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EP4409017A1 - Levure génétiquement modifiée du genre yarrowia pouvant produire de la vitamine a - Google Patents

Levure génétiquement modifiée du genre yarrowia pouvant produire de la vitamine a

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Publication number
EP4409017A1
EP4409017A1 EP21800999.1A EP21800999A EP4409017A1 EP 4409017 A1 EP4409017 A1 EP 4409017A1 EP 21800999 A EP21800999 A EP 21800999A EP 4409017 A1 EP4409017 A1 EP 4409017A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
amino acid
activity
acid sequence
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21800999.1A
Other languages
German (de)
English (en)
Inventor
Tilen KONTE
Martin KAVSCEK
Tadej MARKUS
Mirjan Svagelj
Stefan Fujs
Mingan SHI
Yunchong XIA
Xiangyu SUN
Jing ZUO
Jia Sun
Zhigang Cai
Guoying Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chifeng Pharmaceutical Co Ltd
Original Assignee
Chifeng Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chifeng Pharmaceutical Co Ltd filed Critical Chifeng Pharmaceutical Co Ltd
Publication of EP4409017A1 publication Critical patent/EP4409017A1/fr
Pending legal-status Critical Current

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    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention generally relates to the biotechnology engineering, and specifically to genetically modified yeast, which allow for the production of vitamin A. More specifically, the present invention provides a genetically modified yeast of the genus Yarrowia capable of producing vitamin A. The present invention also provides methods for the production of vitamin A using a genetically modified yeast of the invention.
  • Vitamin A is a common name for a group of fat-soluble compounds: retinol, retinol aldehyde (retinal) , retinoic acid, and retinyl esters (e.g. acetate and fatty acid esters) .
  • Vitamin A group also includes pro-vitamin A carotenoids (e.g. ⁇ -carotene) .
  • Vitamin A has important physiological functions in human body, including visual function (deficiency can cause Xerophthalmia) , maintenance of skin and mucous membrane integrity, nuclear hormone like effect, maintenance and promotion of immune function, promotion of growth and development, maintenance of reproductive function, anti-cell proliferation, Promote the production of hemoglobin and increase the intake of iron in food etc. Vitamin A deficiency can cause serious physiological diseases and metabolic abnormalities, so it is often used as food fortifier and animal feed additive. In recent years, vitamin A as an effective raw material in cosmetics to improve wrinkles and in the treatment of skin diseases has attracted extensive attention.
  • Vitamin A the global market demand for vitamin A was about 27000 tons, of which about 84%is in the feed field, and about 16%is used in medicine, food and beverage. It is estimated that the global market scale of retinoids (including vitamin A and pro-vitamin A) is about 16 billion US dollars. Vitamin A manufacturers are relatively concentrated, and only DSM, BASF, Zhejiang NHU, ADISSO, KINGDOMWAY and Zhejiang Medicine are the six manufacturers in the world.
  • the molecular structure of vitamin A is composed of 20 carbon atoms, containing a ⁇ -ionone ring, a side chain composed of end-to-end isoprenoid units and an end group, which can be hydroxyl (retinol) , aldehyde (retinal) , carboxylic acid (retinoic acid) , or ester (retinyl ester) bound at the carbon atom 15.
  • the main production methods of vitamin A include biological extraction and chemical synthesis.
  • vitamin A mainly exists in animals but not in plants. Therefore, biological extraction method is based mainly on extraction from liver and other tissues rich in vitamin A. The extraction process is not suitable for large-scale production because of its non-centralized material source and complex separation and purification process. So, the main commercial vitamin A in the market comes from chemical synthesis. Roche and BASF synthesis processes are two main synthetic routes of vitamin A in the world. Roche uses C14+C6 process, which uses ⁇ -ionone as the starting material to synthesize vitamin A acetate through Darzens, Grignard, hydrogenation, bromination, dehydrobromination and other reactions. The advantages of this process are mature technology and stable yield, but more than 40 kinds of excipients are used.
  • BASF uses C15+C5 synthesis process route, using ⁇ -ionone as starting material and acetylene to carry out Grignard reaction to produce acetylene- ⁇ -ionol, selective hydrogenation to get ethylene ⁇ -ionol, then after witting reaction, under the catalysis of sodium alcohol, it condenses with C5 aldehyde to produce vitamin A acetate; this process route has few steps and high yield, but the reaction conditions are high and toxic chemicals like Phosgene are used, so it has high requirements for process equipment and is difficult to be realized.
  • CarB phytoene dehydrogenase gene
  • carRP bifunctional phytoene synthase/lycopene cyclase gene
  • MVA mevalonate pathway related enzymes GGS1, ERG3 and HMG.
  • ⁇ -carotene is the precursor of vitamin A synthesis. It can be converted into retinal by ⁇ -carotene oxygenase. Retinal can be converted into retinol by retinol dehydrogenase and retinol can also be esterified to produce more stable vitamin A esters.
  • Choi et al. [4] constructed the ⁇ -carotene metabolic pathway in E. coli, and then gradually extended to the retinol and retinol palmitate metabolic pathway; the production of retinol palmitate titer reached 69.96 mg/L by fed batch fermentation. Liang Sun et al.
  • [5] introduced a ⁇ -carotene metabolic pathway and ⁇ -carotene15, 15-dioxygenase (BCMO) gene into Saccharomyces cerevisiae, which can use xylose as carbon source, developed the fed batch fermentation strategy with dodecane in situ extraction process and finally the titer of vitamin A reached up to 3.35 g/L in 3L bioreactor.
  • BCMO 15-dioxygenase
  • DuPont (US8846374B2) developed a strain with overexpression of heterologous crtY lycopene cyclase gene) , crtZ (carotenoid hydroxylase gene) , and crtW (carotenoid ketolase gene) in Yarrowia lipolytica, and in which ⁇ -carotene content can account for 72%of the total carotenoids.
  • DSM (US9297031 B2) obtained a ⁇ -carotene producing strain by modifying the heterologous expression of phytoene synthase gene, phytoene dehydrogenase gene and geranylgeranyldiphosphate (GGPP) synthase gene in Yarrowia lipolytica; ⁇ -carotene reached up to 5%of dry cell weight after adopting the fed batch fermentation strategy using glucose and oil as carbon source in 2L bioreactor.
  • GGPP geranylgeranyldiphosphate
  • DSM (CN111107832A, CN111108194A and CN111107834A) further introduced ⁇ -carotene oxidase gene, retinol dehydrogenase gene and acetyl-transferase gene into ⁇ -carotene producing host cells to further synthesize retinal, retinol or retinol ester and improve its single component by proportion and stereoselectivity.
  • the object of the present invention is to provide means allowing a more efficient production of vitamin A. More particularly, it is an object of the present invention to provide means allowing the production of vitamin A at higher nominal yield.
  • engineered yeast strains notably of the genus Yarrowia, having one of more modifications as detailed herein.
  • engineered yeast strains surprisingly show unusually high titers of vitamin A in the fermentation broth.
  • the present invention thus provides in a first aspect a genetically modified yeast of the genus Yarrowia capable of producing vitamin A.
  • the present invention provides a genetically modified yeast of the genus Yarrowia, which a) has been modified to have an increased protein expression of a polypeptide having geranylgeranyl diphosphate synthase activity compared to an otherwise identical yeast that does not carry said modification, b) expresses a heterologous polypeptide having phytoene desaturase (lycopene-forming) activity, c) expresses at least one heterologous polypeptide having 15-cis-phytoene synthase activity, preferably expresses a first heterologous polypeptide having 15-cis-phytoene synthase activity and a second heterologous polypeptide having 15-cis-phytoene synthase activity, d) expresses at least one heterologous polypeptide having lycopene beta-cyclase activity, preferably expresses a first polypeptide having lycopene beta-cyclase activity and a second heterologous polypeptide having
  • the present invention further provides in a second aspect a method for producing vitamin A, comprising cultivating a genetically modified yeast according to invention under suitable culture conditions in a suitable culture medium.
  • the present invention may be summarized by the following items:
  • Genetically modified yeast of the genus Yarrowia preferably capable of producing vitamin A.
  • the genetically modified yeast according to item 1 which is characterized by one or more traits selected from higher biomass production, lower citrate production and lower elongation of cells in bioprocess (less viscosity) compared to Yarrowia lipolytica strain W29.
  • the genetically modified yeast according to item 1 or 2 which is a yeast of the species Yarrowia lipolytica.
  • polypeptide having geranylgeranyl diphosphate synthase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 22; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 22.
  • polypeptide having geranylgeranyl diphosphate synthase activity is selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having geranylgeranyl diphosphate synthase activity.
  • yeast according to any one of items 5 to 7, wherein the increase in protein expression of the polypeptide having geranylgeranyl diphosphate synthase activity is achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding said polypeptide.
  • the genetically modified yeast according to any one of items 1 to 14, which expresses a heterologous polypeptide having phytoene desaturase (lycopene-forming) activity.
  • polypeptide having phytoene desaturase (lycopene-forming) activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 23 to 45; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23 to 45.
  • polypeptide having phytoene desaturase (lycopene-forming) activity is selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 23; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 23.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having phytoene desaturase (lycopene-forming) activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having phytoene desaturase (lycopene-forming) activity, and a transcriptional terminator sequence.
  • the genetically modified yeast according to any one of items 1 to 21, which expresses a first heterologous polypeptide having 15-cis-phytoene synthase activity.
  • the genetically modified yeast according item 22 or 23, wherein the first and/or second polypeptide having 15-cis-phytoene synthase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 46 to 74; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 to 74.
  • the genetically modified yeast according item 22 or 23, wherein the first and/or second polypeptide having 15-cis-phytoene synthase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 60 to 74; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 60 to 74.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said first heterologous polypeptide having 15-cis-phytoene synthase activity.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said second heterologous polypeptide having 15-cis-phytoene synthase activity.
  • the genetically modified yeast according to any one of items 1 to 31, which expresses a first heterologous polypeptide having lycopene beta-cyclase activity.
  • the genetically modified yeast according item 32 or 33, wherein the first and/or second polypeptide having lycopene beta-cyclase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 46 to 59 and 75 to 85;and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 to 59 and 75 to 85.
  • the genetically modified yeast according item 32 or 33, wherein the first and/or second polypeptide having lycopene beta-cyclase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 75 to 85; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 75 to 85.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said first heterologous polypeptide having lycopene beta-cyclase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said first heterologous polypeptide having lycopene beta-cyclase activity, and a transcriptional terminator sequence.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said second heterologous polypeptide having lycopene beta-cyclase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said second heterologous polypeptide having lycopene beta-cyclase, and a transcriptional terminator sequence.
  • the genetically modified yeast according to any one of items 1 to 21, which expresses a first heterologous polypeptide having both 15-cis phytoene synthase and lycopene cyclase activity.
  • the genetically modified yeast according item 42 or 43, wherein the first and/or second polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 46 to 59; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 to 59.
  • the genetically modified yeast according to item 42 or 43, wherein the first polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity is selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 46; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 46.
  • the genetically modified yeast according to any one of items 43 to 45, wherein the second polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity is selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 50; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 50.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said first heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said first heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity, and a transcriptional terminator sequence.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said second heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said second heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity, and a transcriptional terminator sequence.
  • the genetically modified yeast according to any one of items 1 to 52, which expresses a heterologous polypeptide having ⁇ -carotene-15, 15-dioxygenase activity (BLH) .
  • polypeptide having ⁇ -carotene-15, 15-dioxygenase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 86 to 113; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 113.
  • polypeptide having ⁇ -carotene-15, 15-dioxygenase activity is selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 86; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 86.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having ⁇ -carotene-15, 15-dioxygenase activity.
  • the genetically modified yeast according to item 56 wherein the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having ⁇ -caroten-15, 15-dioxygenase activity, and a transcriptional terminator sequence.
  • the genetically modified yeast according to item 56 wherein the exogenous nucleic acid molecule comprises at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having ⁇ -caroten-15, 15-dioxygenase activity, and a transcriptional terminator sequence.
  • the genetically modified yeast according to any one of items 56 to 58, wherein the exogenous nucleic acid molecule comprises at least three transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having ⁇ -caroten-15, 15-dioxygenase activity, and a transcriptional terminator sequence.
  • yeast comprises a first exogenous nucleic acid molecule comprising at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having ⁇ -caroten-15, 15-dioxygenase activity, and a transcriptional terminator sequence; and a second exogenous nucleic acid molecule comprising at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having ⁇ -caroten-15, 15-dioxygenase activity, and a transcriptional terminator sequence.
  • the genetically modified yeast according to items1 to 66 which has been modified to have an increased protein expression of a polypeptide having triacylglycerol lipase activity compared to an otherwise identical yeast that does not carry said modification.
  • polypeptide having polypeptide having triacylglycerol lipase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 114 to 147; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 114 to 147.
  • polypeptide having polypeptide having triacylglycerol lipase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 114; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 114.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having triacylglycerol lipase activity.
  • the genetically modified yeast according to any one of items 71 to 73, wherein the exogenous nucleic acid molecule is stably integrated into the genome of the yeast.
  • yeast according to any one of items 66 to 69, wherein the increase in protein expression of the polypeptide having triacylglycerol lipase activity is achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding said polypeptide.
  • the genetically modified yeast according to item 80 wherein the at least one endogenous gene encoding said at least one polypeptide having diacylglycerol O-acyltransferase activity has been inactivated by deletion of part of or the entire gene sequence.
  • the at least one endogenous gene encoding said polypeptide having diacylglycerol O-acyltransferase activity is the gene DGA1 and/or DGA2.
  • the genetically modified yeast according to items1 to 87 which has been modified to have an increased protein expression of a polypeptide having sterol esterase activity compared to an otherwise identical yeast that does not carry said modification.
  • polypeptide having sterol esterase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 148 to 159; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 148 to 159.
  • polypeptide having polypeptide having sterol esterase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 148; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 148.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having sterol esterase activity.
  • the genetically modified yeast according to any one of items 92 to 94, wherein the exogenous nucleic acid molecule is a vector.
  • yeast according to any one of items 87 to 90, wherein the increase in protein expression of the polypeptide having sterol esterase activity is achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding said polypeptide.
  • the genetically modified yeast according to any one of items 1 to 97, which has been modified to have an increased protein expression of a polypeptide having long-chain fatty acid CoA ligase activity or a polypeptide having medium-chain acyl-CoA ligase activity compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast according to item 98 which has been modified to have an increased protein expression of a polypeptide having long-chain fatty acid CoA ligase activity.
  • the genetically modified yeast according to item 98 or 99, wherein the polypeptide having long-chain fatty acid CoA ligase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 162 to 187; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 162 to 187.
  • polypeptide having long-chain fatty acid CoA ligase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 162; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 162.
  • the genetically modified yeast according to item 98 which has been modified to have an increased protein expression of a polypeptide having medium-chain acyl-CoA ligase activity.
  • polypeptide having medium-chain acyl-CoA ligase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 188 to 197; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 188 to 197.
  • polypeptide having medium-chain acyl-CoA ligase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 188; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 188.
  • polypeptide having long-chain fatty acid CoA ligase activity is a polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity.
  • polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 198 to 212; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 198 to 212.
  • polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 198; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 198.
  • the genetically modified yeast according to any one of items 98 to 107, wherein the increase in protein expression of the polypeptide having long chain fatty acid CoA ligase activity or the polypeptide having medium-chain acyl-CoA ligase activity is achieved by increasing the number of copies of a nucleotide sequence encoding said polypeptide.
  • the genetically modified yeast according to item 108 wherein the increase in the number of copies of the nucleotide sequence encoding said polypeptide is achieved by introducing into the yeast at least one exogenous nucleic acid molecules comprising at least one nucleotide sequence encoding said polypeptide having long chain fatty acid CoA ligase activity or said polypeptide having medium-chain acyl-CoA ligase activity.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having long chain fatty acid CoA ligase activity or said polypeptide having medium-chain acyl-CoA ligase activity.
  • the genetically modified yeast according to any one of items 109 to 111, wherein the exogenous nucleic acid molecule is a vector.
  • the genetically modified yeast according to any one of items 109 to 111, wherein the exogenous nucleic acid molecule is integrated into the genome of the yeast.
  • the genetically modified yeast according to any one of items 98 to 109, wherein the increase in protein expression of the polypeptide having long chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity is achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding said polypeptide.
  • the genetically modified yeast according to any one of items 1 to 114, which has been modified to have an increased protein expression of at least one enzymatically active polypeptide involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is selected from the group consisting of: a polypeptide having hydroxymethylglutaryl-CoA reductase activity, a polypeptide having acetyl-CoA C-acetyltransferase activity, a polypeptide having hydroxymethylglutaryl-CoA synthase activity, a polypeptide having mevalonate kinase activity, a polypeptide having phosphomevalonate kinase activity, a polypeptide having diphosphomevalonate decarboxylase activity, a polypeptide having isopentenyl-diphosphate Delta-isomerase activity, and a polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity.
  • polypeptide having hydroxymethylglutaryl-CoA reductase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 213 to 236; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 213 to 236.
  • polypeptide having hydroxymethylglutaryl-CoA reductase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 213 to 232; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 213 to 232.
  • polypeptide having hydroxymethylglutaryl-CoA reductase activity is selected from the group consisting of: i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 213 or 214 (truncated) (YltHMG) ; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 213 or 214.
  • polypeptide having hydroxymethylglutaryl-CoA reductase activity is selected from the group consisting of: i) a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 213; and ii) a polypeptide comprising, or consisting of, an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 213.
  • polypeptide having hydroxymethylglutaryl-CoA reductase activity is selected from the group consisting of: i) a polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO: 214; and ii) a polypeptide comprising, or consisting of, an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 214.
  • polypeptide having acetyl-CoA C-acetyltransferase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 237 to 246; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 237 to 246.
  • polypeptide having acetyl-CoA C-acetyltransferase activity is selected from the group consisting of:i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 237; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 237.
  • the genetically modified yeast according to any one of items 116 to 124, wherein the polypeptide having hydroxymethylglutaryl-CoA synthase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 247 to 256; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 247 to 256.
  • the genetically modified yeast according to any one of items 116 to 124, wherein the polypeptide having hydroxymethylglutaryl-CoA synthase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 247; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 247.
  • polypeptide having mevalonate kinase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 257 to 266; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 257 to 266.
  • the genetically modified yeast according to any one of items 116 to 126, wherein the polypeptide having mevalonate kinase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 257; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 257.
  • polypeptide having phosphomevalonate kinase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 267 to 276; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 267 to 276.
  • the genetically modified yeast according to any one of items 116 to 128, wherein the polypeptide having phosphomevalonate kinase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 267; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 267.
  • polypeptide having diphosphomevalonate decarboxylase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 277 to 286; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 277 to 286.
  • polypeptide having diphosphomevalonate decarboxylase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 277; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 277.
  • the genetically modified yeast according to any one of items 116 to 132, wherein the polypeptide having isopentenyl-diphosphate Delta-isomerase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 287 to 296; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 287 to 296.
  • polypeptide having isopentenyl-diphosphate Delta-isomerase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 287; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 287.
  • the genetically modified yeast according to any one of items 116 to 134, wherein the polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 297 to 306; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 297 to 306.
  • polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 297; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of SEQ ID NO: 297.
  • the genetically modified yeast according to any one of items 115 to 136, wherein the increase in protein expression of the at least one enzymatically active polypeptide involved in the mevalonate pathway is achieved by increasing the number of copies of a nucleotide sequence encoding said polypeptide.
  • yeast comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said at least one enzymatically active polypeptide involved in the mevalonate pathway.
  • the genetically modified yeast according to item 138 or 139 wherein the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said at least one enzymatically active polypeptide involved in the mevalonate pathway, and a transcriptional terminator sequence.
  • the genetically modified yeast according to any one of items 138 to 140, wherein the exogenous nucleic acid molecule is a vector.
  • the genetically modified yeast according to any one of items 138 to 140, wherein the exogenous nucleic acid molecule is integrated into the genome of the yeast.
  • the genetically modified yeast according to any one of items 115 to 136, wherein the increase in protein expression of the at least one enzymatically active polypeptide involved in the mevalonate pathway is achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding said polypeptide.
  • Method for producing vitamin A comprising cultivating a genetically modified yeast according to any one of items 1 to 143 under suitable culture conditions in a suitable culture medium.
  • Vitamin A obtainable by the method of item 144 or 145.
  • Figure 1 Strain lineage development of genetically modified Y. lipolytica capable of producing vitamin A.
  • A Lineage of Y. lipolytica W29 wild type strain descendants.
  • B Lineage of Y. lipolytica YB-392 wild type strain descendants.
  • Figure 2 Example of expression cassette with ⁇ -carotene producing GGS1, crtYB and carB genes, Hyg selection marker and zeta integration sequence.
  • Figure 3 Example of two expression cassettes for ⁇ -carotene production with Leu2 selection marker and zeta integration sequence.
  • A GGS1, crtYB and crtI genes
  • B GGS1, crtYB and carB genes.
  • Figure 4 Expression cassette with tHMGR gene, Nat selection marker and zeta integration sequence.
  • Figure 5 Example of expression cassettes for ⁇ -carotene 15, 15’ -dioxygenase genes (A–BLH, B –GfBCO, C –HsBCO) , Ura3 selection marker and zeta integration sequence.
  • Figure 6 Example of expression cassette carrying two genes for ⁇ -carotene-15, 15’ -dioxygenase (BLH) and one gene for HMG-CoA reductase (tHMGR) , Leu2 selection marker and zeta integration sequence.
  • BSH ⁇ -carotene-15, 15’ -dioxygenase
  • tHMGR HMG-CoA reductase
  • Figure 7 Example of expression cassette with Nat selection marker and zeta integration sequence and (A) additional GGS1 and crtYB genes or (B) additional GGS1 and crtI genes.
  • Figure 8 Example of expression cassette with 2x BLH gene, Leu2 selection marker and zeta integration sequence.
  • Figure 9 Example of expression cassette with YlFAA1 and YlTGL4 genes, Nat selection marker and zeta integration sequence.
  • Figure 10 Example of expression cassettes for HMG-CoA reductase genes, Nat selection marker and zeta integration sequence.
  • Figure 11 Production of ⁇ -carotene in YPD60 production medium after 72 hours by VA128 and VA143.
  • VA128 strain is descendant of YB-392 wild type and VA143 strain is descendant of W29 wild type.
  • Figure 12 Production of ⁇ -carotene in YPO100 production medium after 144 hours by VA1749, VA2036 and VA2070, which carry YlGGS1+carB+crtYB, YlGGS1+crtI+crtYB and YlGGS1+carB+carRP expression cassette for ⁇ -carotene biosynthesis, respectively.
  • Figure 13 Production of ⁇ -carotene in YPD60 after 96 hours by VA128 strain with native mevalonate pathway and by VA270 strain with overexpressed truncated HMG-CoA reductase.
  • Figure 14 Production of vitamin A and ⁇ -carotene in YPD60 production medium with different variants of ⁇ -carotene 15, 15'-dioxygenase gene after 72 hours.
  • VA189, VA191 and VA162 carry overexpressed GfBCO, HsBCO and BLH gene, respectively.
  • Figure 15 Production of ⁇ -carotene and vitamin A after 72 hours in YPD60 by ⁇ -carotene producing strain VA436 and by vitamin A producing strain VA588.
  • Figure 16 Production of vitamin A and ⁇ -carotene in YPO100 production medium after 120 hours by VA588 and VA811, which carries additional expression cassette for ⁇ -carotene production.
  • Figure 17 Production of ⁇ -carotene and vitamin A in YPO100 production medium after 120 hours in strain VA1493, which has overexpressed two additional copies of BLH genes, and its predecessor VA811.
  • Figure 18 Production of vitamin A and ⁇ -carotene in YPO100 production medium after 144 hours by VA1493 and VA2116, which is overexpressing native FAA1 and TGL4 genes.
  • Figure 19 Production of vitamin A and ⁇ -carotene in YPO100 production medium after 144 hours.
  • VA1493 is predecessor to VA2190, VA2215 and VA2225, which carry expression cassettes for 3x YlHMG, 3x PmHMG, and 3x RpHMG, respectively.
  • Figure 20 Production of vitamin A and ⁇ -carotene during a bioprocess with strain VA2116.
  • engineered yeast strains which are capable of producing vitamin A at higher nominal yield.
  • engineered yeast strains according to the present invention surprisingly show unusually high titers of vitamin A of in the fermentation broth.
  • the present invention thus provides in a first aspect a genetically modified yeast of the genus Yarrowia capable of producing vitamin A, which comprises one or more of the modifications detailed herein. More particularly, the present invention provides a genetically modified yeast of the genus Yarrowia, which a) has been modified to have an increased protein expression of a polypeptide having geranylgeranyl diphosphate synthase activity compared to an otherwise identical yeast that does not carry said modification, b) expresses a heterologous polypeptide having phytoene desaturase (lycopene-forming) activity, c) expresses at least one polypeptide having 15-cis-phytoene synthase activity, preferably expresses a first heterologous polypeptide having 15-cis-phytoene synthase activity and a second heterologous polypeptide having 15-cis-phytoene synthase activity, d) expresses at least one heterologous polypeptide having lycopene beta
  • the genetically modified yeast of the present invention has been modified to have an increased protein expression of a polypeptide having geranylgeranyl diphosphate synthase activity compared to an otherwise identical yeast that does not carry said modification.
  • “increased protein expression” it is meant that the amount of the polypeptide having geranylgeranyl diphosphate synthase activity produced by the thus modified yeast is increased compared to an otherwise identical yeast that does not carry said modification. More particularly, by “increased expression” it is meant that the amount of the polypeptide having geranylgeranyl diphosphate synthase activity produced by the thus modified yeast is increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%at least about 9000%or at least about 10000%, compared to an otherwise
  • an increase in protein expression may be achieved by any suitable means well-known to those skilled in the art.
  • an increase in protein expression may be achieved by increasing the number of copies of a nucleotide sequence encoding the polypeptide having geranylgeranyl diphosphate synthase activity in the yeast, such as by introducing into the yeast at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having geranylgeranyl diphosphate synthase activity.
  • the genetically modified yeast of the present invention comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having geranylgeranyl diphosphate synthase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having geranylgeranyl diphosphate synthase activity, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule may comprise at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • An increase in protein expression may also be achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding the polypeptide having geranylgeranyl diphosphate synthase activity, e.g. by replacing the native promoter with a promoter that enables higher expression and overproduction of polypeptide compared to the native promoter.
  • strong promoters are given further below.
  • Polypeptides having geranylgeranyl diphosphate synthase activity are encoded in the genomes of a wide range of organisms, including Yarrowia species such as Yarrowia lipolytica.
  • the polypeptide having geranylgeranyl diphosphate synthase activity may be derived from the same species as the yeast in which it is expressed or may be derived from a species different to the one in which it is expressed (i.e. it is heterologous) .
  • the polypeptide having geranylgeranyl diphosphate synthase activity is derived from the same species as the yeast in which it is expressed.
  • the having geranylgeranyl diphosphate synthase activity is derived from a species different from the one in which it is expressed (i.e. it is heterologous) .
  • a polypeptide having geranylgeranyl diphosphate synthase activity for use according to the invention may for instance be a polypeptide having geranylgeranyl diphosphate synthase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 22; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 22.
  • the polypeptide having geranylgeranyl diphosphate synthase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 22.
  • the polypeptide having geranylgeranyl diphosphate synthase activity comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 22.
  • the polypeptide having geranylgeranyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 1. According to some embodiments, the polypeptide having geranylgeranyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 1.
  • the polypeptide having geranylgeranyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 1. According to some embodiments, the polypeptide having geranylgeranyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 1.
  • Geranylgeranyl diphosphate synthase activity can, for example, be determined with the measurements of geranylgeranyl diphosphate accumulation in vitro or in yeast biomass using an enzyme assay and/or a suitable spectrophotometric, HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining geranylgeranyl diphosphate synthase activity has been described, e.g. by Barja and Rodr ⁇ guez-Concep Terms [6] .
  • the genetically modified yeast of the present invention may be (further) modified to express a heterologous polypeptide having phytoene desaturase (lycopene-forming) activity.
  • the genetically modified yeast of the present invention is characterized in that it (further) expresses a heterologous polypeptide having phytoene desaturase (lycopene-forming) activity.
  • polypeptide having phytoene desaturase (lycopene-forming) activity employed according to the invention will be heterologous to the yeast, which means that said polypeptide is normally not found in or made (i.e. expressed) by the yeast, but is derived from a different species.
  • Polypeptides having phytoene desaturase (lycopene-forming) activity are encoded in the genomes of a wide range of organisms, including Mucor species such as Mucor lusitanicus or Mucor circinelloides.
  • a polypeptide having phytoene desaturase (lycopene-forming) activity for use according to the invention may for instance be a polypeptide having phytoene (lycopene-forming) desaturase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 23 to 45; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23 to 45.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises an amino acid sequence, which has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23 to 45.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises an amino acid sequence of any one of SEQ ID NOs: 23 to 45.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 23.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 23.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 23. According to some embodiments, the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 23.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 24.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 24.
  • the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 24. According to some embodiments, the polypeptide having phytoene desaturase (lycopene-forming) activity comprises the amino acid sequence of SEQ ID NO: 24.
  • phytoene desaturase (lycopene-forming) activity can, for example, be determined with the measurements of lycopene accumulation in vitro or in yeast biomass using an enzyme assay and/or a suitable spectrophotometric HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining phytoene desaturase (lycopene-forming) activity has been described, e.g. by Koschmieder et al. [7] .
  • a genetically modified yeast of the present invention may comprise at least one exogenous nucleic acid molecule which comprises at least one nucleotide sequence encoding said polypeptide.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule may comprise at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the genetically modified yeast of the present invention may be (further) modified to expresses at least one heterologous polypeptide having 15-cis-phytoene synthase activity and/or lycopene beta-cyclase activity.
  • the genetically modified yeast of the present invention is characterized in that it (further) expresses at least one heterologous polypeptide having 15-cis-phytoene synthase activity and/or lycopene beta-cyclase activity.
  • the genetically modified yeast of the present invention expresses at least one heterologous polypeptide having 15-cis-phytoene synthase activity.
  • polypeptide having 15-cis-phytoene synthase activity employed according to the invention will be heterologous to the yeast, which means that said polypeptide is normally not found in or made (i.e. expressed) by the yeast, but is derived from a different species.
  • Polypeptides having 15-cis-phytoene synthase activity are encoded in the genomes of a wide range of organisms.
  • heterologous polypeptide having 15-cis-phytoene synthase activity is expressed in the genetically modified yeast of the present invention, the two or more polypeptides having 15-cis-phytoene synthase activity differ in amino acid composition, and preferably are derived from different species.
  • the genetically modified yeast of the present invention expresses a first heterologous polypeptide having 15-cis-phytoene synthase activity and second heterologous polypeptide having 15-cis-phytoene synthase activity.
  • the genetically modified yeast of the present invention expresses at least one heterologous polypeptide having lycopene beta-cyclase activity.
  • polypeptide having lycopene beta-cyclase activity employed according to the invention will be heterologous to the yeast, which means that said polypeptide is normally not found in or made (i.e. expressed) by the yeast, but is derived from a different species.
  • Polypeptides having lycopene beta-cyclase activity are encoded in the genomes of a wide range of organisms.
  • heterologous polypeptide having lycopene beta-cyclase activity is expressed in the genetically modified yeast of the present invention, the two or more polypeptides having lycopene beta-cyclase activity differ in amino acid composition, and preferably are derived from different species.
  • the genetically modified yeast of the present invention expresses a first heterologous polypeptide having lycopene beta-cyclase activity and second heterologous polypeptide having lycopene beta-cyclase activity.
  • polypeptides having both 15-cis-phytoene synthase and lycopene cyclase activity are encoded in the genomes of a wide range of organisms.
  • the present invention contemplates the expression of at least one heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity.
  • heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity is expressed in the genetically modified yeast of the present invention, the two or more polypeptides having both 15-cis-phytoene synthase and lycopene cyclase activity differ in amino acid composition, and preferably are derived from different species.
  • the genetically modified yeast of the present invention expresses a first heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity and a second heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity.
  • a polypeptide having 15-cis-phytoene synthase activity for use according to the invention may for instance be a polypeptide having 15-cis-phytoene synthase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 46 to 74; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 to 74.
  • a mono-functional polypeptide having 15-cis-phytoene synthase activity for use according to the invention may for instance be a polypeptide having 15-cis-phytoene synthase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 60 to 74; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 60 to 74.
  • a polypeptide having lycopene beta-cyclase activity for use according to the invention may for instance be a polypeptide having lycopene beta-cyclase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 46 to 59 and 75 to 85; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 to 59 and 75 to 85.
  • a mono-functional polypeptide having lycopene beta-cyclase activity for use according to the invention may for instance be a polypeptide having lycopene beta-cyclase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 75 to 85; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 75 to 85.
  • a bi-functional polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity for use according to the invention may for instance be a polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 46 to 59; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 to 59.
  • the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 46.
  • the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 46.
  • the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 46. According to some embodiments, the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46.
  • the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 50.
  • the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 50.
  • the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 50. According to some embodiments, the heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50.
  • the first heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 46
  • the second heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 50.
  • the first heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 46
  • the second heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 50.
  • the first heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 46
  • the second heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 50.
  • the first heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 46
  • the second heterologous polypeptide having both 15-cis-phytoene synthase and lycopene cyclase activity comprises the amino acid sequence of SEQ ID NO: 50.
  • 15-cis-phytoene synthase activity can, for example, be determined with the measurements of 15-cis-phytoene accumulation in vitro or in yeast biomass using an enzyme assay and/or a suitable spectrophotometric, HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining lycopene cyclase activity has been described, e.g. by Popebach et al. [8] .
  • Lycopene cyclase activity can, for example, be determined with the measurements of beta-carotene accumulation in vitro or in yeast biomass using an enzyme assay and/or a suitable spectrophotometric, HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining lycopene cyclase activity has been described, e.g. by Yu et al. [9] .
  • a genetically modified yeast of the present invention may comprise at least one exogenous nucleic acid molecule, such as a DNA molecule, which comprises at least one nucleotide sequence encoding said polypeptide.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule may comprise at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • Retinal forms with cleavage of ⁇ -carotene by the enzyme ⁇ -caroten-15, 15-dioxygenase Surprisingly, the present inventors have found that retinal is converted further by Yarrowia native enzymes to retinol, retinyl esters, dehydroretinol and retinoic acid. Especially, the conversion of retinal to retinol by Yarrowia was unexpected.
  • the genetically modified yeast of the present invention may be (further) modified to express a heterologous polypeptide having ⁇ -carotene-15, 15-dioxygenase activity (BLH) .
  • the genetically modified yeast of the present invention is characterized in that (further) expresses a heterologous polypeptide having ⁇ -carotene-15, 15-dioxygenase activity (BLH) .
  • polypeptide having ⁇ -carotene-15, 15-dioxygenase activity (BLH) employed according to the invention will be heterologous to the yeast, which means that said polypeptide is normally not found in or made (i.e. expressed) by the yeast, but is derived from a different species.
  • Polypeptides having ⁇ -carotene-15, 15-dioxygenase activity (BLH) are encoded in the genomes of a wide range of organisms.
  • a polypeptide having ⁇ -carotene-15, 15-dioxygenase activity for use according to the invention may for instance be a polypeptide having ⁇ -carotene-15, 15-dioxygenase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 86 to 113; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 113.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises an amino acid sequence, which has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 113.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises an amino acid sequence of any one of SEQ ID NOs: 86 to 113.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 86.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 86.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 86. According to some embodiments, the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO: 86.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 100.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 100.
  • the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 100. According to some embodiments, the polypeptide having ⁇ -carotene-15, 15-dioxygenase activity comprises the amino acid sequence of SEQ ID NO:100.
  • ⁇ -carotene-15, 15-dioxygenase activity can, for example, be determined with the measurements of beta-carotene conversion to retinal in vitro or in yeast biomass using an enzyme assay and/or a suitable spectrophotometric, HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining ⁇ -carotene-15, 15-dioxygenase activity has been described, e.g. by Kim et al. [10] .
  • a genetically modified yeast of the present invention may comprise at least one exogenous nucleic acid molecule which comprises at least one nucleotide sequence encoding said polypeptide.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule comprises more than one transcriptional unit.
  • improved production of retinoids was achieved with multiple copies of BLH gene introduced in the yeast strain.
  • a genetically modified yeast of the present invention comprises at least one exogenous nucleic acid molecule which comprises at least two transcriptional unit each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • a genetically modified yeast of the present invention comprises at least one exogenous nucleic acid molecule which comprises at least three transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • retinoids can further be achieved by introducing two or more exogenous nucleic acid molecules, each comprising more than one transcriptional unit comprising a nucleotide sequence encoding a polypeptide having ⁇ -carotene-15, 15-dioxygenase activity.
  • a genetically modified yeast of the present invention comprises at least two exogenous nucleic acid molecules each comprising at least two transcriptional units comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • a genetically modified yeast of the present invention comprises a first exogenous nucleic acid molecule comprising at least two transcriptional units comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence, and a second exogenous nucleic acid molecule comprising at least two transcriptional units comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • a genetically modified yeast of the present invention comprises a first exogenous nucleic acid molecule comprising two transcriptional units comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence, and a second exogenous nucleic acid molecule comprising two transcriptional units comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • a genetically modified yeast of the present invention comprises a first exogenous nucleic acid molecule comprising two transcriptional units comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence, and a second exogenous nucleic acid molecule comprising three transcriptional units comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule (s) may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule (s) may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule (s) may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule (s) may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • exogenous nucleic acid molecules may have been introduced simultaneously or separately.
  • TAGs intracellular storage TAGs as an attractive target.
  • the most important enzymes involved in the first steps of intracellular TAG catabolism are the triacylglycerol lipases, which catalyse the breakdown of TAGs to FFAs, and long-chain fatty acid CoA ligases, which catalyse the activation of free fatty acids to acyl CoA.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have reduced lipid content, such as reduced triacylglycerol content, compared to an otherwise identical yeast that does not carry said modification.
  • reduced lipid content it is meant that the amount of intracellular lipids produced by the thus modified yeast is reduced compared to an otherwise identical yeast that does not carry said modification. More particularly, by “reduced lipid content” it is meant that the amount of triacylglycerols produced by the thus modified yeast is reduced by at least about 10 %, and preferably by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%or 100%, or any percentage, in whole integers between 10%and 100%, compared to an otherwise identical yeast that does not carry said modification.
  • the amount of intracellular lipids in a given cell can be determined by any suitable quantification technique known in the art, such as by adapted folch lipid extraction method described in B
  • a reduction of lipid content may be achieved by reducing the triacylglycerol content within the yeast cell.
  • reduced triacylglycerol content it is meant that the amount of intracellular triacylglycerols produced by the thus modified yeast is reduced compared to an otherwise identical yeast that does not carry said modification. More particularly, by “reduced triacylglycerol content” it is meant that the amount of triacylglycerols produced by the thus modified yeast is reduced by at least about 10 %, and preferably by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%or 100%, or any percentage, in whole integers between 10%and 100%, compared to an otherwise identical yeast that does not carry said modification.
  • the amount of triacylglycerols in a given cell can be determined by any suitable quantification technique known in
  • a reduction of triacylglycerol content may be achieved by increasing the protein expression of a polypeptide having triacylglycerol lipase activity.
  • the genetically modified yeast of the invention has been modified to have an increased protein expression of a polypeptide having triacylglycerol lipase activity compared to an otherwise identical yeast that does not carry said modification.
  • “increased protein expression” it is meant that the amount of the polypeptide having triacylglycerol lipase activity produced by the thus modified yeast is increased compared to an otherwise identical yeast that does not carry said modification. More particularly, by “increased expression” it is meant that the amount of the polypeptide having triacylglycerol lipase activity produced by the thus modified yeast is increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%at least about 9000%or at least about 10000%, compared to an otherwise identical yeast that does not carry
  • an increase in protein expression may be achieved by any suitable means well-known to those skilled in the art.
  • an increase in protein expression may be achieved by increasing the number of copies of a nucleotide sequence encoding the polypeptide having triacylglycerol lipase activity in the yeast, such as by introducing into the yeast at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having triacylglycerol lipase activity.
  • the genetically modified yeast of the present invention comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having triacylglycerol lipase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having triacylglycerol lipase activity, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule may comprise at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • An increase in protein expression may also be achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding the polypeptide having triacylglycerol lipase activity, e.g. by replacing the native promoter with a promoter that enables higher expression and overproduction of polypeptide compared to the native promoter.
  • strong promoters are given further below.
  • Polypeptides having triacylglycerol lipase activity are encoded in the genomes of a wide range of organisms, including Yarrowia species such as Yarrowia lipolytica.
  • the polypeptide having triacylglycerol lipase activity may be derived from the same species as the yeast in which it is expressed or may be derived from a species different to the one in which it is expressed (i.e. it is heterologous) .
  • the polypeptide having triacylglycerol lipase activity is derived from the same species as the yeast in which it is expressed.
  • the having triacylglycerol lipase activity is derived from a species different from the one in which it is expressed (i.e. it is heterologous) .
  • a polypeptide having triacylglycerol lipase activity for use according to the invention may for instance be a polypeptide having triacylglycerol lipase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 114 to 147; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 114 to 147.
  • the polypeptide having triacylglycerol lipase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 114 to 147.
  • the polypeptide having triacylglycerol lipase activity comprises an amino acid sequence of any one of SEQ ID NOs: 114 to 147.
  • the polypeptide having triacylglycerol lipase activity comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 114. According to some embodiments, the polypeptide having triacylglycerol lipase activity comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 114.
  • the polypeptide having triacylglycerol lipase activity comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 114. According to some embodiments, the polypeptide having triacylglycerol lipase activity comprises the amino acid sequence of SEQ ID NO: 114.
  • Triacylglycerol lipase activity can, for example, be determined with the measurements of triacylglycerol content reduction in yeast biomass using a suitable gravimetric, HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining triacylglycerol lipase activity has been described, e.g. by Bhutada et al. [11] .
  • a reduction of lipid content may be achieved by increasing the protein expression of a polypeptide having sterol esterase activity.
  • the genetically modified yeast of the invention has been (further) modified to have an increased protein expression of a polypeptide having sterol esterase activity compared to an otherwise identical yeast that does not carry said modification.
  • “increased protein expression” it is meant that the amount of the polypeptide having sterol esterase activity produced by the thus modified yeast is increased compared to an otherwise identical yeast that does not carry said modification. More particularly, by “increased expression” it is meant that the amount of the polypeptide having sterol esterase activity produced by the thus modified yeast is increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%at least about 9000%or at least about 10000%, compared to an otherwise identical yeast that does not carry said modification.
  • an increase in protein expression may be achieved by any suitable means well-known to those skilled in the art.
  • an increase in protein expression may be achieved by increasing the number of copies of a nucleotide sequence encoding the polypeptide having sterol esterase activity in the yeast, such as by introducing into the yeast at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having sterol esterase activity.
  • the genetically modified yeast of the present invention comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having sterol esterase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide having sterol esterase activity, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule may comprise at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • An increase in protein expression may also be achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding the polypeptide having sterol esterase activity, e.g. by replacing the native promoter with a promoter that enables higher expression and overproduction of polypeptide compared to the native promoter.
  • strong promoters are given further below.
  • Polypeptides having sterol esterase activity are encoded in the genomes of a wide range of organisms, including Yarrowia species such as Yarrowia lipolytica.
  • the polypeptide having sterol esterase activity may be derived from the same species as the yeast in which it is expressed or may be derived from a species different to the one in which it is expressed (i.e. it is heterologous) .
  • the polypeptide having sterol esterase activity is derived from the same species as the yeast in which it is expressed.
  • the having triacylglycerol lipase activity is derived from a species different from the one in which it is expressed (i.e. it is heterologous) .
  • a polypeptide having sterol esterase activity for use according to the invention may for instance be a polypeptide having sterol esterase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 148 to 159; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 148 to 159.
  • the polypeptide having sterol esterase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 148 to 159.
  • the polypeptide having sterol esterase activity comprises an amino acid sequence of any one of SEQ ID NOs: 148 to 159.
  • the polypeptide having sterol esterase activity comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 148. According to some embodiments, the polypeptide having sterol esterase activity comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 148. According to some embodiments, the polypeptide having sterol esterase activity comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 148. According to some embodiments, the polypeptide having sterol esterase activity comprises the amino acid sequence of SEQ ID NO: 148.
  • Sterol esterase activity can, for example, be determined with the measurements of sterol ester content reduction in yeast biomass using a suitable gravimetric, HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining sterol esterase activity has been described, e.g. by Zweytick et al. [12] .
  • a reduction of triacylglycerol content may be achieved by reducing the expression and/or activity of an endogenous polypeptide having diacylglycerol O-acyltransferase activity.
  • the genetically modified yeast of the invention may be modified to have a decreased expression of at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the invention may be modified to have a decreased expression level of the endogenous gene (s) encoding said at least one endogenous polypeptide compared to an otherwise identical yeast that does not carry said modification.
  • the expression level of the endogenous gene may, for example, be decreased by at least 50%, such as by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%or at least 100%compared to the otherwise identical yeast.
  • the endogenous gene (s) encoding said at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity has been inactivated, such as by deletion of part of or the entire gene sequence.
  • the endogenous gene encoding said at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity has been inactivated by introducing or expressing in the yeast a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by a double-strand break, the endogenous gene encoding said polypeptide.
  • a rare-cutting endonuclease to be used in accordance with the present invention to inactivate the endogenous gene may, for instance, be a transcription activator-like effector (TALE) nuclease, meganuclease, zinc-finger nuclease (ZFN) , or RNA-guided endonuclease.
  • TALE transcription activator-like effector
  • the CRISPRi system was developed as a tool for targeted repression of gene expression or for blocking targeted locations on the genome.
  • the CRISPRi system consists of the catalytically inactive, “dead” Cas9 protein (dCas9) and a guide RNA that defines the binding site for the dCas9 to DNA.
  • the endogenous gene encoding said polypeptide is inactivated by introducing or expressing in the yeast an RNA-guided endonuclease, such as a catalytically inactive Cas9 protein, and a single guide RNA (sgRNA) specifically hybridizing (e.g. binding) under cellular conditions with the genomic DNA encoding a said enzyme.
  • an RNA-guided endonuclease such as a catalytically inactive Cas9 protein, and a single guide RNA (sgRNA) specifically hybridizing (e.g. binding) under cellular conditions with the genomic DNA encoding a said enzyme.
  • the expression of said at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity is decreased by way of inhibition.
  • RNAi interfering RNA molecules
  • miRNA microRNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • the expression of said at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity is decreased (e.g., inhibited) by transcriptional and/or translational repression of the endogenous gene encoding said polypeptide.
  • the expression of said at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity is inhibited by introducing or expressing in the yeast an inhibitory nucleic acid molecule.
  • the inhibitory nucleic acid molecule may be introduced by way of an exogenous nucleic acid molecule comprising a nucleotide sequence encoding said inhibitory nucleic acid molecule operably linked to a promoter, such as an inducible promoter, that is functional in the yeast to cause the production of said inhibitory nucleic acid molecule.
  • the inhibitory nucleic acid molecule is one that specifically hybridizes (e.g. binds) under cellular conditions with cellular mRNA and/or genomic DNA encoding the endogenous enzyme. Depending on the target, transcription of the encoding genomic DNA and/or translation of the encoding mRNA is/are inhibited.
  • the inhibitory nucleic acid molecule is an antisense oligonucleotide, ribozyme, or interfering RNA (RNAi) molecule.
  • RNAi interfering RNA
  • such nucleic acid molecule comprises at least 10 consecutive nucleotides of the complement of the cellular mRNA and/or genomic DNA encoding the polypeptide or enzyme of interest (e.g., the cellular mRNA and/or genomic DNA encoding the polypeptide.
  • the inhibitory nucleic acid is an antisense oligonucleotide.
  • antisense oligonucleotide is a nucleic acid molecule (either DNA or RNA) , which specifically hybridizes (e.g. binds) under cellular conditions with the cellular mRNA and/or genomic DNA encoding the polypeptide.
  • the inhibitory nucleic acid molecule is a ribozyme, such as a hammerhead ribozyme.
  • a ribozyme molecule is designed to catalytically cleave the mRNA transcript to prevent translation of the polypeptide.
  • the inhibitory nucleic acid molecule is an interfering RNA (RNAi) molecule.
  • RNA interference is a biological process in which RNA molecules inhibit expression, typically destroying specific mRNA.
  • exemplary types of RNAi molecules include microRNA (miRNA) , small interfering RNA (siRNA) , and short hairpin RNA (shRNA) .
  • miRNA microRNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • the RNAi molecule is a miRNA.
  • the RNAi molecule is a siRNA.
  • the RNAi molecule is a shRNA.
  • the genetically modified yeast of the invention has been modified to have a decreased activity of at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity compared to an otherwise identical yeast that does not carry said modification.
  • a decrease of the activity of the at least one polypeptide having diacylglycerol O-acyltransferase activity may be achieved by any suitable means known in the art.
  • the activity may be decreased by introducing one or more mutations in the active site of the polypeptide resulting in the reduction or loss of activity.
  • the activity of the at least one endogenous polypeptide having diacylglycerol O-acyltransferase activity is decreased by at least one active-site mutation resulting in the reduction or loss of activity.
  • At least one active-site mutation may, for example, be at least one non-conservative amino acid substitution.
  • the endogenous gene encoding said polypeptide having diacylglycerol O-acyltransferase activity is the gene DGA1 and/or DGA2.
  • the at least one polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene is selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 160 and 161; and ii) a polypeptide comprising an amino acid sequence, which has at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 160 and 161.
  • the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 160.
  • the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 160.
  • the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 160 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 160. According to some embodiments, the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 160.
  • the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 161. According to some embodiments, the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 161.
  • the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 161. According to some embodiments, the polypeptide having diacylglycerol O-acyltransferase activity encoded by the endogenous gene comprises the amino acid sequence of SEQ ID NO: 161.
  • the genetically modified yeast of the present invention may be (further) modified to have an increased protein expression of a polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention has been (further) modified to have an increased protein expression of a polypeptide having long-chain fatty acid CoA ligase activity compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention has been (further) modified to have an increased protein expression of a polypeptide having medium-chain acyl-CoA ligase activity compared to an otherwise identical yeast that does not carry said modification.
  • “increased protein expression” it is meant that the amount of the polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity produced by the thus modified yeast is increased compared to an otherwise identical yeast that does not carry said modification.
  • “increased expression” it is meant that the amount of the polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity produced by the thus modified yeast is increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%at least about 9000%or at least about 10000%, compared to an otherwise identical yeast that does not carry said modification.
  • the amount of protein in a given cell can be determined by any suitable quantification technique known in the art, such as ELISA, Immunohistochemistry,
  • an increase in protein expression may be achieved by any suitable means well-known to those skilled in the art.
  • an increase in protein expression may be achieved by increasing the number of copies of a nucleotide sequence encoding the polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity in the yeast, such as by introducing into the yeast at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity.
  • the genetically modified yeast of the present invention comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule may comprise at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • An increase in protein expression may also be achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding the polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity, e.g. by replacing the native promoter with a promoter that enables higher expression and overproduction of polypeptide compared to the native promoter.
  • strong promoters are given further below.
  • Polypeptides having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity are encoded in the genomes of a wide range of organisms, including Yarrowia species such as Yarrowia lipolytica.
  • the polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity may be derived from the same species as the yeast in which it is expressed or may be derived from a species different to the one in which it is expressed (i.e. it is heterologous) .
  • the polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity is derived from the same species as the yeast in which it is expressed. According to some embodiments, the polypeptide having long-chain fatty acid CoA ligase activity or medium-chain acyl-CoA ligase activity is derived from a species different from the one in which it is expressed (i.e. it is heterologous) .
  • a polypeptide having long-chain fatty acid CoA ligase activity for use according to the invention may for instance be a polypeptide having long-chain fatty acid CoA ligase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 162 to 187; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 162 to 187.
  • the polypeptide having long-chain fatty acid CoA ligase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 162 to 187.
  • the polypeptide having long-chain fatty acid CoA ligase activity comprises an amino acid sequence of any one of SEQ ID NOs: 162 to 187.
  • the polypeptide having long-chain fatty acid CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 162. According to some embodiments, the polypeptide having long-chain fatty acid CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 162.
  • the polypeptide having long-chain fatty acid CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 162. According to some embodiments, the polypeptide having long-chain fatty acid CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 162.
  • Long-chain fatty acid CoA ligase activity can, for example, be determined with the measurements of acyl-CoA accumulation in yeast biomass using the in vitro assay and/or a suitable gravimetric, HPLC, LC-MS, or GC-MS method.
  • An exemplary method for determining long-chain fatty acid CoA ligase activity has been described, e.g. by Bhutada et al. [11] .
  • a polypeptide having medium-chain acyl-CoA ligase activity for use according to the invention may for instance be a polypeptide having medium-chain acyl-CoA ligase activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 188 to 197; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 188 to 197.
  • the polypeptide having medium-chain acyl-CoA ligase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 188 to 197.
  • the polypeptide having medium-chain acyl-CoA ligase activity comprises an amino acid sequence of any one of SEQ ID NOs: 188 to 197.
  • the polypeptide having medium-chain acyl-CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 188.
  • the polypeptide having medium-chain acyl-CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 188.
  • the polypeptide having medium-chain acyl-CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 188 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 188. According to some embodiments, the polypeptide having medium-chain acyl-CoA ligase activity comprises the amino acid sequence of SEQ ID NO: 188.
  • Medium-chain fatty acid CoA ligase activity can, for example, be determined with an in-vitro test of inocorporation of medium chain fatty acies CoAs into triglycerides.
  • An exemplary method for determining medium-chain fatty acid CoA ligase activity has been described, e.g. by Knoll et al. [13] .
  • the polypeptide having long-chain fatty acid CoA ligase activity may also be a polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity.
  • Polypeptides having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity are encoded in the genomes of a wide range of organisms, including Yarrowia species such as Yarrowia lipolytica.
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity may be derived from the same species as the yeast in which it is expressed or may be derived from a species different to the one in which it is expressed (i.e. it is heterologous) .
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity is derived from the same species as the yeast in which it is expressed.
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity is derived from a species different from the one in which it is expressed (i.e. it is heterologous) .
  • a polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity for use according to the invention may for instance be a polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity selected from the group consisting of: i) a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 198 to 212; and ii) a polypeptide comprising an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 198 to 212.
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 198 to 212.
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity comprises an amino acid sequence of any one of SEQ ID NOs: 198 to 212.
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity comprises the amino acid sequence of SEQ ID NO: 198 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 198.
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity comprises the amino acid sequence of SEQ ID NO: 198 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 198.
  • the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity comprises the amino acid sequence of SEQ ID NO: 198 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 198. According to some embodiments, the polypeptide having both long-chain fatty acid CoA ligase activity and long-chain fatty acid transporter activity comprises the amino acid sequence of SEQ ID NO: 198.
  • the genetically modified yeast of the present invention may be (further) modified to have an increased protein expression of at least one enzymatically active polypeptide involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of at least one enzymatically active polypeptide involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of at least two enzymatically active polypeptides involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of at least three enzymatically active polypeptides involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of at least four enzymatically active polypeptides involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of at least five enzymatically active polypeptides involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of at least six enzymatically active polypeptides involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of at least seven enzymatically active polypeptides involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • the genetically modified yeast of the present invention is characterized in that it has been (further) modified to have an increased protein expression of eight enzymatically active polypeptides involved in the mevalonate pathway compared to an otherwise identical yeast that does not carry said modification.
  • “increased protein expression” it is meant that the amount of the enzymatically active polypeptide involved in the mevalonate pathway (e.g., a polypeptide having hydroxymethylglutaryl-CoA reductase activity) produced by the thus modified yeast is increased compared to an otherwise identical yeast that does not carry said modification.
  • the enzymatically active polypeptide involved in the mevalonate pathway e.g., a polypeptide having hydroxymethylglutaryl-CoA reductase activity
  • the amount of the enzymatically active polypeptide involved in the mevalonate pathway (e.g., a polypeptide having hydroxymethylglutaryl-CoA reductase activity) produced by the thus modified yeast is increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%at least about 9000%or at least about 10000%, compared to an otherwise identical yeast that does not carry said modification.
  • the amount of protein in a given cell can be determined by any suitable quantification technique known in the art
  • an increase in protein expression may be achieved by any suitable means well-known to those skilled in the art.
  • an increase in protein expression may be achieved by increasing the number of copies of a nucleotide sequence encoding the at least one enzymatically active polypeptide involved in the mevalonate pathway in the yeast, such as by introducing into the yeast at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said at least one enzymatically active polypeptide involved in the mevalonate pathway.
  • the genetically modified yeast of the present invention comprises at least one exogenous nucleic acid molecule comprising at least one nucleotide sequence encoding said at least one enzymatically active polypeptide involved in the mevalonate pathway.
  • the exogenous nucleic acid molecule comprises at least one transcriptional unit comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the exogenous nucleic acid molecule may comprise at least two transcriptional units each comprising, from 5’ to 3’, a promoter that is functional in the yeast to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • the transcriptional units may have the same type of promoter or different types of promoter.
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of such vector, such as an expression cassette comprised by such vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule may be stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast (e.g., by random or targeted insertion) .
  • An increase in protein expression may also be achieved by increasing the strength of the promoter operably linked to a nucleotide sequence encoding the at least one enzymatically active polypeptide involved in the mevalonate pathway, e.g. by replacing the native promoter with a promoter that enables higher expression and overproduction of polypeptide compared to the native promoter.
  • strong promoters are given further below.
  • Enzymatically active polypeptide involved in the mevalonate pathway are encoded in the genomes of a wide range of organisms, including Yarrowia species such as Yarrowia lipolytica.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway e.g., a polypeptide having hydroxymethylglutaryl-CoA reductase activity
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway (e.g., a polypeptide having hydroxymethylglutaryl-CoA reductase activity) is derived from the same species as the yeast in which it is expressed. According to some embodiments, the at least one enzymatically active polypeptide involved in the mevalonate pathway (e.g., a polypeptide having hydroxymethylglutaryl-CoA reductase activity) is derived from a species different from the one in which it is expressed (i.e. it is heterologous) .
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway may be a polypeptide selected from the group consisting of: a polypeptide having hydroxymethylglutaryl-CoA reductase activity, a polypeptide having acetyl-CoA C-acetyltransferase activity, a polypeptide having hydroxymethylglutaryl-CoA synthase activity, a polypeptide having mevalonate kinase activity, a polypeptide having phosphomevalonate kinase activity, a polypeptide having diphosphomevalonate decarboxylase activity, a polypeptide having isopentenyl-diphosphate Delta-isomerase activity, and a polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity.
  • HMG-CoA 3-hydroxy-3-methylglutaryl-CoA
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity may be a full-length polypeptide. However, it is also contemplated to employ a N-terminal truncated version of such polypeptide which remains catalytically active. Usually, the truncation removes the HMG's transmembrane domain, which is responsible for its membrane localisation.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 213 to 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises an amino acid sequence of any one of SEQ ID NOs: 213 to 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 213 to 232.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises an amino acid sequence of any one of SEQ ID NOs: 213 to 232.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 213 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 213.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 213 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 213.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 213 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 213.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises the amino acid sequence of SEQ ID NO: 213.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity consists of the amino acid sequence of SEQ ID NO: 213.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 214.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 214.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 214 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 214.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises the amino acid sequence of SEQ ID NO: 214.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity consists of the amino acid sequence of SEQ ID NO: 214.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 233 to 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises an amino acid sequence of any one of SEQ ID NOs: 233 to 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 236 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 236 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises, or consisting of, the amino acid sequence of SEQ ID NO: 236 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity comprises the amino acid sequence of SEQ ID NO: 236.
  • the polypeptide having hydroxymethylglutaryl-CoA reductase activity consists of the amino acid sequence of SEQ ID NO: 236.
  • Hydroxymethylglutaryl-CoA reductase activity can, for example, be determined with the measurement of mevalonate accumulation in vitro or in yeast biomass using an enzyme assay and/or a suitable spectrophotometric, HPLC, LC-MS, or GC-MS method.
  • An exemplary assay for determining hydroxymethylglutaryl-CoA reductase activity is the HMG-CoA Reductase Activity Assay Kit (ab204701) from Abcam plc.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having acetyl-CoA C-acetyltransferase activity.
  • the polypeptide having acetyl-CoA C-acetyltransferase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 237 to 246.
  • the polypeptide having acetyl-CoA C-acetyltransferase activity comprises an amino acid sequence of any one of SEQ ID NOs: 237 to 246.
  • the polypeptide having acetyl-CoA C-acetyltransferase activity comprises the amino acid sequence of SEQ ID NO: 237 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 237. According to some embodiments, the polypeptide having acetyl-CoA C-acetyltransferase activity comprises the amino acid sequence of SEQ ID NO: 237 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 237.
  • the polypeptide having acetyl-CoA C-acetyltransferase activity comprises the amino acid sequence of SEQ ID NO: 237 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 237. According to some embodiments, the polypeptide having acetyl-CoA C-acetyltransferase activity comprises the amino acid sequence of SEQ ID NO: 237.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having hydroxymethylglutaryl-CoA synthase activity.
  • the polypeptide having hydroxymethylglutaryl-CoA synthase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 247 to 256.
  • the polypeptide having hydroxymethylglutaryl-CoA synthase activity comprises an amino acid sequence of any one of SEQ ID NOs: 247 to 256.
  • the polypeptide having hydroxymethylglutaryl-CoA synthase activity comprises the amino acid sequence of SEQ ID NO: 247 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 247. According to some embodiments, the polypeptide having hydroxymethylglutaryl-CoA synthase activity comprises the amino acid sequence of SEQ ID NO: 247 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 247.
  • the polypeptide having hydroxymethylglutaryl-CoA synthase activity comprises the amino acid sequence of SEQ ID NO: 247 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 247. According to some embodiments, the polypeptide having hydroxymethylglutaryl-CoA synthase activity comprises the amino acid sequence of SEQ ID NO: 247.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having mevalonate kinase activity.
  • the polypeptide having mevalonate kinase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 257 to 266.
  • the polypeptide having mevalonate kinase activity comprises an amino acid sequence of any one of SEQ ID NOs: 257 to 266.
  • the polypeptide having mevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 257 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 257.
  • the polypeptide having mevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 257 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 257.
  • the polypeptide having mevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 257 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 257.
  • the polypeptide having mevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 257.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having phosphomevalonate kinase activity.
  • the polypeptide having phosphomevalonate kinase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 267 to 276.
  • the polypeptide having phosphomevalonate kinase activity comprises an amino acid sequence of any one of SEQ ID NOs: 267 to 276.
  • the polypeptide having phosphomevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 267 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 267. According to some embodiments, the polypeptide having phosphomevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 267 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 267.
  • the polypeptide having phosphomevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 267 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 267. According to some embodiments, the polypeptide having phosphomevalonate kinase activity comprises the amino acid sequence of SEQ ID NO: 267.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having diphosphomevalonate decarboxylase activity.
  • the polypeptide having diphosphomevalonate decarboxylase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 277 to 286.
  • the polypeptide having diphosphomevalonate decarboxylase activity comprises an amino acid sequence of any one of SEQ ID NOs: 277 to 286.
  • the polypeptide having diphosphomevalonate decarboxylase activity comprises the amino acid sequence of SEQ ID NO: 277 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 277. According to some embodiments, the polypeptide having diphosphomevalonate decarboxylase activity comprises the amino acid sequence of SEQ ID NO: 277 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 277.
  • the polypeptide having diphosphomevalonate decarboxylase activity comprises the amino acid sequence of SEQ ID NO: 277 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 277. According to some embodiments, the polypeptide having diphosphomevalonate decarboxylase activity comprises the amino acid sequence of SEQ ID NO: 277.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having isopentenyl-diphosphate Delta-isomerase activity.
  • the polypeptide having isopentenyl-diphosphate Delta-isomerase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: 287 to 296.
  • the polypeptide having isopentenyl-diphosphate Delta-isomerase activity comprises an amino acid sequence of any one of SEQ ID NOs: 287 to 296.
  • the polypeptide having isopentenyl-diphosphate Delta-isomerase activity comprises the amino acid sequence of SEQ ID NO: 287 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 287. According to some embodiments, the polypeptide having isopentenyl-diphosphate Delta-isomerase activity comprises the amino acid sequence of SEQ ID NO: 287 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 287.
  • the polypeptide having isopentenyl-diphosphate Delta-isomerase activity comprises the amino acid sequence of SEQ ID NO: 287 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 287. According to some embodiments, the polypeptide having isopentenyl-diphosphate Delta-isomerase activity comprises the amino acid sequence of SEQ ID NO: 287.
  • the at least one enzymatically active polypeptide involved in the mevalonate pathway is at least a polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity.
  • the polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity comprises an amino acid sequence, which has at least about 70%, such as at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to the amino acid sequence of any one of SEQ ID NOs: : 297 to 306.
  • the polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity comprises an amino acid sequence of any one of SEQ ID NOs: : 297 to 306.
  • the polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 297 or an amino acid sequence having at least 70%, such as at least 75%, sequence identity with SEQ ID NO: 297.
  • the polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 297 or an amino acid sequence having at least 80%, such as at least 85%, sequence identity with SEQ ID NO: 297.
  • the polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 297 or an amino acid sequence having at least 90%, such as at least 95%, sequence identity with SEQ ID NO: 297. According to some embodiments, the polypeptide having (2E, 6E) -farnesyl diphosphate synthase activity comprises the amino acid sequence of SEQ ID NO: 297.
  • a genetically modified yeast according to the present invention can be produced from any yeast of the genus Yarrowia.
  • a yeast of the genus Yarrowia include Yarrowia lipolytica, Yarrowia bubula, Yarrowia deformans, Yarrowia porcina, Yarrowia yakushimensis and Yarrowia parophonii.
  • Strains of the genus Yarrowia are obtainable from the ARS culture collection (NRRL) .
  • the yeast is of the species Yarrowia lipolytica.
  • production micoroorganisms should exhibit high biomass and low production of side products in order to quickly channel all available carbon source into the target product.
  • viscosity and oxygen transfer many times become limiting factors, especially when elongated pseudo hyphal growth is observed.
  • a less elongated, yeast-like cell morphology enables faster growth rates and via better oxygen transfer and mixing of fermentation broth.
  • the yeast of the genus Yarrowia to be used should be one characterized by one, two or three traits selected from higher biomass production, lower citrate production and lower elongation of cells in bioprocess (less viscosity) compared to Yarrowia lipolytica strain W29.
  • a non-limiting example of such yeast is the Yarrowia lipolytica strain YB392.
  • the genetically modified yeast is derived from the Yarrowia lipolytica strain YB392.
  • Yarrowia lipolytica strain W29 is obtainable from the ARS culture collection (NRRL) under deposition number Y-63746.
  • Yarrowia lipolytica strain YB392 is obtainable from the ARS culture collection (NRRL) under deposition number YB-392.
  • Citrate production may for instance be determined in accordance with any of the following protocol:
  • citric acid manufactured by Sigma Aldrich, Cat. No.: 251275-500G
  • 50mL centrifuge tube Add up to 50mL ddH2O and mix until completely dissolved. Sterilize through 0.2 ⁇ m filter directly into sterile 50mL centrifuge tube. Store sterile 10g/L citrate solution at 4 °C.
  • Elongation of cells in bioprocess may for instance be determined by microscopy.
  • a genetically modified yeast of the invention may be modified to express one or more polypeptides as detailed herein, which means that one or more exogenous nucleic acid molecules, which comprise (s) a nucleotide sequence or nucleotide sequences encoding said polypeptide or polypeptides has been introduced in the yeast.
  • a genetically modified yeast of the present invention may comprise at least one exogenous nucleic acid molecule which comprises at least one nucleotide sequence encoding the polypeptide in question.
  • Techniques for introducing an exogenous nucleic acid molecule into a yeast cell include transformation (e.g., heat shock or natural transformation) , transfection, conjugation, electroporation, microinjection, microparticle bombardment etc..
  • the exogenous nucleic acid molecule may be a DNA construct, such as an expression cassette or a vector.
  • the exogenous nucleic acid molecule may thus be a vector, such as an expression vector, or part of a vector, such as an expression cassette comprised by a vector. Normally, such a vector remains extrachromosomal within the yeast cell which means that it is found outside of the nucleus of the yeast cell.
  • the exogenous nucleic acid molecule is stably integrated into the genome of the yeast cell.
  • the exogenous nucleic acid molecule may be an expression cassette stably integrated into the genome of the yeast.
  • the genome integration may be achieved by random integration or targeted integration. Means for stable integration into the genome of a yeast cell, e.g., by homologous recombination, are well known to the skilled person.
  • the at least one exogenous nucleic acid molecule may comprise at least one transcriptional unit comprising suitable regulatory elements such as a promoter that is functional in the yeast cell to cause the production of an mRNA molecule and that is operably linked to a nucleotide sequence encoding said polypeptide, and a transcriptional terminator sequence.
  • Promoters useful in accordance with the invention are any known promoters that are functional in a given yeast cell to cause the production of an mRNA molecule. Many such promoters are known to the skilled person. Such promoters include promoters normally associated with genes found in yeast, and can be derived from a yeast cell. The use of promoters for protein expression is generally known to those of skilled in the art of molecular biology, for example, see Sambrook et al., Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. The promoter employed may be inducible, such as a temperature-inducible promoter.
  • inducible used in the context of a promoter means that the promoter only directs transcription of an operably linked nucleotide sequence if a stimulus is present, such as a change in temperature or the presence of a chemical substance ( “chemical inducer” ) .
  • chemical induction refers to the physical application of an exogenous or endogenous substance (incl. macromolecules, e.g., proteins or nucleic acids) to a host cell. This has the effect of causing the target promoter present in the host cell to increase the rate of transcription.
  • the promoter employed may be constitutive.
  • constitutive used in the context of a promoter means that the promoter is capable of directing transcription of an operably linked nucleotide sequence in the absence of a stimulus (such as heat shock, chemicals, etc. ) .
  • Non-limiting examples of promoters functional in yeast include both constitutive and inducible promoters such as TEF promoter (including TEF1 and TEF2 promoters) , AMO2 promoter, EXP1 promoter, GAPDH promoter, ILV5 promoter, LIP2 promoter, POX2 promoter, TEF1, TEF2, and YAT1EYK1 promoter. Synthetic promoters are also suitable such as HP4D promoter and HP8D promoter.
  • the exogenous nucleic acid molecule may further comprise at least one regulatory element selected from a 5’ untranslated region (5’ UTR) and 3’ untranslated region (3’ UTR) .
  • 5’ UTRs and 3’ UTRs derived from eukaryotes are well known to the skilled person.
  • Such regulatory elements include 5’ UTRs and 3’ UTRs normally associated with other genes, and/or 5’ UTRs and 3’ UTRs isolated from any yeast.
  • Transcriptional terminators useful in accordance with the invention are any known terminators that are functional in a given yeast cell to cause termination of transcription (i.e. it defines the end of a transcriptional unit) .
  • Such terminators include terminators normally associated with genes found in a yeast, and can be derived from a yeast cell.
  • suitable transcriptional terminators include CYC1 terminator, AMO2 terminator, MSC2 terminator, SYN25 terminator, XPR2 terminator, ADH1 terminator, STE2 terminator, Tmini terminator, TEF1 terminator, PGK1 terminator and CYCE1 terminator.
  • each polypeptides described herein to be expressed by the modified yeast cell of the invention may be expressed from an individual exogenous nucleic acid molecule, it is also contemplated by the present invention that two or more of the polypeptides described herein are expressed from the same exogenous nucleic acid molecule. Accordingly, a single nucleic acid molecule, such as an expression cassette, may comprise the coding sequences for two or more of the polypeptides described herein, which is then introduced into the yeast cell.
  • the present invention also provides methods for producing for producing vitamin A, comprising cultivating a genetically modified yeast according to the present invention under suitable culture conditions in a suitable culture medium.
  • the method may further comprise collecting the vitamin A from the culture medium.
  • the culture medium employed may be any conventional medium suitable for culturing a yeast cell in question, and may be composed according to the principles of the prior art.
  • the medium will usually contain all nutrients necessary for the growth and survival of the respective yeast, such as carbon and nitrogen sources and other inorganic salts.
  • Suitable media e.g. minimal or complex media, are available from commercial suppliers or may be prepared according to published receipts, e.g. the American Type Culture Collection (ATCC) Catalogue of strains.
  • suitable media for culturing yeast cells are Yeast extract + peptone (YP) and synthetic complete (SC) , all of which may be supplemented with vitamins, minerals, amino acids and other nutrients required by the particular yeast being cultured.
  • the medium for culturing yeast cells may also be any kind of minimal media such as Yeast minimal media or synthetic minimal (SD) media.
  • the carbon source may be any suitable carbon substrate know in the art, and particularly any carbon substrate commonly used in the cultivation of microorganisms and/or fermentation.
  • suitable fermentable carbon substrates include carbohydrates (e.g., C5 sugars such as arabinose or xylose, or C6 sugars such as glucose) , glycerol, glycerine, acetate, dihydroxyacetone, one-carbon source, methanol, methane, animal fats, plant fats, oils, including animal oils, plant oils, microbial oils and synthetic oils, fatty acids, lipids, phospholipids, glycerolipids, monoglycerides, diglycerides, triglycerides, hydrocarbons, renewable carbon sources, polypeptides (e.g., a microbial or plant protein or peptide) , yeast extract, component from a yeast extract, peptone, casamino acids or any combination of two or more of the foregoing.
  • an oil such as
  • ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used.
  • minerals potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used.
  • the cultivation can be preferably performed under aerobic conditions, such as by a shaking culture, and by a stirring culture with aeration, at a temperature of about 20 to about 45 °C, such as about 25 to 35 °C, such as at about 30°C. According to some embodiments, the cultivation is performed at a temperature of about 25 to 35 °C, such as at about 30°C.
  • the pH of the culture is usually above 3, such as in a range from about 5 to about 8, preferably from about 5.5 to about 7.5, more preferably from about 6.8 to about 7.2. According to some embodiments, the cultivation is performed at a pH from about 5 to about 8.
  • the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. The cultivation may be carried out for a period in the range from 10 to 70 h, such as in the range of 10 to 24 h or 10 to 48 h.
  • the vitamin A can be collected by a conventional method for the isolation and purification of chemical compounds from a medium.
  • Well-known purification procedures include, but are not limited to, centrifugation or filtration, precipitation, ion exchange, chromatographic methods such as e.g. ion-exchange chromatography or gel filtration chromatography, and crystallization methods.
  • the present invention thus provides a vitamin A obtainable by a method as detailed herein.
  • vitamin A means an organic compound comprising a beta-ionone ring to which an isoprenoid chain is attached.
  • Non-limiting examples of “vitamin A” are retinol including any isoforms thereof (such as all-trans-retinol) , retinol aldehyde (retinal) including any isoform thereof (such as 11-cis-retinal) , retinoic acid including any isoform thereof (such as all-trans retinoic acid) , retinyl esters (e.g. acetate and fatty acid esters) , and pro-vitamin A carotenoids (e.g. ⁇ -carotene) .
  • retinol is preferred.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 1 to 22.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 23 to 45.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 46 to 74.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 46 to 59 and 75 to 85.
  • Non-limiting examples of such bifunctional polypeptides are provided in SEQ ID NOs: 46 to 59.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 86 to 113.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 114 to 147.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 148 to 159.
  • a polypeptide having diacylglycerol O-acyltransferase activity is encoded by the gene DGA1 or DGA2, and has, e.g., the amino acid sequence of ID NO: 160 or 161, respectively.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 162 to 187.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 188 to 197.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 198 to 212.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 213 to 232 (NADP + as acceptor) and SEQ ID NOs: 233 to 236 (NAD + as acceptor) .
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 237 to 246.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 247 to 256.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 257 to 266.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 267 to 276.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 277 to 286.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 287 to 296.
  • Non-limiting examples of such polypeptides are provided in SEQ ID NOs: 297 to 306.
  • Polypeptide and “protein” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc. ) . Included within this definition are D-and L-amino acids, and mixtures of D-and L-amino acids.
  • Nucleic acid or “polynucleotide” are used interchangeably herein to denote a polymer of at least two nucleic acid monomer units or bases (e.g., adenine, cytosine, guanine, thymine) covalently linked by a phosphodiester bond, regardless of length or base modification.
  • bases e.g., adenine, cytosine, guanine, thymine
  • Recombinant or “non-naturally occurring” when used with reference to, e.g., a yeast, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.
  • Non-limiting examples include, among others, recombinant yeast cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.
  • Heterologous or “exogenous” as used herein in the context of a gene or nucleic acid molecule refer to a gene or nucleic acid molecule (i.e. DNA or RNA molecule) that does not occur naturally as part of the genome of the yeast in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature. Thus, a “heterologous” or “exogenous” gene or nucleic acid molecule is not endogenous to the yeast and has been exogenously introduced into the yeast.
  • a “heterologous” gene or nucleic acid molecule, such as DNA molecule may be from a different organism, a different species, a different genus, or a different kingdom, as the host DNA.
  • Heterologous as used herein in the context of a polypeptide means that a polypeptide is normally not found in or made (i.e. expressed) by the host yeast, but derived from a different organism, a different species, a different genus, or a different kingdom.
  • ortholog refers to genes, nucleic acid molecules encoded thereby, i.e., mRNA, or proteins encoded thereby that are derived from a common ancestor gene but are present in different species.
  • “decreased expression level” of a gene it is meant that the amount of the transcription product, respectively the amount of the polypeptide encoded by said gene produced by the modified yeast is decreased compared to an otherwise identical yeast that does not carry said modification. More particularly, by “decreased expression level” of a gene it is meant that the amount of the transcription product, respectively the amount of the polypeptide encoded by said gene produced by the modified yeast is decreased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%or at least 100%, compared to an otherwise identical yeast that does not carry said modification.
  • the level of expression of a gene can be determined by well-known methods, including PCR, Southern blotting, and the like.
  • the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like.
  • the amount of the polypeptide encoded by the gene can be measured by well-known methods, including ELISA, Immunohistochemistry, or Western Blotting, and the like.
  • Expression of a gene can be decreased by introducing a mutation into the gene in the genome of the modified yeast so that the intracellular activity of the polypeptide encoded by the gene is decreased as compared to an otherwise identical yeast that does not carry the said mutation.
  • Mutations, which result in a decreased expression of the gene include the replacement of one nucleotide or more to cause an amino acid substitution in the polypeptide encoded by the gene (missense mutation) , the introduction of a stop codon (nonsense mutation) , deletion, or insertion of nucleotides to cause a frameshift, insertion of a drug-resistance gene, or deletion of a part of the gene or the entire gene (Qiu and Goodman, 1997; Kwon et al., 2000) .
  • the expression can also be decreased by modifying an expression regulating sequence such as the promoter etc.
  • Expression of the gene can also be decreased by gene replacement (Datsenko and Wanner, 2000) , such as the "lambda-red mediated gene replacement" .
  • the lambda-red mediated gene replacement is a particularly suitable method to inactive one or more genes as described herein.
  • “Inactivating” , “inactivation” and “inactivated” when used in the context of a gene, means that the gene in question no longer expresses a functional protein. It is possible that the modified DNA region is unable to naturally express the gene due to the deletion of a part of or the entire gene sequence, the shifting of the reading frame of the gene, the introduction of missense/nonsense mutation (s) , or the modification of an adjacent region of the gene, including sequences controlling gene expression, such as a promoter, enhancer, attenuator, ribosome-binding site, etc.
  • a gene of interest is inactivated by the deletion of a part of or the entire gene sequence, such as by gene replacement.
  • Inactivation may also be accomplished by introducing or expressing a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by the double-strand break, the gene of interest.
  • a “rare-cutting endonuclease” within the context of the present invention includes transcription activator-like effector (TALE) nucleases, meganucleases, zinc-finger nucleases (ZFN) , and RNA-guided endonucleases.
  • TALE transcription activator-like effector
  • the presence or absence of a gene in the genome of a yeast can be detected by well-known methods, including PCR, Southern blotting, and the like.
  • the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like.
  • the amount of the protein encoded by the gene can be measured by well-known methods, including SDS-PAGE followed by an immunoblotting assay (Western blotting analysis) , and the like.
  • “increased expression level” of a gene it is meant that the amount of the transcription product, respectively the amount of the polypeptide encoded by said gene produced by the modified yeast is increased compared to an otherwise yeast that does not carry said modification. More particularly, by “increased expression level” of a gene it is meant that the amount of the transcription product, respectively the amount of the polypeptide encoded by said gene produced by the modified yeast is increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%at least about 9000%or at least about 10000%, compared to an otherwise
  • the level of expression of a gene can be determined by well-known methods, including PCR, Southern blotting, and the like.
  • the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like.
  • the amount of the polypeptide encoded by the gene can be measured by well-known methods, including ELISA, Immunohistochemistry, or Western Blotting, and the like.
  • “increased expression level” of a polypeptide it is meant that the amount of the polypeptide in question produced by the modified yeast is increased compared to an otherwise identical yeast that does not carry said modification. More particularly, by “increased expression level” of a polypeptide it is meant that the amount of the polypeptide in question produced by the modified yeast is increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%at least 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, at least about 4000%, at least about 5000%, at least about 6000%, at least about 7000%, at least about 8000%at least about 9000%or at least about 10000%, compared an otherwise identical yeast that does not carry said modification.
  • an increase in polypeptide expression may be achieved by any suitable means well-known to those skilled in the art.
  • an increase in polypeptide expression may be achieved by increasing the number of copies of the gene or genes encoding the polypeptide in the microorganism, such as by introducing into the yeast an exogenous nucleic acid molecule, such as a vector, comprising the gene or genes encoding the polypeptide operably linked to a promoter that is functional in the yeast to cause the production of an mRNA molecule.
  • An increase in polypeptide expression may also be achieved by the integration of at least a second copy of the gene or genes encoding the polypeptide into the genome of the yeast.
  • An increase in polypeptide expression may also be achieved by increasing the strength of the promoter (s) operably linked to the gene or genes encoding the polypeptide.
  • “decreased” means that the expression of said polypeptide in a modified yeast is reduced compared to the expression of said polypeptide in an otherwise identical yeast that does not carry said modification (control) .
  • the expression of a polypeptide in a modified yeast may be reduced by at least about 10 %, and preferably by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%or 100%, or any percentage, in whole integers between 10%and 100%, compared to the expression of said polypeptide in an otherwise identical yeast that does not carry said modification (control) .
  • “decreased” , “decreasing” or “decrease of” expression of a polypeptide means that the amount of the polypeptide in the modified yeast is reduced by at least about 10 %, and preferably by at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%or 100%, or any percentage, in whole integers between 10%and 100%, compared to the amount of said polypeptide in an otherwise identical yeast that does not carry said modification (control) .
  • the expression or amount of a polypeptide in a yeast can be determined by any suitable means known in the art, including techniques such as ELISA, Immunohistochemistry, Western Blotting, or Flow Cytometry.
  • “decreased” , “decreasing” or “decrease of” activity of a polypeptide means that the catalytic activity of said polypeptide in a modified yeast is reduced compared to the catalytic activity of said polypeptide in an otherwise identical yeast that does not carry said modification (control) .
  • the activity of a polypeptide in a modified yeast may be reduced by at least about 10 %, and preferably by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%or 100%, or any percentage, in whole integers between 10%and 100%, compared to the expression of said polypeptide in an otherwise identical yeast that does not carry said modification (control) .
  • the activity of a polypeptide in a yeast can be determined by any suitable protein and enzyme activity assay.
  • inhibitor of the enzyme refers to any chemical compound, natural or synthetic, that inhibits the catalytic activity of the enzyme.
  • An inhibitor of the enzyme does not necessarily need to achieve 100%or complete inhibition.
  • an inhibitor of the enzyme can induce any level of inhibition.
  • substitution refers to the modification of the polypeptide by replacing one amino acid residue with another, for instance, the replacement of a Serine residue with a Glycine or Alanine residue in a polypeptide sequence is an amino acid substitution.
  • substitution refers to a modification of the polynucleotide by replacing one nucleotide with another, for instance, the replacement of cytosine with thymine in a polynucleotide sequence is a nucleotide substitution.
  • Constant substitution when used with reference to a polypeptide, refers to a substitution of an amino acid residue with a different residue having a similar side chain, and thus typically involves the substitution of the amino acid in the polypeptide with amino acids within the same or similar class of amino acids.
  • an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.
  • Non-conservative substitution when used with reference to a polypeptide, refers to a substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., serine for glycine) , (b) the charge or hydrophobicity, or (c) the bulk of the side chain.
  • an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid, and a hydrophilic amino acid substituted with a hydrophobic amino acid.
  • “Expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • the "regulatory region" of a gene refers to a nucleic acid sequence that affects the expression of a coding sequence. Regulatory regions are known in the art and include, but are not limited to, promoters, enhancers, transcriptional terminators, polyadenylation sites, matrix attachment regions, and/or other elements that regulate the expression of a coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • plasmid refers to a circular double-stranded nucleic acid loop into which additional nucleic acid segments can be ligated.
  • Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors” .
  • Certain other vectors are capable of facilitating the insertion of an exogenous nucleic acid molecule into a genome of a yeast. Such vectors are referred to herein as “transformation vectors” .
  • vectors of utility in recombinant nucleic acid techniques are often in the form of plasmids as they are the most commonly used form of a vector.
  • a vector may however also be a yeast artificial chromosome (YAC) .
  • yeast artificial chromosome YAC
  • Large numbers of suitable plasmids and YACs are known to those of skill in the art and commercially available.
  • promoter refers to a sequence of DNA, usually upstream (5') of the coding region of a structural gene, which controls the expression of the coding region by providing recognition and binding sites for RNA polymerase and other factors, which may be required for initiation of transcription. The selection of the promoter will depend upon the nucleic acid sequence of interest.
  • a suitable “promoter” is generally one, which is capable of supporting the initiation of transcription in a yeast of the invention, causing the production of an mRNA molecule.
  • “Strong” promoters include, for example, natural promoters as well as artificial promoters active in Yarrowia species, such as TEF promoters (including TEF1 and TEF2 promoters) , HP4D promoter or HP8D promoter, or similar, that enable efficient expression and overproduction of proteins.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.
  • a promoter sequence is "operably-linked” to a gene (i.e. a nucleotide sequence encoding a polypeptide) when it is in sufficient proximity to the transcription start site of a gene to regulate transcription of the gene.
  • Identity refers to sequence identity between two nucleotide or amino acid sequences. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the nucleotide or amino acid sequences, respectively.
  • Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis. ) , and can be used with, e.g., default setting.
  • percentage of sequence identity is used herein to refer to a comparison between an amino acid sequence and a reference amino acid sequence.
  • the “%sequence identity” is calculated from the two amino acid sequences as follows: The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program) , using the default BLOSUM62 matrix with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (for each additional null in the gap) . After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference amino acid sequence.
  • Reference sequence or “reference amino acid sequence” refers to a defined sequence to which another sequence is compared.
  • a reference amino acid sequence may, for example, be an amino acid sequence set forth in SEQ ID NO: 1.
  • the term "about” means plus or minus 10%of the numerical value of the number with which it is being used.
  • the selected amino acid sequences were used for generating codon-optimized nucleotide sequences for gene expression in Y. lipolytica by using GENEius (Eurofins) .
  • the synthetic DNA fragment was designed with short adapter sequences at both ends of the fragment for easier compatibility with further assembly of expression cassettes.
  • Table 1 Genes involved in biosynthesis of ⁇ -carotene and vitamin A.
  • Example 2 Assembly of expression cassettes for production of ⁇ -carotene and transformation into Y. lipolytica strains YB-392 and W29
  • ⁇ -carotene expression cassette with Hyg as selection marker (SEQ ID NO: 309) was assembled using Golden Gate technique with 3 transcription units (TUs) containing the Y. lipolytica GGPP synthase gene GGS1 (SEQ ID NO: 1) , M. lusitanicus's gene for bifunctional lycopene cyclase/phytoene synthase carRP (SEQ ID NO: 50) , and M. lusitanicus's gene for phytoene dehydrogenase carB (SEQ ID NO: 23) .
  • TUs Golden Gate technique with 3 transcription units
  • the synthesized GGS1 (SEQ ID NO: 1) , M. lusitanicus carRP (SEQ ID NO: 50) and M. lusitanicus carB (SEQ ID NO: 23) genes were assembled into the Golden Gate parts as described in Lee et al., 2015. TUs for all three genes were assembled using strong TEF (SEQ ID NO: 312) or HP8D (SEQ ID NO: 313) promoters and CYC1 terminator (SEQ ID NO: 314) . All three TUs were assembled with Hyg selection marker (SEQ ID NO: 309) and zeta sequences (SEQ ID NO: 310, SEQ ID NO: 311) for random genomic integration. After the assembly the mixture was transformed in E.
  • coli DH10 ⁇ strain by electroporation and cultivated on appropriate selection media. Several colonies were then grown in liquid culture with appropriate selection for plasmid extraction with Pure Yield plasmid extraction kit (Promega, USA) .
  • the plasmids were digested with NotI restriction enzyme (NEB, USA) . After restriction digest, the digested DNA was run on 0.8%agarose gel and correct fragments were excised and cleaned up with Wizard SV Gel and PCR Clean-up system according to the protocol provided by the manufacturer. Approximately 50 ng of the linearized expression plasmid was used for transformation into Y.
  • lipolytica strains YB-392 or W29 with deleted LEU2 and URA3 genes –both deletions were obtained using conventional method with homologous recombination and negative screening. Transformation was performed using standard lithium acetate yeast transformation protocol. Transformed strains were grown on agar medium with appropriate selection. Obtained transformants were subjected to fermentation and ⁇ -carotene production analysis.
  • Example 3 Assembly of alternative expression cassettes for production of ⁇ -carotene and transformation into Y. lipolytica
  • ⁇ -carotene expression cassettes were assembled with Y. lipolytica’s GGPP synthase gene YlGGS1 (SEQ ID NO: 1) , P. rhodozyma's gene for bifunctional lycopene cyclase/phytoene synthase crtYB (SEQ ID NO: 46) and gene for phytoene dehydrogenase from either P. rhodozyma or M. lusitanicus –namely crtI (SEQ ID NO: 30) or carB (SEQ ID NO: 23) , respectively.
  • Genes were assembled into the Golden Gate parts as described in Lee et al., 2015.
  • TUs for all genes were assembled using strong TEF (SEQ ID NO: 312) or HP8D (SEQ ID NO: 313) promoter and CYC1 terminator (SEQ ID NO: 314) .
  • TUs were assembled in expression cassettes with Leu2 selection marker (SEQ ID NO: 307) and zeta sequences (SEQ ID NO: 310, SEQ ID NO: 311) for random genomic integration.
  • Example 4 Assembly of tHMGR overexpression cassettes, and transformation into Y. lipolytica
  • Expression cassette with Y. lipolytica’s truncated gene for hydroxymethylglutaryl-CoA (HMG-CoA) reductase –tHMGR (SEQ ID NO: 214) was assembled using golden Gate cloning as described before.
  • TU was assembled using strong TEF promoter (SEQ ID NO: 312) and CYC1 terminator (SEQ ID NO: 314) .
  • TUs were combined with Nat selection marker (SEQ ID NO: 315) and zeta sequences (SEQ ID NO: 310, SEQ ID NO: 311) for random genomic integration. After the assembly the mixture was transformed in E. coli DH10 ⁇ strain by electroporation and cultivated on appropriate selection media.
  • VA270 strain was constructed by this example.
  • Example 5 Assembly of expression cassettes for evaluation of ⁇ -carotene 15, 15'-dioxygenase candidate genes, and transformation into ⁇ -carotene producing Y. lipolytica
  • Vitamin A production cassettes with Ura3 selection marker were assembled using Golden Gate with 1 TU containing the uncultured marine bacterium 66A03 BLH gene (SEQ ID NO: 86; Fig. 2) , Fusarium fujikuroi GfBCO1 gene (SEQ ID NO: 100) , or Homo sapiens HsBCO1 gene (SEQ ID NO: 107) .
  • the synthesized genes (SEQ ID NO: 86, 100, 107) were assembled into the Golden Gate part plasmids. All three TUs were assembled with strong TEF promoter (SEQ ID NO: 312) and CYC1 terminator (SEQ ID NO: 314) .
  • TUs were combined with Ura3 selection marker (SEQ ID NO: 308) and zeta sequences (SEQ ID NO: 310, SEQ ID NO: 311) for random genomic integration.
  • VA162, VA189 and VA191 were constructed by this example.
  • Example 6 Assembly of combined BLH+BLH+tHMGR expression cassettes, and transformation into Y. lipolytica
  • TUs for all three genes were assembled using strong TEF (SEQ ID NO: 312) or HP8D (SEQ ID NO: 313) promoters and CYC1 terminator (SEQ ID NO: 314) . All three TUs were assembled with Leu2 selection marker (SEQ ID NO: 307) and zeta sequences (SEQ ID NO: 310, SEQ ID NO: 311) for random genomic integration.
  • Strain VA588 was constructed by this example.
  • Example 7 Assembly of additional expression cassettes for production of ⁇ -carotene, and transformation into Y. lipolytica
  • E. coli DH10 ⁇ strain After the assembly the mixture was transformed in E. coli DH10 ⁇ strain by electroporation and cultivated on appropriate selection media. Several colonies were grown in liquid culture with appropriate selection for plasmid extraction with Pure Yield plasmid extraction kit (Promega, USA) . The plasmids were digested with NotI restriction enzyme (NEB, USA) . After restriction digest, the digested DNA was run on 0.8%agarose gel and correct fragments were cleaned with Wizard SV Gel and PCR Clean-up system according to the protocol provided by the manufacturer. Approximately 50 ng of expression plasmid was transformed in Y. lipolytica VA588 strain using standard lithium acetate yeast transformation. Transformed strains were grown on agar medium using appropriate selection. Obtained transformants were subjected to fermentation and ⁇ -carotene production analysis.
  • NotI restriction enzyme NEB, USA
  • VA811 strain was constructed by this example.
  • Example 8 Assembly of additional BLH expression cassettes for higher vitamin A conversion, and transformation into Y. lipolytica
  • An additional vitamin A production cassette was assembled with Leu2 as selection marker (SEQ ID NO: 307) using Golden Gate technique with 2 TUs containing two copies of the uncultured marine bacterium 66A03 BLH gene (SEQ ID NO: 86) .
  • TUs for all genes were assembled using strong TEF (SEQ ID NO: 312) or HP8D (SEQ ID NO: 313) promoters and CYC1 terminator (SEQ ID NO: 314) . All TUs were assembled with Leu2 selection marker (SEQ ID NO: 307) and zeta sequences (SEQ ID NO: 310, SEQ ID NO: 311) for random genomic integration.
  • Strain VA1493 was constructed by this example.
  • Example 9 Assembly of YlFAA1 and YlTGL4 overexpression cassettes, and transformation in Y. lipolytica
  • Expression cassette with Y. lipolytica's native FAA1 (SEQ ID NO: 162) and TGL4 (SEQ ID NO: 114) genes was assembled with Golden Gate technique as described before. Both TUs were assembled with strong synthetic HP8D promoter (SEQ ID NO: 313) and CYC1 terminator (SEQ ID NO: 314) . Next, both TUs were used to assemble expression cassette with Nat selection marker (SEQ ID NO: 315) and zeta sequences (SEQ ID NO: 310, SEQ ID NO: 311) for random genomic integration.
  • VA2116 strain was constructed by this example.
  • Example 10 Assembly of 3x YlHMG, 3x PmHMG, and 3x RpHMG overexpression cassette and transformation into Y. lipolytica
  • Three expression cassettes were assembled, each carrying three copies of single gene encoding hydroxymethylglutaryl-CoA reductase.
  • Genes used for these cassettes were Y. lipolytica’s native gene for type 1 hydroxymethylglutaryl-CoA reductase YlHMG (SEQ ID NO: 213) , and two genes for type 2 hydroxymethylglutaryl-CoA reductase, one from Pseudomonas mevalonii PmHMG (SEQ ID NO: 234) and one from Ruegeria pomeroyi RpHMG (SEQ ID NO: 236) .
  • Each of three cassettes were assembled with Golden Gate technique as described before.
  • Strains VA2190, VA2215 and VA2225 were constructed by this example by transforming 3x YlHMG, 3x PmHMG and 3x RpHMG cassettes, respectively.
  • Example 11 Cultivation of Y. lipolytica strains for production of ⁇ -carotene and vitamin A
  • frozen stock solution 20%glycerol frozen at -80 °C
  • a colony of cells was inoculated into the seed medium with appropriate selection and grown at 28 °C for 18-24 h at 220 RPM. Inoculation into 48-well deep-well plate containing 1 mL of YPO100 production medium or YPD60 was done to a final cell density (OD 600 ) of 0.05. Production cultures were incubated at 28-30°C for up to 168 h at 220 RPM and 70%humidity. The fermented broth was sampled and analyzed as described in Example 12. The titer of ⁇ -carotene, retinal, retinol and retinyl esters was measured using HPLC as described in Example 12.
  • Table 2 Composition of the solid seed medium.
  • Table 3 Composition of the vegetative seed medium.
  • Yeast Nitrogen Base without amino acids and ammonium sulphate (Sigma, Y1251-100G) and ammonium sulphate (Sigma, 11226-5KG) were mixed with 800 mL of distilled water and autoclaved for 30 min at 121°C.
  • MES buffer was prepared by dissolving 92, 6 g 2- (N-morpholino) ethanesulfonic acid (Glentham, GB1243) in 450 mL of distilled water. pH was adjusted to 5.7 and distilled water was added to correct the volume to 500 mL. Solution was filter sterilized.
  • Amino acids without leucine were prepared by dissolving 1,92 g Synthetic Drop-out Medium Supplements without leucine (Sigma, Y1376-20G) in 40 mL of water by mixing. Solution was filter sterilized.
  • Table 4 Composition of the YPD60 medium.
  • Table 5 Composition of the YPO100 production medium.
  • Bacteriological peptone and Yeast extract were mixed with 800 mL of distilled water and autoclaved 30 min, 121°C. Distilled water was added to correct the volume of medium to 1 L. Sunflower oil was autoclaved 30 min, 121°C and added to medium.
  • the fermented production medium was sampled and immediately frozen at -20°C.
  • the fermented production medium was diluted 1: 8 with ethyl acetate as extraction buffer in vials containing glass beads using a FastPrep cell homogenizer (5 cycles of 30 s homogenization, 30 s on ice) . After homogenization cell debris was centrifuged and upper phase was transferred to new vial and immediately analyzed.
  • the samples were analyzed by the Thermo Surveyor HPLC.
  • the method was based on Zorbax SB-C8, 4.6x150 mm, 5 um particle size column kept at 60°C with mobile phase A: 80%MetOH 20%H 2 O and mobile phase B –acetonitrile with starting conditions: 67%A, linear gradient increase in B %to 99%at 4 min, and 5 min stabilization to initial conditions, at 1.5 mL /min flow rate.
  • the PDA detector was detecting at 327, 388 and 455 nm. Detected analytes are described in Table 6. Sum of different retinoic compounds, excluding all-trans retinoic acid and all-trans-13, 14-dihydro retinol, was calculated to evaluate total production of vitamin A.
  • Table 6 Detected analytes using HPLC detection method.
  • Example 13 Initial production of ⁇ -carotene in Y. lipolytica strains
  • Figure 11 shows production of ⁇ -carotene in VA128 and VA143 strains harboring the same expression cassette for ⁇ -carotene production.
  • Example 14 Production of ⁇ -carotene using alternative ⁇ -carotene production cassettes in Y. lipolytica strain
  • Figure 12 shows production of ⁇ -carotene in YB-392 strain transformed with different expression cassettes for ⁇ -carotene production. Best performing combination was the combination of YlGGS1, carB and carRP genes.
  • Example 15 Increase in ⁇ -carotene production with overexpression of tHMGR
  • Figure 13 shows titer of ⁇ -carotene in strain VA128 with and without overexpression of tHMGR gene.
  • VA128 is parent strain to VA270, which has tHMGR overexpression.
  • Example 16 Production of vitamin A in ⁇ -carotene producing strains with different ⁇ -carotene 15, 15'-dioxygenase candidate genes
  • Figure 14 shows conversion of ⁇ -carotene to retinoids by different ⁇ -carotene 15, 15’ -dioxygenase genes. The best yields and conversion of more than 90 %was achieved with BLH gene (SEQ ID NO: 86) derived from uncultured marine bacterium.
  • Example 15 Production of vitamin A in ⁇ -carotene producing strain with the BLH+BLH+tHMGR expression cassette
  • Figure 15 shows production of ⁇ -carotene and vitamin A by the VA588 strain, which harbors BLH+BLH+tHMGR expression cassette in addition to YlGGS1+carB+carRP expression cassette.
  • VA588 was decided to use strain VA588 for further production studies and for improvement of retinol production.
  • Example 16 Production of vitamin A in strains with additional ⁇ -carotene production cassettes
  • Figure 16 shows production of vitamin A and ⁇ -carotene in strain VA811, which has additional YlGGS1+crtYB expression cassette for ⁇ -carotene production, and its parent strain.
  • strain VA811 has additional YlGGS1+crtYB expression cassette for ⁇ -carotene production, and its parent strain.
  • Example 17 Production of vitamin A in strains with additional BLH expression cassettes
  • Figure 17 shows production of vitamin A and ⁇ -carotene in strain VA1493, which has additional 2x BLH expression cassette for higher vitamin A conversion, and its parent strain VA811. Alongside better titers we also observed better conversion of ⁇ -carotene to vitamin A. We decided to use VA1493 strain in further production studies and for improvement of vitamin A production.
  • Example 18 Production of and vitamin A in strains with overexpressed YlFAA1 and YlTGL4 genes
  • Figure 18 shows production of vitamin A and ⁇ -carotene in strain VA2116, which has overexpressed native YlFAA1 and YlTGL4 genes, and its parent strain VA1493.
  • strain VA-2116 has overexpressed native YlFAA1 and YlTGL4 genes, and its parent strain VA1493.
  • titer reached more than 3.5 g/L vitamin A. This is the highest shaker-scale vitamin A titer reported to date.
  • Example 19 Production of vitamin A in strains with overexpressed 3x YlHMG, 3x PmHMG, or 3x RpHMG genes
  • Figure 19 shows production of vitamin A and ⁇ -carotene in strain VA2190 (3x YlHMG) , VA2215 (3x PmHMG) , VA2225 (3x RpHMG) and their parent strain VA1493.
  • VA2190 (3x YlHMG)
  • VA2215 (3x PmHMG)
  • VA2225 (3x RpHMG)
  • VA1493 3x RpHMG
  • Bioprocess fermentations were performed in the bioreactor production medium, based on yeast extract, bacteriological peptone, and sunflower oil as a carbon source. Vitamin A and ⁇ -carotene analysis was performed as described in Example 12.
  • Table 7 Production of vitamin A with Y. lipolytica strains in bioreactor scale.
  • Figure 20 shows production of vitamin A and ⁇ -carotene during a bioprocess with strain VA2116. DCW and pO 2 parameters are also shown. The production of vitamin A increased during fermentation and reached 20 g/L or 0,20 g/g DCW after 11 days. This is the highest reported bioreactor vitamin A titer to date, which is 4-5 fold higher than the one described by Liang Sun et al. [5] for vitamin A production in S. cerevisiae.

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Abstract

La présente invention concerne de manière générale le génie biotechnologique, et particulièrement les levures génétiquement modifiées, permettant la production de vitamine A. Plus particulièrement, la présente invention procure une levure génétiquement modifiée du genre Yarrowia capable de produire de la vitamine A. La présente invention procure également des procédés de production de vitamine A utilisant une levure génétiquement modifiée de l'invention.
EP21800999.1A 2021-09-27 2021-09-27 Levure génétiquement modifiée du genre yarrowia pouvant produire de la vitamine a Pending EP4409017A1 (fr)

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