WO2023143872A2 - Method for producing lipids using yarrowia lipolytica - Google Patents

Method for producing lipids using yarrowia lipolytica Download PDF

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WO2023143872A2
WO2023143872A2 PCT/EP2023/025040 EP2023025040W WO2023143872A2 WO 2023143872 A2 WO2023143872 A2 WO 2023143872A2 EP 2023025040 W EP2023025040 W EP 2023025040W WO 2023143872 A2 WO2023143872 A2 WO 2023143872A2
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seq
set forth
protein
oleaginous yeast
amino acid
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WO2023143872A3 (en
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Wei Xia
Bernard Louis Robert PORA
Xia Wang
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Roquette Freres
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/0102—Diacylglycerol O-acyltransferase (2.3.1.20)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express lipid- droplet associated protein.
  • the present invention relates to a method for producing lipids such as docosahexaenoic acid (or DHA), DHA-enriched triacylglycerol, or phosopholipids by culturing an oleaginous yeast.
  • lipids such as docosahexaenoic acid (or DHA), DHA-enriched triacylglycerol, or phosopholipids by culturing an oleaginous yeast.
  • Lipids constitute one of the three major families of macronutrients, along with proteins and carbohydrates. Among the lipids, triglycerides and phospholipids are of particular interest.
  • Triglycerides also called triacylglycerols, triacylglycerides or TAGs
  • TAGs triacylglycerides
  • Triglycerides represent approximately 95% of the dietary lipids ingested by human beings. In the body, they are present mainly in adipose tissues and constitute the main form of energy storage.
  • Phospholipids are amphiphilic lipids, i.e., lipids consisting of a polar (hydrophilic) “head” and two aliphatic (hydrophobic) “tails”. Phospholipids are structural lipids since they are constituents of cell membranes of which they provide, inter alia, the fluidity.
  • phospholipids are phosphoglycerides, the head of which is organized around a glycerol-3 -phosphate residue esterified with a polar molecule, and the two tails of which are the aliphatic chains of two fatty acids.
  • the other phospholipids are sphingomyelins, which derive structurally from sphingosine and not from glycerol, sphingosine constituting one of the two aliphatic tails.
  • the first phospholipids isolated from live tissues were characterized from egg yolk lecithin; they were more particularly phosphatidylcholines. This is, moreover, why phosphatidylcholines are also known as lecithins.
  • Phosphatidylcholines are naturally produced by the liver. They are an important constituent of bile, in which they emulsify the fats present in the duodenum. They are also necessary, in addition to bile salts, for preventing lipid droplets from re-agglutinating.
  • phosphatidylcholines participate in the membranes of cells and serve to preserve their viscoelasticity. They are an essential component of the nervous system and constitute close to 30% of the dry weight of the brain and 15% of the nerves.
  • Triglycerides and phospholipids are composed predominantly of fatty acids which are both provided by the diet and, for some of them, synthesized by the organism.
  • the biochemical classification (based on the number of double bonds contained in the fatty acid molecule) distinguishes saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs).
  • SFAs saturated fatty acids
  • MUFAs monounsaturated fatty acids
  • PUFAs polyunsaturated fatty acids
  • the set of indispensable and “conditionally” indispensable fatty acids constitutes the essential fatty acids.
  • the other fatty acids are termed non-essential.
  • the non-indispensable fatty acids comprise, in particular, eicosapentaenoic acid (EPA) of the omega 3 fatty acid family, oleic acid, the predominant monounsaturated fatty acid in our diet, and saturated fatty acids, such as lauric acid, myristic acid or palmitic acid.
  • EPA eicosapentaenoic acid
  • saturated fatty acids such as lauric acid, myristic acid or palmitic acid.
  • omega 6 fatty acids or n-6 PUFAs
  • LA linoleic acid
  • omega 3 fatty acids or n-3 PUFAs
  • ALA alpha-linolenic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • omega 6 arachidonic acid or ARA
  • omega 3 such as EP A, or DHA
  • the “5” and “6” thus correspond to the number of unsaturations of the carbon chain presented respectively by EPA and by DHA.
  • DHA of the omega 3 fatty acid family, is a fatty acid that the organism can synthesize from alpha-linolenic acid, or which is provided by the consumption of oily fish (tuna, salmon, herring, etc.).
  • DHA plays an important role in the structure of membranes and in the development and function of the brain and retina.
  • Fish oils are used mainly as a source of omega 3 fatty acids, such as DHA and EPA, but they are also found in oils of microalgae where they are extracted either as a mixture, or separately, as is the case for example with the oils derived from certain selected strains, such as those of the Schizochytrium genus, which contain only traces of EPA but have high DHA contents.
  • the Applicant has also developed a strain of Schizochytrium mangrovei which produces DHA and which has the particularity of producing very few hypercholesterolemic saturated fatty acids (less than 6% of lauric and myristic acids, which those skilled in the art know are the most hypercholesterolemic known), and more than 40% of palmitic acid (the % understood here to be by weight of total fatty acids).
  • Said strain and its use for producing lipids is described for example in international publication WO2014/122158.
  • Oleaginous yeasts such as Yarrowia lipolytica for Y. lipolytica) a have also been engineered and used as a host to produce high levels of n-3 PUFA such as DHA, EPA and ALA. .
  • lipid-soluble carotenoids lycopene, beta-carotenen, astaxanthin. Therefore, engineered strains of Yarrowia lipolytica with increased lipid contents can be used for enhancing the production of functional lipids and carotenoids.
  • Xue et al. (Nature Biotechnology, 2013, vol 31:8, p 734-740) describe metabolic engineering of the oleaginous yeast Yarrowia lipolytica in order to increase the production of omega-3 eicosapentaenoic acid by said yeast.
  • Xie et al. (Appl Microbiol Biotechnol 2015, 99 : 1599-1610) describe a Yarrowia platform for producing tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acids in mixtures in the cellular lipid profiles.
  • tailored omega-3 EPA, DHA
  • ARA, GLA omega-6
  • the present invention relates to a method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6.
  • the invention also relates to a host cell transformed with an expression vector encoding the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6, wherein said host cell is an oleaginous yeast.
  • the invention also concerns the Yarrowia lipolytica strain deposited on 9 December 2021 under the Budapest Treaty before the China Center for Type Culture Collection (No. 299, Bayi Road, Wuchang District, Wuhan City, 430072, Hubei province, Wuhan University) as CCTCC M 20211578 .
  • the invention also relates to the use of a nucleic acid comprising the sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3 and/or SEQ ID NO:5 for transforming an oleaginous yeast cell.
  • a first object of the present invention is a method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6.
  • LDAP Lipid Droplet-Associated Protein
  • the mRNA sequence of Idap is set forth in SEQ ID NO: 1 :
  • the protein sequence of LDAP is set forth in SEQ ID NO:2:
  • the sequence of the DGAT2-1 mRNA is set forth in SEQ ID NO:3:
  • the sequence of the DGAT2-1 protein is set forth in SEQ ID NO:4:
  • the sequence of the DGAT2-2 mRNA is set forth in SEQ ID NO: 5:
  • the sequence of the DGAT2-2 protein is set forth in SEQ ID NO:6:
  • Suitable yeast include any yeast species generally employed in order to produce high yields of lipids.
  • the oleaginous yeast is selected in the group consisting of the genera Yarrow ia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces.
  • the oleaginous yeast is selected in the group consisting of the species Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrow ia lipolytica.
  • said oleaginous yeast is a Yarrowia lipolytica yeast.
  • Any suitable methods for expressing a given nucleic acid can be employed.
  • the oleaginous yeast can further be transformed to express other genes known to increase the production of lipids, such as those described in Zhu et al. (Current Opinion in Biotechnology, 2015, 36:65-72).
  • the invention also relates to a host cell transformed with an expression vector encoding the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6, wherein said host cell is an oleaginous yeast.
  • the invention also concerns the Yarrowia lipolytica strain deposited on 9 December 2021 under the Budapest Treaty before the China Center for Type Culture Collection (No. 299, Bayi Road, Wuchang District, Wuhan City, 430072, Hubei province, Wuhan University) as CCTCC M 20211578.
  • the invention also relates to the use of a nucleic acid comprising the sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3 and/or SEQ ID NO:5 for transforming an oleaginous yeast cell.
  • Example 1 Isolation from the S mansrovei strain D5 of “Lipid-Droplet Associated Protein”
  • Cells were collected at 5000 g for 10 min and washed twice with 30 mL of phosphate buffered saline (PBS). After incubating in 30 ml of buffer A (25 mM tricine, 250 mM sucrose, pH 7.8) on ice for 20 min, cells were homogenized. The cell homogenate was centrifuged in a 50-mL tube at 6000 g for 10 min to remove cell debris and unbroken cells. The supernatant fraction (10 mL) overlaid with 2 mL of buffer B (20 mM HEPES, 100 mM KC1, 2 mM MgC12, pH 7.4) was centrifuged at 38 000 rpm for 1 h at 4°C (Beckman SW40).
  • buffer A 25 mM tricine, 250 mM sucrose, pH 7.8
  • buffer B 20 mM HEPES, 100 mM KC1, 2 mM MgC12, pH 7.4
  • the white band containing Lipid Droplets (LDs) at the top of the gradient was collected using a 200 ⁇ L pipette tip and transferred to a 1.5 mL Eppendorf tube. LDs were washed with 200 ⁇ L of Buffer B for three times. The purity of LDs was confirmed by observing staining. Then, total proteins were extracted from LDs and were analyzed by proteomics.
  • LDs Lipid Droplets
  • LDAP LDAP
  • the sequence of the Idap mRNA is set forth in SEQ ID NO: 1.
  • Example 2 Production of engineered Yarrowia lipolytica expressing Idap
  • the inventors used a vector pYLXP’2 for overexpressing of Idap in Y. lipolytica. It is a standard procedure for producing engineered Y. lipolytica, described in Lv Y, Edwards H, Zhou J, Xu P. Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica. ACS Synthetic Biology. 2019 Mar;8(3):568-576. DOI: 10.1021/acssynbio.8b00535. PMID: 30695641.
  • the resulting transformed strains was isolated and further characterized for lipid production.
  • Example 3 Increase in biomass and overall lipid production
  • Y. lipolytica does not naturally produce DHA, it can only synthesize usual fatty acids, such as C18:0, C18:1 and C18:2.
  • the oleaginous Y. lipolytica as a host because it can produce abundant acetyl-CoA, which is the precursor for the biosynthesis of functional lipids and carotenoids.
  • only oleaginous microbes can form obvious LDs and it has been demonstrated that TAG are mainly deposited in LDs in Y. lipolytica.
  • LDAP probably participated in the accumulation of lipids in LDs. The results showed that, when Idap was overexpressed, LDs were be enlarged or stabilized, and more TAG was deposited and generated in Y.
  • LDAP-5 and LDAP-12 represented two selected engineered strains. Data shown here were in triplicate.
  • Strain LDAP-12 (also called pLDAP) was deposited on 9 December 2021 under the Budapest Treaty before the CCTCC collection under deposit number CCTCC M 20211578.
  • Example 4 Engineered Yarrowia lipolytica expressing Idap and/or dgat
  • DGAT2 Yarrowia lipolytica strains overexpressing diacylglycerol acyltransferase
  • DGAT2 is the limiting enzyme involved in the biosynthesis of TAG
  • the inventors found two diacylglycerol acyltransferase (DGAT2) genes from the genome of D5. These two DGAT2 encoding genes were codon-optimized and synthesized. Overexpressing either dgat2-1 or dgat2-2 led to an increase of total fatty acids (TFA). The biomass was slightly decreased.
  • Table 3 Biomass and TFA production in original Polf strain and strains overexpressing the various genes according to the invention. Results are presented as average ⁇ standard deviation Polf, Y. lipolytica, MATa, leu2-270, ura3-302, xpr2-322, axp-2, (Leu2-, Ura3-), LDAP, Polf harboring pYLXP': : ldap: DGAT2-1, Polf harboring dgat2-, DGAT2-2, Polf harboring pYLXP’ : :dgat2-2; LDAP-DGAT2-1, pYLXP’ : :ldap: :dgat2-1, LDAP-DGAT2- 2, pYLXP’ ::ldap::dgat2-2.
  • the inventors have shown that Yarrowia lipolytica yeast expressing the Idap or dgat2-l or dgat2-2 genes from S. mangrovei could be

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Abstract

The present invention relates to a method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express lipid-droplet associated protein and/or diacylglycerol acyltransferase.

Description

METHOD FOR PRODUCING LIPIDS USING YARROWIA LIPOLYTICA
FIELD OF THE INVENTION
The present invention relates to a method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express lipid- droplet associated protein.
PRIOR ART
The present invention relates to a method for producing lipids such as docosahexaenoic acid (or DHA), DHA-enriched triacylglycerol, or phosopholipids by culturing an oleaginous yeast.
Lipids constitute one of the three major families of macronutrients, along with proteins and carbohydrates. Among the lipids, triglycerides and phospholipids are of particular interest.
Triglycerides (also called triacylglycerols, triacylglycerides or TAGs) are glycerides in which the three hydroxyl groups of glycerol are esterified with fatty acids. They are the main constituent of vegetable oil and animal fats.
Triglycerides represent approximately 95% of the dietary lipids ingested by human beings. In the body, they are present mainly in adipose tissues and constitute the main form of energy storage.
Phospholipids are amphiphilic lipids, i.e., lipids consisting of a polar (hydrophilic) “head” and two aliphatic (hydrophobic) “tails”. Phospholipids are structural lipids since they are constituents of cell membranes of which they provide, inter alia, the fluidity.
Most phospholipids are phosphoglycerides, the head of which is organized around a glycerol-3 -phosphate residue esterified with a polar molecule, and the two tails of which are the aliphatic chains of two fatty acids.
The other phospholipids are sphingomyelins, which derive structurally from sphingosine and not from glycerol, sphingosine constituting one of the two aliphatic tails. The first phospholipids isolated from live tissues were characterized from egg yolk lecithin; they were more particularly phosphatidylcholines. This is, moreover, why phosphatidylcholines are also known as lecithins.
Phosphatidylcholines are naturally produced by the liver. They are an important constituent of bile, in which they emulsify the fats present in the duodenum. They are also necessary, in addition to bile salts, for preventing lipid droplets from re-agglutinating.
As phospholipids, phosphatidylcholines participate in the membranes of cells and serve to preserve their viscoelasticity. They are an essential component of the nervous system and constitute close to 30% of the dry weight of the brain and 15% of the nerves.
Triglycerides and phospholipids are composed predominantly of fatty acids which are both provided by the diet and, for some of them, synthesized by the organism.
The biochemical classification (based on the number of double bonds contained in the fatty acid molecule) distinguishes saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs).
From the physiological point of view, the following are distinguished: indispensable fatty acids, required for development and correct functioning of the human body, but which the body is not able to produce;
“conditionally” indispensable fatty acids, which are essential for normal growth and the physiological functions of cells, but which can be produced from their precursor if it is provided by the diet, and which are therefore rigorously required if their indispensable precursor is absent; and non-indispensable fatty acids.
The set of indispensable and “conditionally” indispensable fatty acids constitutes the essential fatty acids.
The other fatty acids are termed non-essential.
The non-indispensable fatty acids comprise, in particular, eicosapentaenoic acid (EPA) of the omega 3 fatty acid family, oleic acid, the predominant monounsaturated fatty acid in our diet, and saturated fatty acids, such as lauric acid, myristic acid or palmitic acid.
Polyunsaturated fatty acids are classified according to the position of the first double bond, starting from the final methyl function. Thus, in the nomenclature, for omega “x” or “nx”, “x” corresponds to the position of the first unsaturation. Two major families of essential fatty acids are distinguished: omega 6 fatty acids (or n-6 PUFAs), of which the precursor and the major representative is linoleic acid (LA), and omega 3 fatty acids (or n-3 PUFAs), such as alpha-linolenic acid (ALA) and derivatives thereof such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).
The majority of the polyunsaturated fatty acids of biological interest belong to the omega 6 family (arachidonic acid or ARA) or omega 3 family (such as EP A, or DHA).
In addition, in the nomenclature, the carbon number constituting the chain is also defined: thus, EPA is described as C20:5 and DHA as C22:6.
The “5” and “6” thus correspond to the number of unsaturations of the carbon chain presented respectively by EPA and by DHA.
DHA, of the omega 3 fatty acid family, is a fatty acid that the organism can synthesize from alpha-linolenic acid, or which is provided by the consumption of oily fish (tuna, salmon, herring, etc.).
DHA plays an important role in the structure of membranes and in the development and function of the brain and retina.
Fish oils are used mainly as a source of omega 3 fatty acids, such as DHA and EPA, but they are also found in oils of microalgae where they are extracted either as a mixture, or separately, as is the case for example with the oils derived from certain selected strains, such as those of the Schizochytrium genus, which contain only traces of EPA but have high DHA contents.
Commercial preparations of biomasses of microalgae rich in DHA are available. Mention may thus be made, for example, of products of the Algamac range, sold by Aquafauna BioMarine Inc., proposed for nutrition in rotifer aquaculture, or products sold by DSM under the brand name DHA Gold™.
The Applicant has also developed a strain of Schizochytrium mangrovei which produces DHA and which has the particularity of producing very few hypercholesterolemic saturated fatty acids (less than 6% of lauric and myristic acids, which those skilled in the art know are the most hypercholesterolemic known), and more than 40% of palmitic acid (the % understood here to be by weight of total fatty acids). Said strain and its use for producing lipids is described for example in international publication WO2014/122158. Oleaginous yeasts such as Yarrowia lipolytica for Y. lipolytica) a have also been engineered and used as a host to produce high levels of n-3 PUFA such as DHA, EPA and ALA. . .) and of lipid-soluble carotenoids (lycopene, beta-carotenen, astaxanthin...). Therefore, engineered strains of Yarrowia lipolytica with increased lipid contents can be used for enhancing the production of functional lipids and carotenoids.
For example, Xue et al. (Nature Biotechnology, 2013, vol 31:8, p 734-740) describe metabolic engineering of the oleaginous yeast Yarrowia lipolytica in order to increase the production of omega-3 eicosapentaenoic acid by said yeast.
Similarly, Xie et al. (Appl Microbiol Biotechnol 2015, 99 : 1599-1610) describe a Yarrowia platform for producing tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acids in mixtures in the cellular lipid profiles.
Zhu et al. (Current Opinion in Biotechnology, 2015, 36:65-72) provide a review of the recent progress on metabolic engineering of Y. lipolytica for production of biodiesel fuel, functional fatty acids and carotenoids.
In addition to introducing specific enzymes useful in the metabolic pathways for producing lipids such EPA, it is also desirable to increase the overall production of lipids
There is still a need in the art for further methods for increasing the production of desirable lipids by Yarrowia lipolytica.
The Applicant has thus, to its credit, developed such a process, which will be disclosed in more details below.
SUMMARY OF THE INVENTION
The present invention relates to a method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6.
The invention also relates to a host cell transformed with an expression vector encoding the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6, wherein said host cell is an oleaginous yeast. The invention also concerns the Yarrowia lipolytica strain deposited on 9 December 2021 under the Budapest Treaty before the China Center for Type Culture Collection (No. 299, Bayi Road, Wuchang District, Wuhan City, 430072, Hubei Province, Wuhan University) as CCTCC M 20211578 .
Finally, the invention also relates to the use of a nucleic acid comprising the sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3 and/or SEQ ID NO:5 for transforming an oleaginous yeast cell.
DETAILED DESCRIPTION
A first object of the present invention is a method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6.
Indeed, the inventors have surprisingly found that certain genes from the microalgae Schizochytrium mangrovei were able to significantly increase the production of fatty acids in a transformed oleaginous yeast, such as Yarrowia. lipolytica.
In particular, the inventors have isolated and discovered a gene encoding a protein having SEQ ID NO:2, which they have termed “Lipid Droplet-Associated Protein (LDAP)”. When this gene is expressed in an oleaginous yeast such as Y. lipolytica, the overall production of fatty acids is increased.
The mRNA sequence of Idap is set forth in SEQ ID NO: 1 :
Figure imgf000006_0001
Figure imgf000007_0001
The protein sequence of LDAP is set forth in SEQ ID NO:2:
Figure imgf000007_0002
In addition, the inventors have found the sequences of two diacylglycerol acyltransferase (dgat2) genes, which encode proteins named DGAT2-1 and DGAT2-2.
The sequence of the DGAT2-1 mRNA is set forth in SEQ ID NO:3:
Figure imgf000007_0003
Figure imgf000008_0001
The sequence of the DGAT2-1 protein is set forth in SEQ ID NO:4:
Figure imgf000008_0002
Figure imgf000009_0001
The sequence of the DGAT2-2 mRNA is set forth in SEQ ID NO: 5:
Figure imgf000009_0002
The sequence of the DGAT2-2 protein is set forth in SEQ ID NO:6:
Figure imgf000009_0003
Figure imgf000010_0001
The skilled person in the art can readily select a suitable oleaginous yeast. Suitable yeast include any yeast species generally employed in order to produce high yields of lipids. In one embodiment, the oleaginous yeast is selected in the group consisting of the genera Yarrow ia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces.
In a preferred embodiment, the oleaginous yeast is selected in the group consisting of the species Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrow ia lipolytica.
In a preferred embodiment, said oleaginous yeast is a Yarrowia lipolytica yeast.
Any suitable methods for expressing a given nucleic acid can be employed.
Typically, the skilled person knows the method described in the publication by Lv Y, Edwards H, Zhou J, Xu P. Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica. ACS Synthetic Biology. 2019 Mar;8(3):568-576. DOI: 10.1021/acssynbio.8b00535. PMID: 30695641.
In a preferred embodiment, the oleaginous yeast can further be transformed to express other genes known to increase the production of lipids, such as those described in Zhu et al. (Current Opinion in Biotechnology, 2015, 36:65-72).
The invention also relates to a host cell transformed with an expression vector encoding the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6, wherein said host cell is an oleaginous yeast. The invention also concerns the Yarrowia lipolytica strain deposited on 9 December 2021 under the Budapest Treaty before the China Center for Type Culture Collection (No. 299, Bayi Road, Wuchang District, Wuhan City, 430072, Hubei Province, Wuhan University) as CCTCC M 20211578.
Finally, the invention also relates to the use of a nucleic acid comprising the sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3 and/or SEQ ID NO:5 for transforming an oleaginous yeast cell.
The invention will be understood more clearly on reading the examples which follow, which are intended to be purely illustrative and do not in any way limit the scope of the protection.
EXAMPLES
Example 1: Isolation from the S mansrovei strain D5 of “Lipid-Droplet Associated Protein”
Cells were collected at 5000 g for 10 min and washed twice with 30 mL of phosphate buffered saline (PBS). After incubating in 30 ml of buffer A (25 mM tricine, 250 mM sucrose, pH 7.8) on ice for 20 min, cells were homogenized. The cell homogenate was centrifuged in a 50-mL tube at 6000 g for 10 min to remove cell debris and unbroken cells. The supernatant fraction (10 mL) overlaid with 2 mL of buffer B (20 mM HEPES, 100 mM KC1, 2 mM MgC12, pH 7.4) was centrifuged at 38 000 rpm for 1 h at 4°C (Beckman SW40). The white band containing Lipid Droplets (LDs) at the top of the gradient was collected using a 200 μL pipette tip and transferred to a 1.5 mL Eppendorf tube. LDs were washed with 200 μL of Buffer B for three times. The purity of LDs was confirmed by observing staining. Then, total proteins were extracted from LDs and were analyzed by proteomics.
One particular protein, named LDAP, was isolated by mass spectrometry and the corresponding mRNA was sequenced.
The sequence of the Idap mRNA is set forth in SEQ ID NO: 1.
The sequence of the LDAP protein is set forth in SEQ ID NO:2. Example 2: Production of engineered Yarrowia lipolytica expressing Idap
The inventors used a vector pYLXP’2 for overexpressing of Idap in Y. lipolytica. It is a standard procedure for producing engineered Y. lipolytica, described in Lv Y, Edwards H, Zhou J, Xu P. Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica. ACS Synthetic Biology. 2019 Mar;8(3):568-576. DOI: 10.1021/acssynbio.8b00535. PMID: 30695641.
The resulting transformed strains was isolated and further characterized for lipid production.
Example 3: Increase in biomass and overall lipid production
Y. lipolytica does not naturally produce DHA, it can only synthesize usual fatty acids, such as C18:0, C18:1 and C18:2. The oleaginous Y. lipolytica as a host because it can produce abundant acetyl-CoA, which is the precursor for the biosynthesis of functional lipids and carotenoids. In addition, only oleaginous microbes can form obvious LDs and it has been demonstrated that TAG are mainly deposited in LDs in Y. lipolytica. LDAP probably participated in the accumulation of lipids in LDs. The results showed that, when Idap was overexpressed, LDs were be enlarged or stabilized, and more TAG was deposited and generated in Y. lipolytica. Both total lipid titer and yield were greatly enhanced by overexpressing a solo Idap in Y. lipolytica (Table 1 below). LDAP-5 and LDAP-12 represented two selected engineered strains. Data shown here were in triplicate.
Figure imgf000012_0001
Table 1 : Biomass and TFA production in original Polf strain and strains LDAP-5 and LDAP- 12, Results are presented as average ± standard deviation Besides, the inventors noticed that monounsaturated fatty acid C18: 1 were significantly increased in both LDAP-5 and LDAP- 12 (see Table 2), suggesting overexpressing Idap might affect the biosynthesis of fatty acid in Y. lipolytica.
Figure imgf000013_0001
Table 2: Fatty acid profiles in original Polf strain and strains 5 and 12, Results are presented as average ± standard deviation
Strain LDAP-12 (also called pLDAP) was deposited on 9 December 2021 under the Budapest Treaty before the CCTCC collection under deposit number CCTCC M 20211578.
Example 4: Engineered Yarrowia lipolytica expressing Idap and/or dgat
The same methods as in Example 2 were used in order to generate Yarrowia lipolytica strains overexpressing diacylglycerol acyltransferase (DGAT2). DGAT2 is the limiting enzyme involved in the biosynthesis of TAG Indeed, the inventors found two diacylglycerol acyltransferase (DGAT2) genes from the genome of D5. These two DGAT2 encoding genes were codon-optimized and synthesized. Overexpressing either dgat2-1 or dgat2-2 led to an increase of total fatty acids (TFA). The biomass was slightly decreased.
Figure imgf000013_0002
Figure imgf000014_0001
Table 3: Biomass and TFA production in original Polf strain and strains overexpressing the various genes according to the invention. Results are presented as average ± standard deviation Polf, Y. lipolytica, MATa, leu2-270, ura3-302, xpr2-322, axp-2, (Leu2-, Ura3-), LDAP, Polf harboring pYLXP': : ldap: DGAT2-1, Polf harboring dgat2-, DGAT2-2, Polf harboring pYLXP’ : :dgat2-2; LDAP-DGAT2-1, pYLXP’ : :ldap: :dgat2-1, LDAP-DGAT2- 2, pYLXP’ ::ldap::dgat2-2. The inventors have shown that Yarrowia lipolytica yeast expressing the Idap or dgat2-l or dgat2-2 genes from S. mangrovei could be useful in producing fatty acids, in particular DHA.
PCT-SequenceListings BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000015_0002
Figure imgf000015_0001
PCT-SequenceListings BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000016_0001
PCT-SequenceListings_BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000017_0001
PCT-SequenceListings_BR871 - LDAP CLONING YARROWIA LIPOLYTICA WO . txt
Figure imgf000018_0001
PCT-SequenceListings BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000019_0001
PCT-SequenceListings BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000020_0001
PCT-SequenceListings_BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000021_0001
PCT-SequenceListings_BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000022_0001
PCT-SequenceListings BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000023_0001
PCT- Sequence Li st ings_BR871 LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000024_0001
PCT-SequenceListings_BR871 - LDAP CLONING YARROWIA LIPOLYTICA-WO.txt
Figure imgf000025_0001

Claims

CLAIMS :
1. A method for producing lipids comprising culturing an oleaginous yeast, wherein said oleaginous yeast has been transformed to express the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6.
2. A host cell transformed with an expression vector encoding the protein having the amino acid sequence as set forth in SEQ ID NO:2 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:4 and/or the protein having the amino acid sequence as set forth in SEQ ID NO:6, wherein said host cell is an oleaginous yeast.
3. A method according to claim 1 or a host cell according to claim 2, wherein said oleaginous yeast is selected in the group consisting of genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces.
4. A method or host call according to claim 3, wherein the oleaginous yeast is selected in the group consisting of the species Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica.
5. A method or a host cell according to claim 4 wherein said oleaginous yeast is a strain of Yarrowia lipolytica.
6. The Yarrowia lipolytica deposited under the Budapest Treaty on 9 December 2021 as CCTCC M 20211578.
7. Use of a nucleic acid comprising the sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3 and/or SEQ ID NO:5 for transforming an oleaginous yeast cell.
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