CN116555362A - Method for producing lipids using yarrowia lipolytica - Google Patents

Method for producing lipids using yarrowia lipolytica Download PDF

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CN116555362A
CN116555362A CN202210110527.4A CN202210110527A CN116555362A CN 116555362 A CN116555362 A CN 116555362A CN 202210110527 A CN202210110527 A CN 202210110527A CN 116555362 A CN116555362 A CN 116555362A
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oleaginous yeast
protein
yarrowia lipolytica
amino acid
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夏炜
B·L·R·珀拉
万霞
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Roquette Co
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Roquette Co
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Abstract

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

Description

Method for producing lipids using yarrowia lipolytica
Technical Field
The present invention relates to a method for producing lipids comprising culturing oleaginous yeast, wherein the oleaginous yeast has been transformed to express a lipid-associated protein.
Background
The present invention relates to a method for producing lipids, such as docosahexaenoic acid (or DHA), triacylglycerols enriched in DHA or phospholipids, by culturing oleaginous yeast.
Lipids, together with proteins and carbohydrates, constitute one of three major families of macronutrients. Of the lipids, triglycerides and phospholipids are of particular interest.
Triglycerides (also known as triacylglycerols, triacylglycerides or TAGs) are glycerides in which three hydroxyl groups of glycerol are esterified with fatty acids. They are the main components of vegetable oils and animal fats.
Triglycerides constitute about 95% of dietary lipids ingested by humans. In the body, they are mainly present in adipose tissue 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 because they are components of the cell membrane, they provide fluidity, among other things.
Most phospholipids are phosphoglycerides, the head of which is organized around glycerol-3-phosphate residues esterified with polar molecules, and the two tails of which are aliphatic chains of two fatty acids.
Other phospholipids are sphingomyelins, which are structurally derived from sphingosine, which constitutes one of the two aliphatic tails, instead of from glycerol.
The first phospholipid isolated from living tissue is characterized from egg yolk lecithin; they are more particularly phosphatidylcholine. In addition, this is why phosphatidylcholine is also known as lecithin.
Phosphatidylcholine is naturally produced by the liver. They are important components of bile, in that they emulsify the fat present in the duodenum. In addition to bile salts, they are also necessary to prevent lipid droplets from re-coalescing.
As phospholipids, phosphatidylcholine participates in cell membranes and serves to maintain their viscoelastic properties. They are essential components of the nervous system and constitute nearly 30% of the dry brain weight and 15% of the nerves.
Triglycerides and phospholipids are mainly composed of fatty acids, both of which are provided by the diet and, for some of them, are synthesized by the organism.
Biochemical classification (based on the number of double bonds contained in the fatty acid molecule) distinguishes between Saturated Fatty Acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA).
From a physiological point of view, the differences are as follows:
essential fatty acids, which are required for human development and correct function, but which are not produced by the body;
"conditioned" essential fatty acids, which are necessary for the normal growth and physiological function of the cells, but which can be produced from their precursors if provided by diet, and which are therefore strictly necessary if their essential precursors are not present; and
-non-essential fatty acids.
The organization of essential and "conditional" essential fatty acids constitutes the essential fatty acids.
Other fatty acids are referred to as optional.
In particular, the non-essential fatty acids comprise eicosapentaenoic acid (EPA), oleic acid, the main monounsaturated fatty acids in our diet and saturated fatty acids (such as lauric, myristic or palmitic acid) of the omega 3 fatty acid family.
Polyunsaturated fatty acids are classified according to the position of the first double bond starting from the final methyl function. Thus, in terms of ω "x" or "nx," x "corresponds to the location of the first unsaturation.
Two major families of essential fatty acids are distinguished: omega 6 fatty acids (or n-6 PUFAs), where the precursor and primary representation are Linoleic Acid (LA), and omega 3 fatty acids (or n-3 PUFAs), such as alpha-linolenic acid (ALA) and derivatives thereof (e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)).
Most biologically interesting polyunsaturated fatty acids belong to the omega 6 family (arachidonic acid or ARA) or the omega 3 family (such as EPA or DHA).
In addition, in the terminology, the number of carbon atoms constituting the chain is also defined as: thus EPA is described as C20:5 and DHA as C22:6.
Thus, "5" and "6" correspond to the number of unsaturations of the carbon chains represented by EPA and DHA, respectively.
DHA of the omega 3 fatty acid family is a fatty acid that the organism can synthesize from alpha-linolenic acid, or it is provided by edible oily fish (tuna, salmon, herring, etc.).
DHA plays an important role in the structure of the membrane, and in the development and function of the brain and retina.
Fish oils are mainly used as a source of omega 3 fatty acids, such as DHA and EPA, but they are also present in microalgae oils where they are extracted as mixtures or alone, for example from certain selected strains, such as those of schizochytrium (Schizochytrium genus), which contain only trace amounts of EPA but have a high DHA content.
Commercial preparations of DHA-rich microalgae biomass are available. Thus, mention may be made of the Algamac series of products sold, for example, by Aquafauna Bio-Marine Inc., suggesting nutrition for use in rotifer aquaculture, or by DSM under the trade name DHA Gold TM And (5) selling the product.
The applicant has also developed a strain of schizochytrium mangrove (Schizochytrium mangrovei) producing DHA, which has the property of producing very little hypercholesterolemic saturated fatty acids (less than 6% lauric and myristic acids, the most hypercholesterolemic known to the person skilled in the art) and more than 40% palmitic acid (understood herein as% by weight of total fatty acids). Said strains and their use for the production of lipids are described, for example, in international publication WO 2014/122158.
Oleaginous yeasts such as yarrowia lipolytica (Yarrowia lipolytica or y. Lipolytica) have also been engineered and used as hosts to produce high levels of n-3 PUFAs such as DHA, EPA and ala and fat-soluble carotenoids (lycopene, beta-carotene, astaxanthin …). Thus, engineered yarrowia lipolytica strains with increased lipid content can be used to enhance 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 to increase omega-3 eicosapentaenoic acid production by the yeast.
Similarly, xie et al (Appl Microbiol Biotechnol 2015, 99:1599-1610) describe a yarrowia platform for producing custom omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acids in a mixture of cell lipid profiles.
Zhu et al (Current Opinion in Biotechnology,2015, 36:65-72) reviewed recent advances in the metabolic engineering of yarrowia lipolytica for the production of biodiesel fuels, functional fatty acids and carotenoids.
In addition to introducing specific enzymes useful in metabolic pathways for the production of lipids (e.g., EPA), there is a need to increase the overall yield of lipids.
There remains a need in the art to add other methods for producing desired lipids by yarrowia lipolytica.
Thus, the applicant's mahonia is in developing such a process, which will be disclosed in more detail below.
Summary of The Invention
The present invention relates to a method for producing lipids, comprising culturing oleaginous yeast, wherein the oleaginous yeast has been transformed to express a protein having an amino acid sequence as shown in SEQ ID NO. 2 and/or a protein having an amino acid sequence as shown in SEQ ID NO. 4 and/or a protein having an amino acid sequence as shown in SEQ ID NO. 6.
The invention also relates to a host cell transformed with an expression vector encoding a protein having the amino acid sequence shown in SEQ ID NO. 2 and/or a protein having the amino acid sequence shown in SEQ ID NO. 4 and/or a protein having the amino acid sequence shown in SEQ ID NO. 6, wherein the host cell is an oleaginous yeast.
The invention also relates to a yarrowia lipolytica (Yarrowia lipolytica) strain which was preserved under the Budapest treaty at day 12 and 9 of 2021 with CCTCC M20211578 at the China center for type culture Collection (eight 299 lines of Wuchang district, wuhan university, wuhan, hubei province, china).
Finally, the invention also relates to the use of nucleic acids comprising the sequences shown in SEQ ID No. 1, SEQ ID No. 3 and/or SEQ ID No. 5 for transforming oleaginous yeast cells.
Detailed Description
A first object of the present invention is a method for producing a lipid, comprising culturing oleaginous yeast, wherein the oleaginous yeast has been transformed to express a protein having an amino acid sequence as shown in SEQ ID NO. 2 and/or a protein having an amino acid sequence as shown in SEQ ID NO. 4 and/or a protein having an amino acid sequence as shown in SEQ ID NO. 6.
Indeed, the inventors have surprisingly found that certain genes from the microalgae schizochytrium mangrove (Schizochytrium mangrovei) are capable of significantly increasing the yield of fatty acids in transformed oleaginous yeast, such as yarrowia lipolytica.
In particular, the present inventors have isolated and found genes encoding proteins having SEQ ID NO. 2, which are referred to as "Lipid Droplet Associated Proteins (LDAP)".
When the gene is expressed in oleaginous yeast (e.g., yarrowia lipolytica), the total yield of fatty acids increases.
The mRNA sequence of ldap is shown in SEQ ID NO: 1:
ATGCCTGCCGCCAAGACCATGCAAGAGTCCAAAGTCCCGG ACGATGACTATGTGCTCCACGAGCCGCACAATGGCCCCGTTGGT AAGCTCATCGACATGAGCTACGAACAGTACTCGGGTGCCAAGG ACATGCTCGCCAAGAACCAGGTGATCGGCAAGCGCCTCACTCC ATTCGTGGACTGGACTGAGAACACCACCAAGGCGATTCTCAGA AAGTCCCCGGTCGCTGTCGACAAGGTCGTCTCGGCCGTCGACAC CCGCGCAGAGCGTGTTGTTAACTTCACCTCTGACAAGGTACAGA CCATTCGTGACACTCCCAAAAATACTCTTGGATATGTTCATGGC AAGCTGAAATCCTTGCGTACTGAGCCCCAGGAAGCCGAGAATG AGGAACCTGGTGTCCGCACCATCATCATCACCGGCAAGTCTGCC GCTGCTGAGCGCCTCAATGTCCTCCTCGATTCTAGCGAGGGGTA CCTCAAGCAGTACCTCCCCATCTCTGATGATGAGAAGGCGCTCA TGGTCAATGGCTCCAAGTCTGAGATCCGCACCGTCGCTACTCGC ACGGTCAACCTTTCCAAGGTGGCCATCTCCAAGGCCATCGAGC GCGCTCTTAACCGCGCCGGTGAAATCAAGGAGCGCACCAAGGA GACCATTCATGTGGATCTCATTCGCTACAACGAGTGGCTTGACA TGAACGTTAAGCAGCCCGTTTCGCGTCGCCTCAAGATTGTCGAC GACAAGCTTGCCATCTCTGAGAAGGTTGACAAGGTCGACCAGA AGGTTGTCAAGCCCATTAAAAACTCCATCAACCGTCGTGTCGAG CTCATTGATGCCAAGGTTGTGACCCCAGTTAAGGAAAAGTTTGT ACTCGTTGTCACTCGTGTCAGCGACACTTACCAGCACAAGGTTG TTGAGCCTCGTGACCAGATCATCCAAATGTTCCGCGAGGAACTT AGCCTCCAGCAAGAAATCGCCAAGAGCAAGTCCGGTGAGGAGG AGCTTACCATTAGTGCTGGCCTCTCTGCCGTCATTGCCGCCGCT AAGTCTCGCCTCGAGAAGGAGTACGAGGTTCGCGTCTCCCCGG CCCTCCACCGCTTCATGGGCCGCGAAAAGGAAGAGGAGATTGA GGACCAGTCTTTCGGTGATGATGTTGAGGAGGACGAGGATGTC GATGAGACCTACTAA
the protein sequence of LDAP is shown as SEQ ID NO. 2:
MPAAKTMQESKVPDDDYVLHEPHNGPVGKLIDMSYEQYSGA KDMLAKNQVIGKRLTPFVDWTENTTKAILRKSPVAVDKVVSAVD TRAERVVNFTSDKVQTIRDTPKNTLGYVHGKLKSLRTEPQEAENE EPGVRTIIITGKSAAAERLNVLLDSSEGYLKQYLPISDDEKALMVN GSKSEIRTVATRTVNLSKVAISKAIERALNRAGEIKERTKETIHVDLI RYNEWLDMNVKQPVSRRLKIVDDKLAISEKVDKVDQKVVKPIKN SINRRVELIDAKVVTPVKEKFVLVVTRVSDTYQHKVVEPRDQIIQM FREELSLQQEIAKSKSGEEELTISAGLSAVIAAAKSRLEKEYEVRVS PALHRFMGREKEEEIEDQSFGDDVEEDEDVDETY
in addition, the present inventors have found the sequences of two diacylglycerol acyltransferase (DGAT 2) genes, which encode proteins known as DGAT2-1 and DGAT 2-2.
The sequence of the DGAT2-1mRNA is shown as SEQ ID NO: 3:
ATGTCTTCAAGCCCCGCCAAAGCTCCTCCTGCTCGCCAGCA AACTGGCAATGGGGTGTCTAGTCGGAGCAGCGTGTCCCAGCGT CGTCGCGTAGAGAAGCTCAAGCAAGGCTATGGCGAGGAGGAGG ATGGAAGCAACACTCCTACAGGCCTGAACACGCCTTACAGCAC ATCCATGGGCTCTATTTCAAGTTATTCGTCTTCTGGAGACTACG CTCAGTCCGAAGGCGAAGGATCTGAGCCTGCTCTGGACCCTGC GGACAGTGTGGATGGCAGTCCAGGCAAGCGGGATTCTGCGTAC AACAAATCGTCCAAAAGACAACTCACGCAAGAAGAGCGCGAAC TCTTCTTGCGTCTTGAGAAGGAATGGCGCGAGGAGGACTCTTGG GCAGAACAACCTGGGTCCTGGTACTCAATGCTTGCGTGGATGCC TGTTCTTATTGCACTTCGCGTGTTTAACGTCTTCCTCTCGATCCT CTTTTGGCCTGTATCATTTGTGGCGCGGGTCTTCTTTGGAAAGG AGATCCACTCGGTTAGTTTTTGGGACGTTCCGCTAGCTCGACGC AAGCAAACTGCAGTGGTCTTGCTTTTCGTTATGCTTTTGCCGAT GATTATGGTAGTCTACTCTTGGACCCTGATCCTCCTCATTTTCCC ACTCACGACCCTTCCCACACTTTCATACCTTATCTGGATTATGTA TATTGACAAGTCGCATGAGACTGGTAAGCGCAAGCCGTTCATG CGTTATTGGAAAATGTGGCGCCACTTTGCAAACTACTTCCCTTT GCGCCTCATCCGTACAACCCCCCTTGATCCGCGCAGGAAATATG TATTTTGCTACCACCCGCACGGGATCATTTCGCTTGGTGCCTTTG GTAACTTCGCAACTGATTCCACTGGTTTCTCGCGCAAGTTTCCA GGCATTGACTTGCGCCTTCTTACTTTGCAGATCAACTTTTATTGC CCAATCATTCGGGAGCTTCTGCTGTATATGGGTCTCTGCTCTGC CGCCAAGAAGTCATGCAATCAAATTCTCCAGCGCGGACCTGGG TCAGCTATCATGCTTGTCGTAGGTGGTGCTGCAGAATCCCTTGA CTCGCAACCTGGTACATATCGTCTCACGCTTGGTCGCAAGGGCT TCGTTCGTGTTGCACTTGACAACGGTGCCGATCTTGTTCCTGTTC TAGGATTTGGTGAGAATGATGTCTTCGATACCGTCTACTTACCA CCCAACTCCTGGGCACGAAATGTTCAAGAGTTCGTTCGAAAGA AGCTTGGCTTTGCGACTCCAATTTTTAGTGGACGAGGTATCTTC CAATACAACATGGGCCTCATGCCGCACCGCAAGCCTATCATTAT TCCCAAGATCCCGGATGAATTAAAGGGCCGTGCTCTCTCTACAA CCGCAGAAGGTGTCGCCTTAGTTGATAAGTACCACGAGAAATA TGTCCGGGCTCTGCGTGAGCTATGGAACTTGTACAAGGAGCGCT GGGCTGTTCACCGCCAAGGTTCTCTTTTAATTCAGAAGTAA
the sequence of the DGAT2-1 protein is shown as SEQ ID NO. 4:
MSSSPAKAPPARQQTGNGVSSRSSVSQRRRVEKLKQGYGEEE DGSNTPTGLNTPYSTSMGSISSYSSSGDYAQSEGEGSEPALDPADS VDGSPGKRDSAYNKSSKRQLTQEERELFLRLEKEWREEDSWAEQP GSWYSMLAWMPVLIALRVFNVFLSILFWPVSFVARVFFGKEIHSVS FWDVPLARRKQTAVVLLFVMLLPMIMVVYSWTLILLIFPLTTLPTL SYLIWIMYIDKSHETGKRKPFMRYWKMWRHFANYFPLRLIRTTPL DPRRKYVFCYHPHGIISLGAFGNFATDSTGFSRKFPGIDLRLLTLQI NFYCPIIRELLLYMGLCSAAKKSCNQILQRGPGSAIMLVVGGAAES LDSQPGTYRLTLGRKGFVRVALDNGADLVPVLGFGENDVFDTVY LPPNSWARNVQEFVRKKLGFATPIFSGRGIFQYNMGLMPHRKPIIIP KIPDELKGRALSTTAEGVALVDKYHEKYVRALRELWNLYKERWA VHRQGSLLIQK
the sequence of the DGAT2-2mRNA is shown as SEQ ID NO. 5:
ATGGATCGCACAGCAGAATGCTCTGCGAAGGAACCTTATT ACTCACTTGACGTATGGCGAATCCCAACCGCTTTGCACGCGTCA AAAGTGGTGCCACAAGATGCAGACGAGGAGACTCGCACAAGGT TAGAAGATGAGGCAAACGAGGTGCTTCTCCGGGAGTTCGCCGT GAAGCATGATGGTCCATTTCCTGAAGACTTTCTAACAGCAAAG GCTGACATCACATTTTCAGAAGAAATCATGGTGCTTTCTGCTTT GCTCGTGACTCTCGGTGGCCCACTCTTTTGGTTTTCGGCTGGCCT CCTCACGTTCATCGCTGCATCATGGACATCAGTTATGTTGTACA TAATCCTGACAGCCTTCCTTGCTTTCCACCCACTCCCTAACTCTA TTCCTGCTTTATGGACTTCGCCTTTGATCATCGCAATGTACAAGT ACTTCTCGTACAGGTTTGTGTGGAAAGGCAACGCACGAGAAAT GCTACGATCGAACAAGCAATTTCTTGGTTCAGGCGTGCCTCACG GTGTGATGCCGTTTGCTAATCTTCTTTGCATCCCTGCGACGAAC TCTGTTCTGTACAGAGGCTGCAATTTTTGGGGTGCCCCAGCGTC AGTTGTGTTTCATACTCCTTTCCTGCGATACTTGTCAATTCTGCA GTGTTGCCATGTTGGCCGCGAAGCAATAATGCGCGAACTTGAA GTTGGGCACTCTGTAGGTTTAGTTGGTGATGGAATTGCTGGAAT CTTTCAATCAAACCATGACGACGAGGTTGTTGCTCTGAAGCACC GCAAGGGCTTGGCGAAGCTTGCGTTACGGACAGGTACTCCTGT GCTTCCTTGCTACTCACTGGGAAATACCGCTGCCTTTTCTGCAT GGTTTGACAGTTATGGAGTCATGGAGTGGCTTTCTCGCAAAGCT CAGGCCTCTATTTTCTTTTATTGGGGTCGGTTTGGCCTACCTATC CCACATCGTGTAAATATTACTATGATTGTAGGCAACATGGTTCT TGTAGAAAAAGTAGACGAGCCATCAGAAGATCAGATCCAAGTC CTTCATGATAAGATTTTGGATGGGTTCCGGGATGCGTTTGACTC GCATAAAGCATCGCTAGGATGGGCGCACGGAAGTTGCGCTTTG TGTAACAAAGCGTTGCTACTGCAGGGCGGAATCTAG
the sequence of the DGAT2-2 protein is shown as SEQ ID NO. 6:
MDRTAECSAKEPYYSLDVWRIPTALHASKVVPQDADEETRTR LEDEANEVLLREFAVKHDGPFPEDFLTAKADITFSEEIMVLSALLV TLGGPLFWFSAGLLTFIAASWTSVMLYIILTAFLAFHPLPNSIPALW TSPLIIAMYKYFSYRFVWKGNAREMLRSNKQFLGSGVPHGVMPFA NLLCIPATNSVLYRGCNFWGAPASVVFHTPFLRYLSILQCCHVGRE AIMRELEVGHSVGLVGDGIAGIFQSNHDDEVVALKHRKGLAKLAL RTGTPVLPCYSLGNTAAFSAWFDSYGVMEWLSRKAQASIFFYWG RFGLPIPHRVNITMIVGNMVLVEKVDEPSEDQIQVLHDKILDGFRD AFDSHKASLGWAHGSCALCNKALLLQGGI
suitable oleaginous yeasts can be readily selected by those skilled in the art. Suitable yeasts include any yeast species commonly used to produce high yields of lipids.
In one embodiment, the oleaginous yeast is selected from the group consisting of: yarrowia (Yarrowia), candida (Candida), rhodotorula (Rhodotorula), rhodosporidium (Rhodosporidium), cryptococcus (Cryptococcus), candida (Trichosporon) and olea (Lipomyces).
In a preferred embodiment, the oleaginous yeast is selected from the group consisting of: oleaginous yeast of the genus s (Lipomyces starkeyi), rhodosporidium toruloides (Rhodosporidium toruloides), rhodotorula glutinis (Rhodotorula glutinis) and yarrowia lipolytica (Yarrowia lipolytica).
In a preferred embodiment, the oleaginous yeast is yarrowia lipolytica.
Any suitable method of expressing a given nucleic acid may be used.
Typically, those skilled in the art are aware of the conditions found in Lv Y, edwards H, zhou J, xu P. Coding 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia Limetica. ACS Synthetic biology.2019Mar;8 (3): 568-576.DOI: 10.1021/acslynbio.8b 00535.PMID:30695641.
In a preferred embodiment, oleaginous yeast may be further transformed to express other genes known to increase lipid production, such as those described by 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 a protein having the amino acid sequence shown in SEQ ID NO. 2 and/or a protein having the amino acid sequence shown in SEQ ID NO. 4 and/or a protein having the amino acid sequence shown in SEQ ID NO. 6, wherein the host cell is an oleaginous yeast.
The invention also relates to a yarrowia lipolytica (Yarrowia lipolytica) strain which was preserved under the Budapest treaty at day 12 and 9 of 2021 with CCTCC M20211578 at the China center for type culture Collection (eight 299 lines of Wuchang district, wuhan university, wuhan, hubei province, china).
Finally, the invention also relates to the use of nucleic acids comprising the sequences shown in SEQ ID No. 1, SEQ ID No. 3 and/or SEQ ID No. 5 for transforming oleaginous yeast cells.
The invention will be more clearly understood from a reading of the following examples, which are merely illustrative and do not limit the scope of the invention in any way.
Detailed Description
Example 1: isolation of merozoite mangrove Strain D5 from "lipid droplet related proteins
Cells were collected at 5000g for 10 min and washed twice with 30mL Phosphate Buffered Saline (PBS). After incubation for 20 min on ice in 30ml buffer a (25 mM tricine, 250mM sucrose, pH 7.8), the cells were homogenized. The cell homogenate was centrifuged at 6000g for 10 min in a 50mL tube to remove cell debris and uncrushed cells. The supernatant fraction (10 mL) covered with 2mL of buffer B (20 mM HEPES, 100mM KCl, 2mM MgCl2, pH 7.4) was centrifuged at 38000rpm for 1h at 4℃to give BeckmanSW 40. A white band containing Lipid Droplets (LD) at the top of the gradient was collected using a 200. Mu.L pipette tip and transferred to a 1.5mL Eppendorf tube. LD was washed three times with 200. Mu.L buffer B. The purity of LD was confirmed by observation of the staining. Then, total proteins were extracted from LD and analyzed by proteomics.
One specific protein, called LDAP, was isolated by mass spectrometry and the corresponding mRNA was sequenced.
The sequence of the ldap mRNA is shown in SEQ ID NO. 1.
The sequence of the LDAP protein is shown as SEQ ID NO. 2.
Example 2: production of ldap expressing engineered yarrowia lipolytica
The inventors used the vector pYLXP'2 to overexpress ldap in yarrowia lipolytica. It is a standard procedure for the production of engineered yarrowia lipolytica as described by Lv Y, edwards H, zhou J, xu P. 26srDNA was combined with the Cre-loxP system for repeated gene integration and efficient marker immobilization in yarrowia lipolytica. ACS Synthetic biology 2019Mar;8 (3) 568-576. DOI:10.1021/acslynbio.8 b00535.PMID 30695641.
The resulting transformed strain was isolated and further characterized for lipid production.
Example 3: increased biomass and total lipid production
Yarrowia lipolytica does not naturally produce DHA, but it can only synthesize common fatty acids, such as C18:0, C18:1 and C18:2. Oleaginous yarrowia lipolytica is a host in that it is capable of producing abundant acetyl-CoA, a precursor of biosynthetically functional lipids and carotenoids. Furthermore, only oleaginous microorganisms can form distinct LD, and TAG has been demonstrated to be deposited primarily in LD in yarrowia lipolytica. LDAP may be involved in the accumulation of lipids in LD. The results indicate that when ldap is overexpressed, LD increases or stabilizes and deposits and produces more TAG in yarrowia lipolytica. By over-expressing ldap alone in yarrowia lipolytica, both total lipid titer and yield were greatly enhanced (table 1 below). LDAP-5 and LDAP-12 represent two selected engineered strains. The data presented herein were repeated three times.
Table 1: biomass and TFA production of the original Polf strain and strains LDAP-5 and LDAP-12. Results are expressed as mean + - Standard deviation representation
Furthermore, the inventors noted a significant increase in monounsaturated fatty acid C18:1 in both LDAP-5 and LDAP-12 (see Table 2), indicating that overexpression of LDAP may affect fatty acid biosynthesis in yarrowia lipolytica.
Table 2: fatty acid profile in original Polf strain and strains 5 and 12. Results are expressed as mean ± standard deviation
The strain LDAP-12 (also called pLDAP) was preserved in CCTCC under the preservation number CCTCC M20211578 by the Budapest treaty at 2021, 12 months and 9 days.
Example 4: engineered yarrowia lipolytica expressing ldap and/or dgat
The same method as in example 2 was used to produce yarrowia lipolytica strain overexpressing diacylglycerol acyltransferase (DGAT 2). DGAT2 is a restriction enzyme involved in TAG biosynthesis.
In fact, the inventors found two diacylglycerol acyltransferase (DGAT 2) genes from the genome of D5. These two genes encoding DGAT2 were codon optimized and synthesized. Overexpression of dgat2-1 or dgat2-2 results in an increase in Total Fatty Acids (TFA).
The biomass is slightly reduced.
Table 3: biomass and TFA production in the original Polf strain and strains overexpressing various genes according to the invention And (5) producing. Results are expressed as mean ± standard deviation
Po1f,Y.lipolytica,MATa,leu2-270,ura3-302,xpr2-322,axp-2, (Leu2 - ,Ura3 - )；LDAP,Po1f harboring pYLXP’::ldap；DGAT2-1,Po1f harboring dgat2-；DGAT2-2,Po1f 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 expressing the ldap or dgat2-1 or dgat2-2 genes from schizochytrium mangrove strains can be used to produce fatty acids, in particular DHA.

Claims (7)

1. A method of producing lipids comprising culturing oleaginous yeast, wherein the oleaginous yeast has been transformed to express a protein having an amino acid sequence as shown in SEQ ID No. 2 and/or a protein having an amino acid sequence as shown in SEQ ID No. 4 and/or a protein having an amino acid sequence as shown in SEQ ID No. 6.
2. A host cell transformed with an expression vector encoding a protein having the amino acid sequence as shown in SEQ ID No. 2 and/or a protein having the amino acid sequence as shown in SEQ ID No. 4 and/or a protein having the amino acid sequence as shown in SEQ ID No. 6, wherein the host cell is oleaginous yeast.
3. The method of claim 1 or the host cell of claim 2, wherein the oleaginous yeast is selected from the group consisting of: yarrowia, candida, rhodotorula, rhodosporidium, cryptococcus, candida and olea.
4. A method or host cell according to claim 3, wherein the oleaginous yeast is selected from the group consisting of: oleaginous yeast of the St.dara, rhodosporidium toruloides, rhodotorula glutinis and yarrowia lipolytica.
5. The method or host cell of claim 4, wherein the oleaginous yeast is a yarrowia lipolytica strain.
6. Yarrowia lipolytica which was preserved under the budapest treaty at 2021, 12 months and 9 days under cctccc M20211578.
7. Use of a nucleic acid comprising a sequence as shown in SEQ ID No. 1, SEQ ID No. 3 and/or SEQ ID No. 5 for transforming oleaginous yeast cells.
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