acid and the major component of the oil from the fruit of oil palms. Until now, recovery of palmitic acid or palm oil from palm trees is a very cheap and commonly used method, even though it is known that extensive palm tree plantations result in deforestation, loss of biodiversity, increased carbon emission (e.g. through burning of carbon-rich soil) - just to name only some of its negative effects.

The use of palm oil or palmitic acid - even if produced in a sustainable manner - particularly when feeding in a bioreactor, leads to further issues, due to its chemical properties: because of its high amount of saturated lipid in the triglyceride, the solid liquid phase of palm oil is semisolid at room temperature (melting point 35°C). This factor adds a complication and cost of heated feed lines and can lead to problems in production. Further palmitic acid has been shown to be insoluble in mixtures of hydrolyzed triglycerides resulting in fouling of feed pumps and inconsistent feeding.

It is also known that consumption of high content of saturated fatty acids can lead to some health issues, such as e.g. high blood cholesterol levels or higher risk of heart disease. Semi-solubility of retinyl palmitate at room temperature (melting point 28-29°C) has the added disadvantage that it is semisolid at room temperature which limits this form's processing when modified into forms for proper human assimilation.

Thus, there is a strong need for providing more sustainable and eco-friendly bio-based production processes for stable forms of vitamin A and for more healthy triglyceride oil sources to be used in the food, feed, cosmetic or pharma industry in replacement of vitamin A palmitate. Particularly, it is desirable to develop a fermentative process using e.g. oleaginous host cells growing on renewable and sustainable carbon sources, i.e. vegetable oil instead of petroleum based oils such as C12-C16 alkanes, without compromising the growth of the host cell or the fermentation conditions, such as it is known for addition of lipases to a bioreaction with high vegetable oil content resulting in negative effects, i.e. rapidly increase in the viscosity leading to emulsification, i.e. development of a cottage cheese like substance in the reactor which would be an obstacle to apply such process on an industrial level.

Surprisingly, we now found a way for sustainable fermentative production of retinyl esters, with the exclusion of retinyl palmitate, i.e. fatty acid retinyl esters (FAREs) in fungal host cells, particularly oleaginous yeast, such as e.g. Yarrowia, wherein the host cell comprises one or more genetic modification(s), i.e. addition and/or enhancement of certain endogenous lipase activities, said host cell being grown on certain vegetable oils, particularly with high content on unsatu rated fatty acids, and wherein an emulsification due to increased viscosity during the fermentation could be avoided.

Particularly, the present invention is directed to a retinoid-producing host cell, such as a retinol-producing host cell, such as a fungal host cell, preferably oleaginous yeast cell such as e.g. Yarrowia, comprising one or more genetic modification(s), i.e. increased activity of endogenous lipases, preferably wherein certain endogenous lipase genes, i.e. genes expressing enzymes with lipase activities, are overexpressed and/or wherein heterologously added lipase genes are overexpressed and optionally wherein furthermore exogenous lipases might be added to the cultivation, particularly comprising overexpression of genes encoding enzymes with lipase activity corresponding to Yarrowia lipolytica LIP2, LIP3, LIP4, LIP8, and combinations thereof, preferably Yarrowia lipolytica LIP8 activity, including but not limited to modification in the activity of an endogenous gene encoding a protein with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to LIP2, LIP3, LIP4, and/or LIP8 of Yarrowia lipolytica accordingto SEQ ID NOs:1, 3, 5, 7, including a lipase encoded by a nucleic acid according to SEQ ID NOs:2, 4, 6, 8.

Suitable endogenous genes encoding proteins having lipase activity to be modified accordingto the present invention might be selected from enzymes with lipase and/or esterase activity. The term "lipase" is used interchangeably herein with the term "esterase" or "enzyme having lipase activity". It refers to enzymes involved in pre-digestion of triglyceride oils such as e.g. vegetable oils as defined herein into glycerol and fatty acids that are normally expressed in oleaginous host cells. Suitable enzymes to be modified in a host cell as defined herein might be selected from endogenous enzymes belonging to EC class 3.1.1. -, including, but not limited to one or more enzyme(s) with activities corresponding to Yarrowia LIP2, LIP3, LIP4, LIP8, TGL1, LIP16, LIP17, LIP18 or e.g. to commercially available lipase composition, such as e.g. products available from Creative Enzymes or Sigma Aldrich typically comprising a mixture of several lipases including but not limited to lipases natively expressed by Candida rugosa and/or Candida cylindracea including enzymes with activities equivalent to said commercially available lipases that are heterologous expressed in said host cell, such as e.g. lipases originated from Candida, Aspergillus, Thermomyces or Rhizopus. As used herein, an enzyme having activity corresponding to the respective LIP activity in Yarrowia includes not only the genes originating from Yarrowia, e.g. Yarrowia lipolytica, such as e.g. Yarrowia LIP2, LIP3, LIP4, LIP8, TGL-1, LIP16, LIP17, LI PI 8 according to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15 or combinations thereof, but also includes enzymes having equivalent enzymatic activity but are originated from another source organism, particularly retinoid/retinol-producing oleaginous host cell, wherein a modification of such equivalent genes would lead to an increase in retinol to retinyl ester conversion as defined herein.

The present invention is directed to a host cell which is modified in certain endogenous lipase activities leading to a percentage of esterified retinol, i.e. fatty acid retinyl esters (FAREs) in a fermentation process and under conditions as defined herein, wherein the percentage of FAREs based on total retinoids produced/present in the host cell is at least about 70%. Suitable host cells to be modified are selected from retinoid-producing host cells, particularly retinol- producing host cells, wherein retinyl ester is formed via enzymatic conversion of retinol catalyzed by enzymes with lipase activity as defined herein, e.g. fungal host cells including oleaginous yeast cells, such as e.g. Rhodosporidium, Lipomyces, Candida or Yarrowia, preferably Yarrowia, more preferably Yarrowia lipolytica, and wherein the modification comprises genetic modification, such as e.g. enhancing/overexpressing activity of genes encoding certain lipases or corresponding enzyme activities from other oleaginous host cells as specified herein, including but not limited to overexpression of the corresponding endogenous genes, expression of heterologous genes within said host cell, and/or addition of suitable heterologous lipases as defined herein. Furthermore, a suitable host cell according to the present invention might be selected from a retinoid-producing host cell, particularly retinol-producing host cell, such as e.g. selected from the oleaginous yeast cells as defined above but also from cells such as e.g. Saccharomyces or Escherichia, particularly retinol- producing Saccharomyces cerevisiae or E. coli, wherein said strains have been genetically modified to enable enhanced production of retinol and or retinyl acetate, such as e.g. disclosed in WO2019/057998 or W02019/058001, with particularly a retinal to retinol conversion of about 90%, including a retinoid mix with a retinol to retinal ratio of about 9:1 based on total retinoids, with particularly a retinol to retinyl acetate ratio of about 80% or more based on total retinoids, said retinoid-producing host cells being cultivated under suitable culture conditions as defined herein with the proviso that said cultivation is a fermentation being carried out in the presence of suitable triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil, wherein exogenous enzymes having activities equivalent to Yarrowia LIP2 and/or LIP3 and/or LIP4 and/or LIP8 are contacted with the strains and/or the fermentatively produced retinoid mix comprising retinol and/or retinyl acetate, with a percentage of at least about 80% based on total retinoids, and wherein retinol and/or retinyl acetate is enzymatically converted into FARES via activity of the lipases as described herein leading to retinoid mix with a percentage of FARES of at least about 70% based on total retinoids.

As defined herein, a "modified host cell" is compared to a "wild-type host cell", i.e., the respective host cell without such modification in the defined enzyme activities, i.e. wherein said corresponding heterologous enzyme is not expressed and active in vivo or wherein the endogenous enzyme activity is not enhanced and thus reflects the wild-type situation.

As defined herein, "overexpressed" in the context of gene expression in a modified host cell means increased expression of a polynucleotide in comparison to the wild-type host cell, and includes furthermore expression of (newly) introduced foreign polynucleotides, i.e. heterologous polynucleotides, which are not present/expressed in the wild-type host cell and/or addition of heterologous enzymes with lipase activities, such as e.g. lipases originated from Candida, Aspergillus, Thermomyces or Rhizopus, preferably from Candida rugosa and/or Candida cylindracea and as defined herein. It might also include the modification of the host cell in a two-step process, wherein firstly the endogenous gene(s) is/are completely inactivated, such as e.g. disrupted or knocked-out, and in a second step, this gene activity is brought back into the host cell but expressed under control of a strong promoter or with more than one gene copy.

The present invention is related to a process wherein the modified host cell as defined herein, i.e. wherein the activity of certain lipases is increased such as e.g. via overexpressing of the corresponding genes, said process comprising fermentation of said modified host cell, particularly retinoid-producing host cell, more particularly retinol-producing host cell, under suitable culture conditions, i.e. conditions that enable the esterification of retinol via activity of said (over)expressed lipases into fatty acid retinyl esters (FAREs), said fermentation being carried out in the presence of suitable triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil.

As used herein, the term "with the proviso that this is not palmitic acid or palm oil" means a triglyceride oil or fatty acid, particularly vegetable oil, not in the scope of the present invention and that should be avoided. Particularly, this excludes a vegetable oil that is rich in saturated fatty acids, such as rich in palmitic acid, i.e. a vegetable oil containing about 40-50% of palmitic acid. It also excludes triglyceride oils or fatty acids with high content of saturated fatty acids, such as about 45-50% saturated fatty acids based on total fatty acid composition.

In one embodiment, the present invention provides a modified host cell, such as modified retinol-producing oleaginous host cell, comprising a modification in a polypeptide with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to SEQ ID NO:7, including but not limited to LIP8 obtainable from Yarrowia lipolytica, wherein the activity of said polypeptide is enhanced such as e.g. via overexpression of the corresponding gene wherein the use of such modified host cell in a fermentation in the presence of triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil, palm oil results in a percentage of at least about 70% retinyl ester based on total retinoids present in the respective host cell as defined herein. Particularly, the host cell is selected from Yarrowia, such as Yarrowia lipolytica, wherein the activity of LIP8 according to SEQ ID NO:7, including a polypeptide encoded by a polynucleotide according to SEQ ID NO:8, is overexpressed, leading to an increase of at least about 20%, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more retinyl ester based on total retinoids in the host cell. LIP8 according to SEQ ID NO:7 is derived from RefSeq YALI0_B09361g. Said modified host cell overexpressing a polypeptide encoding endogenous LIP8 or an equivalent heterologous lipase might comprise further modifications, such as e.g. overexpressing further polynucleotides encoding (endogenous) enzymes including but not limited to enzymes with activities equivalent to Yarrowia LIP2, LIP3, TGL1, LI PI 6, LIP17, LIP18, or LIP4 or commercially available lipase compositions comprising e.g. Candida rugosa and/or Candida cylindracea lipases, including enzymes with at least about 50% identity to a sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and combinations thereof. In one embodiment, the present invention provides a modified host cell, such as modified retinol-producing oleaginous host cell, comprising a modification in a polypeptide with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to SEQ ID NO:1, including but not limited to LIP2 obtainable from Yarrowia lipolytica, wherein the activity of said polypeptide is enhanced such as e.g. via overexpression of the corresponding gene wherein the use of such modified host cell in a fermentation in the presence of triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil, results in a percentage of at least about 70% retinyl ester based on total retinoids present in the respective host cell as defined herein. Particularly, the host cell is selected from Yarrowia, such as Yarrowia lipolytica, wherein the activity of LIP2 according to SEQ ID NO:1, including a polypeptide encoded by a polynucleotide according to SEQ ID NO:2, is overexpressed, leading to an increase of at least about 20%, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more retinyl ester based on total retinoids in the host cell. LIP2 according to SEQ ID NO:1 is derived from RefSeq YALI0_A20350g. Said modified host cell overexpressing a polypeptide encoding endogenous LIP2 or an equivalent heterologous lipase might comprise further modifications, such as e.g. overexpressing further polynucleotides encoding (endogenous) enzymes including but not limited to enzymes with activities equivalent to Yarrowia LIP8, LIP3, TGL1, LI PI 6, LIP17, LIP18, or LIP4 or commercially available lipase compositions comprising e.g. Candida rugosa and/or Candida cylindracea lipases, including enzymes with at least about 50% identity to a sequence selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, and combinations thereof.

In a further embodiment, the present invention provides a modified host cell, such as modified retinol-producing oleaginous host cell, comprising a modification in a polypeptide with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to SEQ ID NO:3, including but not limited to LIP3 obtainable from Yarrowia lipolytica, wherein the activity of said polypeptide is enhanced such as e.g. via overexpression of the corresponding gene wherein the use of such modified host cell in a fermentation in the presence of triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil, results in a percentage of at least about 70% retinyl ester based on total retinoids present in the respective host cell as defined herein. Particularly, the host cell is selected from Yarrowia, such as Yarrowia lipolytica, wherein the activity of LIP3 according to SEQ ID NO:3, including a polypeptide encoded by a polynucleotide according to SEQ ID NO:4, is overexpressed, leading to an increase of at least about 20%, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more retinyl ester based on total retinoids in the host cell. LIP3 according to SEQ ID NO:3 is derived from RefSeq YALI0_B08030g. Said modified host cell overexpressing a polypeptide encoding endogenous LIP3 or an equivalent heterologous lipase might comprise further modifications, such as e.g. overexpressing further polynucleotides encoding (endogenous) enzymes including but not limited to enzymes with activities equivalent to Yarrowia LIP8, LIP2, TGL1, LI PI 6, LIP17, LIP18, or LIP4 or commercially available lipase compositions comprising e.g. Candida rugosa and/or Candida cylindracea lipases, including enzymes with at least about 50% identity to a sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and combinations thereof.

In a further embodiment, the present invention provides a modified host cell, such as modified retinol-producing oleaginous host cell, comprising a modification in a polypeptide with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to SEQ ID NO:5, including but not limited to LIP4 obtainable from Yarrowia lipolytica, wherein the activity of said polypeptide is enhanced such as e.g. via overexpression of the corresponding gene wherein the use of such modified host cell in a fermentation in the presence of triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil, results in a percentage of at least about 70% retinyl ester based on total retinoids present in the respective host cell as defined herein. Particularly, the host cell is selected from Yarrowia, such as Yarrowia lipolytica, wherein the activity of LIP4 according to SEQ ID NO:5, including a polypeptide encoded by a polynucleotide according to SEQ ID NO:6, is overexpressed, leading to an increase of at least about 20%, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more retinyl ester based on total retinoids in the host cell. LIP4 according to SEQ ID NO:5 is derived from RefSeq YALI0_E08492g. Said modified host cell overexpressing a polypeptide encoding endogenous LIP4 or an equivalent heterologous lipase might comprise further modifications, such as e.g. overexpressing further polynucleotides encoding (endogenous) enzymes including but not limited to enzymes with activities equivalent to Yarrowia LIP8, LIP2, TGL1, LIP16, LIP17, LIP18, or LIP3 or commercially available lipase compositions comprising e.g. Candida rugosa and/or Candida cylindracea lipases, including enzymes with at least about 50% identity to a sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11,13, 15, and combinations thereof.

According to further embodiments, the present invention provides a modified host cell, such as modified retinol-producing oleaginous host cell, comprising a modification in a polypeptide with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to SEQ ID NOs:9, 11, 13 or 15, including but not limited to an enzyme selected from the group consisting of TGL1, LIP16, LIP17, LIP18, and combinations thereof obtainable from Yarrowia lipolytica, wherein the activity of said polypeptide(s) is enhanced such as e.g. via overexpression of the corresponding gene(s) wherein the use of such modified host cell in a fermentation in the presence of triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil, results in a percentage of at least about 70% retinyl ester based on total retinoids present in the respective host cell as defined herein. Particularly, the host cell is selected from Yarrowia, such as Yarrowia lipolytica, wherein the activity of said lipase(s) according to SEQ ID NOs:9, 11, 13, or 15, including polypeptide(s) encoded by polynucleotide(s) according to SEQ ID NOs:10, 12, 14 or 16, is overexpressed, leading to an increase of at least about 20%, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more retinyl ester based on total retinoids in the host cell. TGL1 according to SEQ ID NO:9 is derived from RefSeq YALI0_E32035g. LIP16 according to SEQ ID NO:11 is derived from RefSeq YALI0_D18480g. LIP17 according to SEQ ID NO:13 is derived from RefSeq YALI0_F32131g. LIP18 according to SEQ ID NO:15 is derived from RefSeq YALI0_B20350g. Said modified host cell overexpressing a polypeptide encoding endogenous TGL1, LIP16, LIP17, LIP18, combinations thereof or equivalent heterologous lipase(s) might comprise further modifications, such as e.g. overexpressing further polynucleotides encoding (endogenous) enzymes including but not limited to enzymes with activities equivalent to Yarrowia LIP8, LIP2, LIP3, LIP4 or commercially available lipase compositions comprising e.g. Candida rugosa and/or Candida cylindracea lipases, including enzymes with at least about 50% identity to a sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7 and combinations thereof.

Preferably, a modified host cell according to the present invention comprises a modification in an enzyme with activity of an enzyme with at least about 50% identity to LIP8 according to SEQ ID NO:7 such as obtainable from Yarrowia or an enzyme from another host cell with activity equivalent to Yarrowia LIP8 as defined herein, leading to a percentage of retinyl ester based on total retinoids of at least about 70%, such as in the range of about 70-90% or more, such as e.g. in a process wherein the host cell is grown in the presence of triglyceride oils or fatty acids as carbon source, particularly vegetable oil, with the proviso that this is not palmitic acid or palm oil. The percentage of retinyl ester might be furthermore increased, such as e.g. by at least about 10% based on total retinoids, such as e.g. in a process wherein the host cell is grown in the presence of triglyceride oils as carbon source as defined herein, with combination of further modifications in the endogenous enzyme activity in the host cell, Particularly preferred are combination with further modifications, such as e.g. modification in the activity of an enzyme with at least about 50% identity to LIP2 and/or LIP3 and/or LIP4 accordingto SEQ ID NO:1 or 3 or 5 such as obtainable from Yarrowia or enzymes from another host cell with activities equivalent to Yarrowia LIP2 and/or LIP3 and/or LIP4 and/or commercially available lipase compositions comprising e.g. Candida rugosa and/or Candida cylindracea lipases. Further increase in retinyl ester percentage based on total retinoids might be possible via introduction of one or more modifications in the activity of one or more enzyme(s) with at least about 50% identity to an enzyme selected from the group consisting of TGL1, LI PI 6, LIP17, LIP18 and combinations thereof accordingto SEQ ID NOs:9, 11, 13, 15 such as obtainable from Yarrowia or enzymes from another host cell with activities equivalent to an enzyme selected for the group consisting of Yarrowia TGL1, LI PI 6, LIP17, and LIP18.

As used herein, "activity" of an enzyme, particularly lipase activity, including activity of lipases as defined herein, is defined as "specific activity" i.e. its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate, such as e.g. the formation of FARES from retinol as defined herein. An enzyme, e.g. a lipase, is active, if it performs its catalytic activity in vivo, i.e. within the host cell as defined herein or within a system in the presence of a suitable substrate. The skilled person knows how to measure enzyme activity, in particular activity of lipases as defined herein, including but not limited to enzyme with activities corresponding to Yarrowia LIP2 and/or LIP3 and/or LIP8 and/or TGL1 and/or LIP16 and/or LIP17 and/or LIP18 and/or LIP4 and/or activity of e.g. Candida rugosa and/or Candida cylindracea lipases as comprised in commercially available lipase compositions. Analytical methods to evaluate the capability of lipases as defined herein involved in formation of FARES are known in the art and include measurement via HPLC and the like. Further, general hydrolase enzyme activity can be monitored by hydrolysis of nitrophenyl esters with steric, oleic or other interesting esters, since the hydrolysis can be monitored by evolution at 340nm using a spectrophotometer and converting the evolution of moles product over time using Beer's law to find the specific activity. With regards to activity of LIP2, LIP3, LIP4, LIP8, TGL1, LIP16, LIP17, LIP18 and/or e.g. Candida rugosa and/or Candida cylindracea lipase compositions as defined herein, the skilled person might measure the formation of FARES from conversion of retinol using a modified host cell in comparison to the formation of FARES from conversion of retinol using a non-modified or wild-type host cell. As used herein, an enzyme, particularly a lipase as defined herein, having "increased" activity means an increase in its specific activity, i.e. enhanced/overexpressed ability to catalyze formation of a product from a given substrate, such as in the presence of triglyceride oils, particularly vegetable oil with the proviso that this is not palmitic acid or palm oil, such as e.g. corn oil, olive oil, sunflower oil, rapeseed oil or free fatty acids like oleic acid, as carbon source, including enhanced or overexpressed activity of the respective

(endogenous) gene encoding such lipases, particularly enhancement by at least about 10% as compared to enzyme activity in the respective wild-type or non- modified suitable host cell as defined herein. Such enhancement might be achievable e.g. via modification of copy number and mRNA message by insertion of 3’ promoter elements, preferably TEF1 promoter or mRNA message stability by adjustment of codon code, copy number, 5’ untranslated region, optimization of targeting sequences, and PRO domains that lead to enhanced activity. Also, this includes evolution or library screening to find variants with enhanced activity.

In one particular embodiment, the present invention is directed to a modified host cell as defined herein capable of retinyl ester formation, wherein formation of retinyl ester is increased during fermentation compared to the formation of retinyl ester using the respective non-modified host cell. As used herein, increased retinyl ester formation means a percentage of at least about 70%, such as e.g. about 75, 80, 85, 90, 95, 98% or more, including 100%, retinyl ester based on total retinoids present in/produced by said modified host cell.

Thus, the present invention is directed to a retinoid-producing modified host cell, particularly retinol-producing host cell, especially a fungal host cell, wherein the percentage of retinyl ester based on the total amount of retinoids produced by said host cell is at least in the range of about 70-90%, such as at least about 70%, such as e.g. about 75, 80, 85, 90, 95, 98% or more, including 100%, as compared to the respective non-modified host cell, and wherein said modification means enhancement or overexpression of endogenous lipase activities, including but not limited to activity corresponding to Yarrowia LIP8 and optionally furthermore to activity corresponding to Yarrowia LIP2 and/or LIP3 and/or LIP4 and/or TGL1 and/or LIP16 and/or LIP17 and/or LIP18, particularly in combination with enhanced activity of commercially available lipase compositions comprising Candida rugosa and/or Candida cylindracea lipases as known in the art as defined herein.

The host cell to be modified according to the present invention might be selected from oleaginous yeast, such as, e.g. Yarrowia lipolytica as disclosed in W02019/058001 or WO2019/057999, wherein the formation of retinyl ester from beta-carotene is optimized via heterologous expression of beta-carotene oxidases (BCO), retinol dehydrogenase (RDH) and/or lipase (LIP). Particularly, a modified host cell as defined herein might be expressing a BCO originated from Drosophila melanogaster, RDH originated from Homo sapiens or Yarrowia, and overexpressing enzymes with activities equivalent to Yarrowia LIP2, and/or LIP3, and/or LIP4, and/or LIP8, and/or TGL-1, and/or LIP16, and/or LIP17, and/or LI PI 8, and/or heterologous lipase enzymes originated from Candida, Aspergillus, Thermomyces or Rhizopus, such as e.g. via use of commercially available lipase compositions. To enhance the conversion of beta-carotene into retinal into retinol and/or retinyl acetate into retinyl ester produced by the host cell as defined herein, said enzymes might comprise one or more mutations leadingto improved conversion of retinol into retinyl ester.

Introduction of modification(s) in the retinoid-producing host cell, particularly retinol-producing host cell, in order to produce more or increased copies of genes and/or proteins, such as lipases and respective genes as defined herein, including generation of modified suitable host cell capable of retinyl ester formation as defined herein with increased activity in enzymes corresponding to Yarrowia LIP8, particularly further combined with increased activity in enzyme(s) correspondingto Yarrowia LIP2 and/or LIP3 and/or LIP4 and/or TGL1 and/or LI PI 6 and/or LIP17 and/or LIP18, together with Candida rugosa and/or Candida cylindracea lipases as known in the art may include the use of strong promoters, or the introduction of one or more mutation(s) (e.g. multiple copies, cis element enhancement or codon optimization, increased copy number) of (parts of) the respective enzymes (as described herein), in particular its regulatory elements, leading to overexpression or addition of said enzyme activity, such as e.g. activation via in vivo mutagenesis, for example by mutation of the catalytic residues or by making mutations or deletions that accentuate protein folding or pre- or pro-sequence cleavage needed to activate the lipase upon secretion by the host cell. The skilled person knows how to genetically manipulate or modify a host cell as defined herein resulting in overexpression or addition of such activity, e.g. lipase activity as defined herein. These genetic manipulations include, but are not limited to, e.g. gene replacement, gene amplification, gene disruption, transfection, transformation using plasmids, viruses, transposons or other vectors. An example of such a genetic manipulation may for instance affect the interaction with DNAthat is mediated by the N-terminal region of enzymes as defined herein or interaction with other effector molecules. In particular, modifications leading to enhanced/overexpressed specific enzyme activity may be carried out in functional, such as functional for the catalytic activity, parts of the proteins. Furthermore, enhancement/overexpression of enzyme specific activity might be achieved by contacting said enzymes with specific activators or other substances that specifically interact with them. In order to identify such activators, the respective enzymes, such as e.g. certain lipases as defined herein, may be expressed and tested for activity in the presence of compounds suspected to enhance their activity.

The generation of a mutation into nucleic acids or amino acids, i.e. mutagenesis, may be performed in different ways, such as for instance by random or side- directed mutagenesis, physical damage caused by agents such as for instance radiation, chemical treatment, or insertion of a genetic element. The skilled person knows how to introduce mutations. Further enhanced mutations can be selected using modern computer systems and Al algorithms. A modified host cell capable of retinyl ester production according to the present invention might comprise further modifications including overexpression or addition of further lipase or esterase activities present in said host cell as long as they result in increasing the percentage of retinyl ester based on the total retinoids produced in fermentation as defined herein without compromisingthe growth of such modified host cell.

Thus, the present invention furthermore includes a process for identification of endogenous lipases or heterologous lipase activities to be modified, such as e.g. via overexpression or addition of the specific enzyme activity, including lipases with activities correspondingto Yarrowia LIP8 and/or LIP2 and/or LIP3 and/or LIP4 and/or TGL and/or LI PI 6 and/or LIP17 and/or LIP18, comprising the step of over-expressing the respective genes one by one in a suitable host cell, such as e.g. retinol-producing host cell, to see if that results in increased FARE production.

A particular embodiment is directed to a process for the identification of suitable endogenous lipases as defined herein comprising (l) providing an oleaginous yeast capable of retinol production,

(2) selection of lipase enzymes based on sequence homology of at least about 50%, such as e.g. 60, 70, 80, 90, 95, 98 or 100% to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15,

(3) overexpression of selected genes and comparison of retinyl ester percentage based on total retinoids,

(4) selection of genes, wherein overexpression leads to at least a percentage of about 70% FARES based on total retinoids present/produced by said host cell.

The terms "sequence identity", "% identity" or "sequence homology" are used interchangeable herein. For the purpose of this invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest identity". If both amino acid sequences which are compared do not differ in any of their amino acids, they are identical or have 100% identity. With regards to enzymes originated from plants, the skilled person knows plant-derived enzymes might contain a chloroplast targeting signal which is to be cleaved via specific enzymes, such as e.g. chloroplast processing enzymes (CPEs).

In one embodiment, the present invention features the use of a modified host cell as defined herein in a fermentation process for production of retinol and retinyl ester, comprising the step of enzymatic conversion of retinal, particularly with a percentage of at least about 65-90% trans-retinal based on the total amount of retinoids produced by such host cell, via action of suitable retinol dehydrogenases (RDHs), as e.g. exemplified in WO2019/057998, with a retinal to retinol conversion in the range of 90%, including a ratio or retinal to retinol in the range of 1:9, based on total retinoids. Optionally, the FARES are isolated and/or further purified from the fermentation medium. Such process might comprise further steps, such as e.g. enzymatic conversion of beta-carotene into retinal via action of suitable BCOs, preferably BCOs with a selectivity towards formation of trans-retinal, more preferably leading to at least about 65-90% trans-isoforms based on the total amount of retinoids produced by said host cell, such as e.g. exemplified in WO2019/057999. Thus, a preferred process for production of retinol and/or retinyl ester using a modified host cell as defined herein comprises the steps of (1) enzymatic conversion of beta-carotene into retinal via action of suitable BCOs, (2) enzymatic conversion of retinal into retinol via action of suitable RDHs, (3) conversion of retinol into a retinyl ester, and optionally (4) isolation and/or purification of retinol from the fermentation medium. In one aspect, the present invention is directed to a fermentation process using such modified host cell defined herein said host cell being grown on triglyceride oils, like for example vegetable oil, with the proviso that the triglyceride oil or fatty acid is not selected from palmitic acid or palm oil, as carbon source, wherein the formation of retinyl ester from conversion of retinol leads to a percentage of at least about 70%, such as e.g. about 75, 80, 85, 90, 95, 98% or more, including 100%, retinyl ester based on total retinoids present in/produced by said modified host cell.

In one embodiment, carbon sources that are in scope of the present invention are selected from vegetable triglyceride oils or fatty acids with low content of saturated fatty acids, i.e. a percentage of less than about 45%, such as e.g. less than 40, 35, 30, 25, 20, 15% of saturated fatty acids.

In a further embodiment, carbon sources that are in the scope of the present invention are selected from vegetable triglyceride oils or fatty acids with a high content of unsaturated fatty acids, i.e. a percentage of more than 50%, such as e.g. about 55, 60, 65, 70, 75, 80, 85 or 90% and more unsaturated fatty acids based on total fatty acids, particularly a vegetable triglyceride oil or fatty acid with about less than 40%, such as e.g. 35, 30, 25, 20, 15, 10 or less palmitic acid based on total fatty acids. In one particular embodiment, carbon sources that are in the scope of the present invention are selected from vegetable triglyceride oils or fatty acids that are liquid at room temperature, i.e. temperatures of about 25°C.

Preferably the triglyceride oils or fatty acids, particularly vegetable oils, within the scope of the present invention are selected from the group consisting of corn oil, olive oil, peanut oil, safflower oil, soybean oil, sunflower oil, rapeseed oil, canola oil, and flaxseed oil or free fatty acids such as e.g. oleic acid, most preferably selected from sunflower oil, olive oil, corn oil, rapeseed oil or oleic acid.

Further, to make the process economically viable to provide a solution to vitamin A markets it is desirable and within the scope of the present invention to produce retinoids including retinyl esters, wherein the retinoid produced is above about 1% mass fraction of the total fermentation.

Thus, in a particular embodiment, the present invention is directed to a process for production of a product selected from the group consisting of retinol, retinyl esters, vitamin A, and a mix comprising retinol, retinyl esters and vitamin A, wherein said mix comprises at least about 70% retinyl esters based on total retinoids, said process comprising the steps of:

(a) providing a retinoid-producing, particularly retinol-producing, host cell capable of formation of retinyl esters, (b) introduction of one or more modification(s) into the genome of said host cell, such as modification(s) into enzyme(s) belonging to the EC class 3.1.1. - having lipase activity, such as e.g. increasing/overexpressing the enzyme activity including but not limited to overexpression of the respective endogenous genes or of suitable heterologous genes as defined herein or via addition of lipase activities, particularly lipase compositions comprising lipases such as originated from Candida rugosa or Candida cylindreacea, particularly overexpression of lipase activity corresponding to Yarrowia LIP2, LIP3, LIP4, LIP8 or combinations thereof and optionally further overexpressing enzyme activity corresponding to Yarrowia TGL1 and/or LIP16 and/or LIP17 and/or LIP18, wherein the modified host cell is (still) able to grow on triglyceride oils or free fatty acids, such as e.g. vegetable oils as defined herein;

(c) optionally introduction of further modification(s) comprising expression of one or more copies of (heterologous) enzymes involved in retinal, retinol, retinyl esters production as known to a person skilled in the art, (d) cultivation of such modified host cell under suitable conditions resulting in formation of retinol and retinyl esters via lipase-catalyzed conversion of retinol into retinyl esters, wherein the modified host cell is grown on triglyceride oils or fatty acids, such as e.g. vegetable oil, as carbon source as defined herein, with the proviso that this is not palmitic acid or palm oil; and (e) optionally isolation and/or further purification of retinyl esters from the cultivation (fermentation) medium.

In one particular preferred embodiment, the fermentation process according to the present invention is performed in the presence of corn oil with an overlay of Isopar, particularly IsoparM, preferably using 1-10% corn oil, such as 2, 3, 4, 5% corn oil and 10 to 50% IsoparM, such as 15, 20, 25, 30% IsoparM.

A product comprising retinyl esters as defined herein obtained via such process might be further used in formulations for food, feed, cosmetic or pharma applications as used in the art.

The modified host cell as defined herein may be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person for the different host cells, including the presence of triglyceride oils or free fatty acids as defined herein, such as e.g. vegetable oil with the proviso that this is not palmitic acid or palm oil, as carbon source. The cultivation/growth of the host cell may be conducted in batch, fed- batch, semi-continuous or continuous mode. Depending on the host cell, preferably, production of retinoids such as e.g. vitamin A and precursors such as retinal, retinol, retinyl esters can vary, as it is known to the skilled person. Cultivation and isolation of beta-carotene and retinoid-producing host cells selected from Yarrowia is described in e.g. W02008/042338.

Depending on the carbon source, i.e. triglyceride oil or free fatty acid, with the proviso that this is not palmitic acid or palm oil, the retinyl ester formed via the process according to the present invention might be selected from retinyl caprylate, retinyl caprate, retinyl laurate, retinyl mysterate, retinyl myristate, retinyl palmitoleate, retinyl sterate, retinyl oleate, retinyl linolate, retinyl linolenate, retinyl arachidinoate, retinyl behenate, retinyl erutate, retinyl lignocerate and retinyl cerotate. Also other natural oxidized forms of aforementioned forms with additional cis double bonds such as omega 3 and omega 6 fatty acids like retinyl eicosapentaenoate and retinyl docosahexaenoic are in the scope of the present invention.

"Retinoids" or a "retinoid-mix" as used herein include vitamin A, precursors and/or intermediates of vitamin A such as beta-carotene cleavage products also known as apocarotenoids, including but not limited to retinal, retinoic acid, retinol, retinoic methoxide, retinyl acetate, retinyl fatty esters, 4-keto-retinoids, 3 hydroxy-retinoids or combinations thereof. Biosynthesis of retinoids is described in e.g. W02008/042338. A host cell capable of production of retinoids in e.g. a fermentation process is known as "retinoid-producing host cell". The genes of the vitamin A pathway and methods to generate retinoid-producing host cells are known in the art (see e.g. W02019/058000), including but not limited to beta-carotene oxidases or retinol dehydrogenases. Suitable beta- carotene oxidases leading to high percentage of trans-retinal are described in e.g. WO2019/057999. A "retinol-producing host cell" as used herein is expressing suitable enzymes catalyzing the conversion of retinal into retinol. A "retinyl ester-producing host cell" as used herein is expressing suitable lipases as defined herein catalyzing the conversion of retinol together with suitable triglyceride oils or free fatty acids as defined herein into retinyl esters. The terms "fatty acids" and "free fatty acids" in connection with specification of the carbon source are interchangeably used herein. "Retinyl fatty esters" or "retinyl esters" or "FARES" as used herein includes long chain retinyl esters. These long chain retinyl esters define hydrocarbon esters that consists of at least about 8, such as e.g. 9, 10, 12, 13, 14, 15, 16, 18, or 20 carbon atoms and up to about 26, such as e.g. 25, 22, 21 or less carbon atoms, with preferably up to about 6 unsaturated bonds, such as e.g. 0, 1, 2, 4, 5, 6 unsaturated bonds with the exclusion of 16:0 retinyl palmitate that is the current industrial form for human health.

"Vitamin A" as used herein may be any chemical form of vitamin A found in aqueous solutions, in solids and formulations, and includes retinol, retinyl acetate and retinyl esters. It also includes retinoic acid, such as for instance undissociated, in its free acid form or dissociated as an anion.

"Retinal" as used herein is known under lUPAC name (2E,4E,6E,8E)-3,7-Dimethyl- 9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenal. It includes both cis- and trans-isoforms, such as e.g. 11-cis retinal, 13-cis retinal, trans-retinal and all- trans retinal. For the purpose of the present invention, the formation of trans- retinal is preferred, which might be generated via the use of stereoselective beta-carotene oxidases, such as described in e.g. WO2019/057999.

Conversion according to the present invention is defined as specific enzymatic activity, i.e. catalytic activity of enzymes described herein, including but not limited to the enzymatic activity of lipases, in particular (endogenous) enzymes belonging to the EC class 3.1.1. - involved in conversion of retinol into retinyl fatty esters.

With regards to the present invention, it is understood that organisms, such as e.g. microorganisms, fungi, algae, or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). Thus, for example, strain Lachancea mirantina is a synonym of strain Zygosaccharomyces sp. IFO 11066, originated from Japan. The following examples are illustrative only and are not intended to limit the scope of the invention in anyway. The contents of all references, patent applications, patents, and published patent applications, cited throughout this application are hereby incorporated by reference, particularly W02019/058001 or WO2019/057999, W02019/058000, W02008/042338, WO2019/057998, W02020/141168 or WO2016/172282. Examples

Example 1: General Methods and Strains

All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998). All genetic manipulations exemplified were performed in Yarrowia lipolytica.

Shake plate assay. Typically, 200pl of 0.075% Yeast extract, 0.25% peptone (0.25X YP) is inoculated with 10mI of freshly grown Yarrowia and overlaid with 200mI of Drakeol 5 (Penreco) mineral oil with either 2% corn oil or 2% oleic acid as a carbon source in Drakeol 5. Additionally, mixtures of free fatty acids and glycerol were used to mimic the hydrolysate of corn oil. Clonal isolates of transformants were grown in 24-well plates (Multitron, 30°C, 800RPM) in above media for 4 days. The mineral oil fraction was removed from the shake plate wells and analyzed by HPLC on a normal phase column, with a photo-diode array detector.

DNA transformation. Strains are transformed by overnight growth on YPD plate media 50mI of cells is scraped from a plate and transformed by incubation in 500mI with 1pg transforming DNA, typically linear DNA for integrative transformation, 40% PEG 3550MW, 100mM lithium acetate, 50mM Dithiothreitol, 5mM Tris-Cl pH 8.0, 0.5mM EDTA for 60 minutes at 40°C and plated directly to selective media or in the case of dominant antibiotic marker selection the cells are out grown on YPD liquid media for 4 hours at 30°C before plating on the selective media. URA3 marker recycling was performed using 5-fluoroorotic acid (FOA). Episomal hygromycin resistance marker (Hyg) plasmids were cured by passage on non-selective media, with identification of Hyg-sensitive colonies by replica plating colonies from non-selective media to hygromycin containing media (100 pg/mL). Selection of the nourseothricin-resistance marker (Nat) was performed on YPD media containing nourseothricin (100 pg/mL). DNA molecular biology. Plasmids MB9523 (SEQ ID NO:17) containing expression systems for DrBCO, and FfRDH, MB9099 (SEQ ID NO:18) containing overexpressed Yarrowia lipolytica LIP8, MB9117 (SEQ ID NO:19) containing overexpressed Yarrowia lipolytica LIP3 and MB9721 (SEQ ID NO:20) for the expression of a chimeric Yarrowia lipolytica LIP2pre-Candida rugosaLIPI protein C; SEQ ID NO:21; SEQ ID NO:24 for CrLIPl), MB9115 (SEQ ID NO:22) containing overexpressed Yarrowia lipolytica LIP2, and MB5082-4 (SEQ ID NO:23) containing overexpressed Yarrowia lipolytica LIP4 were synthesized at Genscript (Piscataway, NJ, USA). All plasmids contain the URA3 for marker selection in Yarrowia lipolytica transformations and are based on the empty plasmid MB5082 (SEQ ID NO:25). For clean gene insertion by random non-homologous end joining of the gene and marker Hindlll/Xbal (MB9721) or Sfil (MB9523), plasmid fragments of interest were purified by gel electrophoresis and Qiagen gel purification column. Clones were verified by sequencing. Typically, genes are synthesized by a synthetic biology at GenScript (Piscataway, NJ). Plasmid list. Plasmid, strains, nucleotide and amino acid sequences to be used are listed in Table 1, 2, and the sequence listing. In general, all non-modified sequences referred to herein are the same as the accession sequence in the database for reference strain CLIB122 (Dujon B, et al, Nature. 2004 Jul 1;430(6995):35-44). Table 1: list of plasmids used for construction of the strains for overexpression of the respective genes indicated as "Insert". "Yl" means Yarrowia lipolytica, "Cr" means Candida rugosa, "BCO" means beta-carotene oxidase and "RDH" means retinol-dehydrogenase. LIP2, 3, 4 and 8 are from Yarrowia lipolytica. For more details, see text.

Table 2: list of Yarrowia lipolytica strains used. Construction of ML17544 is described in Table 2 of W02020/141168. For more details, see text.

Retinoid quantification. Analysis of retinoids were carried out with a C4 reverse phase retinoid method (see below) and C18 as described elsewhere (W02020/141168). The addition of all added intermediates gives the total amount of retinoids.

Fermentation conditions. Fermentations were identical to the previously described conditions using Isopar M overlay and stirred tank in a bench top reactor with 0.5L to 5L total volume (see WO2016/172282, Ex. 5 and 6 but with a different oil), however, they were vegetable oil fed. Typically corn oil was used but other oils can be used to specify the fatty acid side chain distribution in the final retinyl esters, since the oils are assimilated by Yarrowia and delivered to the retinoids by the action of native Yarrowia lipase. Generally, the same results were observed with a fed-batch stirred tank reactor with an increased productivity, which demonstrated the utility of the system to produce retinoids. Preferably, fermentations were batched with 6% glucose and 20% Isopar M and feed was added to achieve 20% dissolved oxygen throughout the feeding program. Fermenters were harvested and compared at 138hrs. In some exemplifications mineral and silicone oils can be used as necessary to specify the nature of the final product. C4 reverse phase chromatography. For exact determination of discrete retinoids the long run reverse phase system was used. Analytes were separated at 230nm and 325nm through the Agilent 1290 instrument with YMC Pro C4, 150 x 3.0mm 3um column (YMC America, Allentown PA) stationary phase, and a 5pl injection loop volume and column and sample tray controlled at 23°C with gradients described in Table 3B. Analytes were detected at 230nm and 325nm and verified the peaks identity with LCMS. The analytes separated as discrete peaks were assigned according to Table 3A.

Table 3A: list of analytes using C4-reverse phase method. The addition of all added intermediates gives the total amount retinoids. "RT" means retention time. For more details, see text.

Table 3B: UPLC Method Gradient with solvent A (acetonitrile); solvent B (water); solvent C (water/acetonitrile/methanesulfonic acid 1000:25:1). For more details, see text.

Method Calibration. Method is calibrated using high purity retinyl acetate received from DSM Nutritional Products, Kaiseraugst, CH. Retinols and retinal are quantitated against retinyl acetate. Dilutions described in Table 3C are prepared as follows. 40 mg of retinyl acetate is weighed into a 100 mL volumetric flask, and dissolved in ethanol, yielding a 400 pg/mL solution. This solution is sonicated as required to ensure dissolution. 5mL of this 400 pg/mL solution is diluted into 50 mL (1/10 dilution, final concentration 40pg/mL), 5mL into 100mL (1/20 dilution, final concentration 20pg/mL), 5mL of 40pg/mL into 50mL (1/10 dilution, final concentration 4pg/mL), 5mL of 20 pg/mL into 50mL (1/10 dilution, 2pg/mL), using 50/50 methanol/ methyl tert-butyl ether(MTBE) as the diluent. All dilutions are done in volumetric flasks. Purity of retinyl acetate is determined by further diluting the 400 mg/mL stock solution 100-fold (using a 2 mL volumetric pipet and a 200 mL volumetric flask) in ethanol. Absorbance of this solution at 325nm using ethanol is taken as the blank, with adjustment of the initial concentration using the equation (Abs * dilution (100) * molecular weight (328.5)/51180 = concentration in mg/mL). Because of quick out- maximization of UV absorbance of retinyl acetate, lower concentrations are better. Consequently, lower concentrations might be better. Retinyl palmitate can also be used as retinyl ester calibration. The separation of the retinyl esters occurs on the column with the longer chain sticking to the C4 mobile phase resulting in a longer retention time and a C+2 ladder of elution on the column.

Table 3C: preparation of calibration standards. [RA] means retinyl acetate. For more explanation, see text.

Samole preparation. Top second phase layer samples from each strain were diluted at a 25-fold dilution or higher into tetrahydrofuran (THF). Fermentation whole broth was prepared using a 2 mL Precellys (Bertin Corp, Rockville, MD) tube, add 25pl of well mixed broth and 975 mΐ of THF. Precellys 3x15x7500 rpm for two cycles with a freeze at -80°C for 10 minutes between cycles. Cell debris was spun down via centrifugation for 1 minute at 13000 rpm. These samples were diluted 10-fold in THF.

Example 2: Addition of heterologous lipases and overexpressed lipases to Yarrowia lipolytica

Commercially available lipases from Candida rugosa (CrLIP Sigma), Candida cyUndracea (CcLIP, Creative Enzymes), Rhizopus niveus (RnLIP, Sigma), and Rhizopus oryzae (RoLIP, Creative enzymes) and others were added to strain

ML18812 and inoculated to 0.25xYEP medium with 2% corn oil as the sole carbon source (Table 4A). Commercially produced Cc and Cr lipase preparations (Creative Enzymes and Sigma Aldrich) are typically a mixture of several lipases natively expressed by Candida rugosa/ Candida cylindracea.

Commercial enzyme preparations were added to the fermentation as directed by the manufacturer. Since the activity of the enzyme preparations are variable we titrated the enzyme starting at a 10mg/ml solution and making tenfold dilutions of these into the final fermentation volume. We report only the dilution with maximal activity for clarity. Empty is the negative control with no insert in the plasmid. Table 4A: retinoid production in Yarrowia lipolytica strain ML18812 ("LIP+") as control with or without addition of the respective lipase. "Total retinoids" is the percentage of retinoids produced compared to total retinoids in the control without addition of lipases ("none"), wherein the total retinoids obtained with the control are set to 100%. "%esters" is the percentage of FARES produced compared to FARE in the control without addition of lipases and which is set to 100%. "SA" means Sigma Aldrich, "Novo" means Novozymes, "Unit" means lipase Unit added per well. For more explanation, see text.

To test the influence of endogenous lipases on production of retinoids in a suitable Yarrowia host, overexpression experiments were carried out, wherein only 1 gene at the time was overexpressed. Lipases were overexpressed as described above (Example 1). Native Yarrowia lipase genes were synthesized and sequence verified by GenScriptthen cloned into the Nhel and Mlul sites of MB5082. The genes are TEF1 promoter driven that allows selection for by complementation of an uracil auxotroph strain (ura3). In some instances the integrated plasmid was cured for the URA3 by FOA treatment and serial addition of multiple lipases were added.

Plasmids containing the respective lipase genes cleaved by Xbal/Hindlll were transformed into retinoid producing strain ML18812 carrying the wild-type lip8 gene (see Example 1) and selected for uracil prototrophy. Clonal isolates of transformations were grown for four days in 0.25X Yeast/ Peptone (YP) with 2% corn oil as a carbon source and a 20% IsoparM oil overlay in the standard shake plate assay and assayed by the previously described UPLC analytical method. At least two individual clonal isolates of transformed Yarrowia strains were tested by shake plate and measured by UPLC assay. The result is depicted in Table 4B, showing the increase in total retinoids and total FARE due to the overexpressed lipase. Best performance on accumulation of retinyl fatty esters and conversion of retinol is achieved with overexpression of LIP8.

Table 4B: Retinoid production in Yarrowia lipolytica strain ML18812 ("LIP+") as control, i.e. insert = none, or cured of marker and transformed with specific plasmid expressing the respective Yarrowia LIP-gene. Column definitions are the same as Table 4A, sequences according to the sequence listings. For more explanation, see text.

Example 3: Feed oil defines retinyl ester forms created by lipase To test the influence of feed oils on production of retinoids in a suitable

Yarrowia retinoid fermentation, addition of various feed oils was combined with overexpression and addition of heterologous lipases. We found that the feed oil could be used to control the form of the retinyl ester. If we feed medium chain triglyceride (MCT) oils containing 6-12 carbons in length we found that the retinyl ester contains those fatty acids that were defined in the feed. Since maximal growth of Yarrowia was not maintained on the feed oil oleic or corn oil growth was used to generate biomass that can be finished with the MCT oil to create retinyl-medium chain esters. Conversely, if we feed vegetable oil that contains primarily triglycerides with C16 and C18 oils we find that those oils are also contained within the retinyl esters produced in the fermentation. Further the chain length preference that is observed in some lipases (Quaglia et al., PLOS One 14(l):e0210100; 2019) can be used to enrich desired chain length using triglyceride of specific chain length /unsaturation and lipase that promotes the specific transesterification of the desired fatty acid. With the Li p8 overexpression we found that ethyl esters of PUFA oils containing EPA, DPA and DHA could be assimilated in the Yarrowia derived retinyl esters.

Claims

1. A retinoid-producing host cell capable of fatty acid retinyl ester (FARE) formation, particularly retinol-producing host cell, such as fungal host cells, preferably oleaginous yeast cell such as e.g. Yarrowia, comprising one or more genetic modification(s), such as overexpression of endogenous and/or heterologous genes expressing lipase activities, preferably overexpression of endogenous enzymes belonging to EC class 3.1.1. -, more preferably comprising overexpression of genes encoding enzymes with lipase activity corresponding to Yarrowia lipolytica LIP2, LIP3, LIP4, LIP8 and combinations thereof.

2. The host cell according to claim 1, further comprising overexpression of genes encoding enzymes with lipase activity corresponding to Yarrowia lipolytica TGL-1, LIP16, LIP17, LIP18 and combinations thereof or heterologous genes expressing lipases originated from Candida, Aspergillus, Thermomyces and/or Rhizopus. 3. The host cell according to claim 1 or 2, wherein the enzymes with lipase activity are selected from the group consisting of protein(s) with at least about 50%, such as 60, 70, 80, 90, 95, 98, or 100% identity to Yarrowia lipolytica LIP2, LIP3, LIP4, TGL1, LIP16, LIP17, LIP18 according to SEQ ID NOs:1,

3, 5, 7, 9, 11, 13 or 15.

4. The host cell according to any one of claims 1 to 3, comprising heterologous expressed lipases from Candida rugosa and/or Candida cylindracea.

5. The host cell according to any one of claims 1 to 4 selected from the group consisting of Rhodosporidium, Lipomyces, Candida, and Yarrowia, preferably Yarrowia, more preferably Yarrowia lipolytica.

6. The host cell according to any one of claims 1 to 5 used in a process for production of retinyl esters, with the proviso that the ester is not selected from retinyl palmitate and wherein the percentage of retinyl esters based on total retinoids present or produced by the host cell is at least about 70%.

7. The host cell according to claim 6, wherein the production of retinyl esters is conducted in the presence of triglyceride oils of fatty acids, particularly vegetable oils, as carbon source, with the proviso that the carbon source is not selected from palm oil or palmitic acid.

8. A process for the production of retinyl esters in a fermentation with a host cell according to any one of claims 1 to 7, comprising cultivating said host cell under suitable culture conditions to allow formation of retinol and retinyl esters, wherein said host cell is grown on triglyceride oils or fatty acids as carbon source, with the proviso that the carbon source is not palm oil or palmitic acid and wherein the percentage of retinyl esters based on total retinoids is at least about 70%.

9. The process according to claim 8, wherein the carbon source is selected from vegetable triglyceride oils or fatty acids with a percentage of less than about 45% of saturated fatty acids.

10. The process according to claim 8 or 9, wherein the carbon source is selected from vegetable triglyceride oils or fatty acids with a percentage of more than about 50% of unsaturated fatty acids.

11. The process accordingto any one of claims 8 to 10, wherein the carbon source is selected from vegetable triglyceride oils or fatty acids that are liquid at room temperature.

12. The process according to any one of claims 8 to 11, wherein the carbon source is selected from the group consisting of corn oil, olive oil, peanut oil, safflower oil, soybean oil, sunflower oil, rapeseed oil, canola oil, flaxseed oil and oleic acid, preferably selected from sunflower oil, olive oil, corn oil, rapeseed oil or oleic acid.

13. The process according to any one of claims 8 to 12, wherein the retinyl esters are isolation and/or further purified from the cultivation (fermentation) medium.

14. A product comprising retinyl esters obtainable by a process accordingto any one of claims 8 to 13 for preparation of formulations for food, feed, pharma or cosmetic applications.