Lysophospholipid production

Abstract

The invention relates to methods and engineered biological systems for producing lysophospholipids (LPLs), including lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPS), particularly those comprising omega‑3 and omega‑6 fatty acids. The invention further provides compositions comprising LPLs, and uses of said compositions in industrial products and pharmaceutical compositions.

Description

[0001] 008933087

[0002] 1

[0003] Lysophospholipid production

[0004] Technical Field

[0005] The present invention relates to methods of producing lysophospholipids (LPL), including LPLs comprising omega-3 fatty acids and / or an omega 6-fatty acids. In particular, the methods of the invention involve enzymes for catalysing LPL production. Reaction mixtures and engineered cells that are capable of performing the LPL production methods are also encompassed.

[0006] Background

[0007] Essential omega-3 fatty acids are vital for brain development and often exist as part of a phospholipid. Specifically, docosahexaenoic acid (DHA) is highly enriched in brain phospholipids, particularly in the phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) pools within membranes. DHA comprises up to 20% or more of the total fatty acid composition of the prefrontal cortex. In humans, brain acquisition of DHA occurs as early as the end of the second trimester, coinciding with the development of the blood brain barrier (BBB) where considerable amounts of membrane phospholipids are required for the growing brain. DHA is continuously acquired from early postnatal days until approximately 2 years of age (Cunnane etal., 2000; Martinez 1992; and Svennerholm 1968).

[0008] Decreased levels of DHA in the developing brain have been associated with negative effects on cognitive function Guesnet and Alessandri 2011; McNamara 2010), and neurodevelopmental disorders (Colombo 2004, Heird and Lapillonne 2005; Martinez 1996). Importantly, DHA cannot be de novo synthesized by the brain and must be transported across the BBB into brain. Historically, both the scientific community and the DHA supplement industry rallied around the concept that DHA fatty acids diffuse across the BBB. This concept was beneficial to the DHA supplement industry because consumer DHA products are of two chemically distinct types: triglyceride-DHA (e.g. fish oil) and phospholipid-DHA (krill oil). These products when ingested and processed by the human gut circulate as lipoprotein triglycerides which were suggested, without strong supporting biochemical evidence, to be taken up at the BBB as DHA fatty acid. Omega fatty acids are a class of saturated fatty acids which have a carbon-carbon double bond in the alkyl chain. The position of the “first” carbon-carbon double bond relative to the methyl end (i.e. the cocarbon) of the alkyl chain determines the species of an Omega fatty acid. For example, Omega-3 (co-3) fatty acids, such as DHA, are a class of saturated fatty acids which have a first carbon-carbon double bond located three carbons from the methyl end of the alkyl chain. Omega-3 fatty acids are considered as essential fatty acids as they cannot be synthesized in adequate amounts by humans and must be obtained through diet. Similarly, omega-6 (co-6) fatty acids have a first carbon-carbon double bond located six carbons from the methyl end of the acyl chain. Omega-6 fatty acids are also considered to be essential fatty acids. Omega-3 fatty acids may have a carbon double bond at both the w-3 and w-6 positions.In 2014, the inventors identified the pathway by which the brain obtains omega-3 fatty acids: a transporter named Major Facilitator Superfamily Domain containing 2a (Mfsd2a) expressed at the BBB transports a specialized lysophospholipid (LPL) named lysophosphatidylcholine (LPC) that is the physiologically relevant form of omega-3 fatty acids that is taken up by the brain via Mfsd2a (Nguyen et al., 2014). LPLs are derived from phospholipids. These studies show that LPC transport is critical for brain growth and myelination. The main sources of plasma LPCs are found in plasma lipoproteins and albumin. LPCs on lipoproteins contain primarily saturated fatty acids — not polyunsaturated fatty acids (PUFAs, e.g. DHA, EPA) — and are generated through the action of lecithin-cholesterol acyltransferase (LCAT), hepatic lipase and endothelial lipase. These lipoprotein-derived LPCs are embedded in the lipoprotein lipid layer and are not readily exchangeable with cellular membranes and hence not a source for uptake by Mfsd2a. In contrast, circulating albumin is the pool of LPCs containing PUFAs (LPC-PUFA) and is the source for uptake of PUFAs by Mfsd2a (Nguyen et al., 2014). While the biochemical origin of the most abundant plasma lipids are known (i.e. lipoprotein lipids, fatty acids, steroid hormones, bile acids, sphingosine-1-phosphate, phosphatidic acid), and despite the fact that LPCs were first discovered associated with albumin in human plasma in 1965 (Switzer and Eder, 1995), the biochemical origin of the critical pool of LPC-PUFA remains unknown. The liver is considered to be the source of plasma LPC and hepatocytes are thought to release LPC-PUFA to be picked up by extracellular albumin. However, prior to the present disclosure, the enzyme(s) that generate plasma LPC-PUFA and other plasma LPLs comprising PUFAs had not been identified.

[0009] Beyond the brain, LPLs comprising omega-3 and omega-6 PUFAs and MUFAs are biologically significant in a range of cellular processes. For example, Lysophosphatidylethanolamine (LPE) and Lysophosphatidylserine (LPS) play pivotal signalling roles as well as being precursors for membrane biogenesis. The significance of LPLs in biological systems is reviewed in Tan et al., Prog Lipid Res. (2020) 80:101068, which is hereby incorporated by reference in its entirety.

[0010] All commercially available sources of omega-3 lipids for dietary supplements and food ingredients come from microalgae, fish and krill. While doses vary, a typical fish oil supplement provides about 1 ,000 mg fish oil, containing 180 mg EPA and 120 mg DHA (National Institutes of Health Office of Dietary Supplements - Omega-3 Fatty Acids). However, despite the most biologically available forms of omega-3 and omega-6 being LPL-Omega-3 / 6, omega-3 supplements currently on the market are in the form of triglycerides (from fish oils) or phospholipids (from krill) and do not comprise significant proportions of biologically available LPLs. Importantly, triglyceride PUFAs are not transported across the BBB by Mfsd2a. As such, for essential fatty acids such as DHA which are ingested as part of a triglyceride or phospholipid to reach the brain, they must first be converted to lysophospholipids, associate with a carrier protein, and then be transported across the BBB. As in most metabolic pathways, product yield can be lost at each step of an enzymatic pathway, thus the quantity of omega fatty acids reaching brain compared to the quantity ingested is potentially considerably less. Accordingly, there is a need for dietary supplements comprising biologically available forms of omega-3 / 6 fatty acids (and other fatty acids) which can be readily transported across the BBB via Mfsd2a.

[0011] 008933087Moreover, LPLs comprising omega PUFAs and MUFAs have a broad range of applications beyond nutritional supplements. LPLs demonstrate a greater hydrophilicity relative to the phospholipids they are derived from, thus providing LPLs with a greater emulsification capacity than phospholipids. As such, LPLs are commonly used as emulsifiers in food products, cosmetics and pharmaceuticals. In agriculture, LPLs are commonly added to a range of animal feeds to improve nutritional absorption, leading to enhance growth rates and greater productivity (Che et al., 2023; and Kinh etal., 2022). In the cosmetics sector, LPLs have been considered a safe emulsifier and have been used extensively. In food production, LPLs are used as emulsifiers (e.g. dairy and bakery products) and in beverages to improve texture and flavour of the products. LPLs allow beverages to maintain their clear colour while comprising oils for flavourings. LPLs also find use as pharmaceutical excipients (van Hoogevest et al., 2014).

[0012] The manufacture of omega-3 containing products, particularly those for the dietary supplement markets, still uses krill oil and thus still relies on harvesting krill from Antarctica. The production of these supplements is not sustainable in the long term and comes with a cost to the environment with harvesting, processing, and shipping having a collateral impact on the environment in Antarctica and along the processing chain. As such, there is also an unmet need for efficient and sustainable lysophospholipid production methods with reduced environmental impact.

[0013] The present invention has been devised in light of the above considerations.

[0014] Summary of the Invention

[0015] Using bioengineering approaches, the inventors have developed a novel method for producing a broad range of lysophospholipids (LPL), including LPC, LPE and LPS, with reduced environmental impact compared to current methods. The methods provided in the present invention utilise enzymes from the TMEM150 protein family. The inventors demonstrate, for the first time, that TMEM150 family proteins are the phospholipases responsible for producing the lysophospholipids in the plasma albumin LPL pool. Of particular importance, the inventors have demonstrated that TMEM150 family proteins are able to generate LPLs comprising essential omega-3 and omega-6 fatty acids, as well as omega-9 fatty acids. Moreover, the inventors have generated cells which have been engineered to express TMEM150 family proteins. The inventors found that cells expressing TMEM150 family proteins have increased production of a broad range of LPLs including LPC, LPE and LPS. Such cells form part of the invention.

[0016] The cells and methods of the invention enable the production of compositions that are enriched for LPLs comprising essential omega-3 and omega-6 fatty acids, as well as omega-9 fatty acids. Such compositions form a part of the invention.

[0017] In a first aspect, the disclosure provides a method of producing a lysophospholipid (LPL), the method comprising providing an enzyme selected from the TMEM150 protein family and contacting the enzyme with a phospholipid to allow the enzyme to hydrolyse a fatty acid from the phospholipid, thereby producing the LP.

[0018] 008933087In some embodiments, the LPL is selected from the LPLs shown in Table 1 , Table 2 or Table 3, or any combination thereof.

[0019] In some embodiments, the enzyme from the TMEM150 protein family is selected from TMEM150B or a functional homologue thereof, TMEM150A or a functional homologue thereof, SFK1 or a functional homologue thereof, TMEM150C or a functional homologue thereof, DRAM1 or a functional homologue thereof, and DRAM2 or a functional homologue thereof, or any combination of the foregoing.

[0020] In some embodiments, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), or any combination thereof. In some embodiments, the phospholipid can comprise a monounsaturated fatty acid (MUFA) and / or a polyunsaturated fatty acid (PUFA). In some embodiments, the phospholipid can comprise a MUFA or a PUFA covalently bound at the sn2 position.

[0021] In some embodiments, the MUFA and / or PUFA is selected from an omega-3 fatty acid, an omega-6 fatty acid, or any combination thereof. In some embodiments, the MUFA is hypogeic acid, oleic acid, elaidic acid, gondoic acid, mead acid, erucic acid, nervonic acid or ximenic acid, or any combination thereof. In some embodiments, the PUFA is selected from linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), or docosapentaenoic acid (DPA), or any combination thereof. In some embodiments, the PUFA is selected from oleic acid, linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). In some embodiments, the PUFA is docosahexaenoic acid (DHA). In some embodiments, the MUFA is oleic acid.

[0022] Unmodified TMEM150 family members show preferential hydrolysis activity towards fatty acids at the sn1 position of LPs. However, TMEM150 family members can hydrolyse fatty acids at the sn2 position. In some embodiments, the TMEM150 enzyme preferentially hydrolyses fatty acids from the sn1 position of phospholipids.

[0023] In some embodiments, the enzyme is not situated in a membrane. In some embodiments, the enzyme is situated in a membrane. In some embodiments, the membrane forms part of a i) cell; ii) liposome or proteoliposome; iii) lipid nanoparticle; or iv) micelle, optionally a detergent micelle. In some embodiments, the micelle is a detergent micelle.

[0024] In some embodiments, the method comprises a step of harvesting the LPL. In some embodiments, the method is performed in a reaction solution, culture medium and / or growth medium. In some embodiments, the method comprises a step of harvesting the LPL from the reaction solution, culture medium and / or growth medium. In some embodiments the harvesting step further comprises homogenizing and / or dissolving the membrane. In some embodiments, the harvesting step further comprises a step of harvesting the LPL from a membrane homogenate or dissolved membrane solution. In some embodiments, the harvesting step comprises contacting the LPL with a transporter moiety which can bind to amphipathic lipids, hydrophobic lipids and / or polar lipids. In some embodiments, the harvesting step comprises contacting the LPL with a transporter moiety which can bind to amphipathic

[0025] 008933087lipids. In some embodiments, the transporter moiety non-covalently binds to the LPL. In some embodiments the transporter moiety is an albumin protein, alpha-fetoprotein protein, a vitamin D-binding protein, cyclodextrin, a-cyclodextrin, p-cyclodextrin, BSA, an apolipoprotein, apolipoprotein A-1, apolipoprotein B, apolipoprotein D, apolipoprotein E, apolipoprotein F, apolipoprotein M, or any combination thereof. In some embodiments, the harvesting step comprises adding a transporter moiety to the reaction solution, culture medium, growth medium, membrane homogenate or dissolved membrane solution. In some embodiments, the transporter moiety non-covalently binds to the LPL.

[0026] In a second aspect, the present invention provides a cell that is engineered to express an enzyme from the TMEM150 protein family. In some embodiments, the cell is engineered to overexpress an enzyme from the TMEM150 protein family. In some embodiments, the cell is engineered to express a heterologous enzyme from the TMEM150 protein family. In some embodiments, the cell is engineered to express an exogenous enzyme from the TMEM150 protein family. In some embodiments, the cell is engineered to overexpress a heterologous enzyme from the TMEM150 protein family. In some embodiments, the cell is engineered to overexpress an exogenous enzyme from the TMEM150 protein family. In some embodiments, the cell is engineered to express a plasmid encoding an enzyme from the TMEM150 protein family. In some embodiments, the cell is engineered to overexpress an endogenously expressed enzyme from the TMEM150 protein family.

[0027] In some embodiments the enzyme from the TMEM150 protein family is selected from TMEM150B or a functional homologue thereof; TMEM150A or a functional homologue thereof; SFK1 or a functional homologue thereof; TMEM150C or a functional homologue thereof; DRAM1 or a functional homologue thereof; or DRAM2 or a functional homologue thereof; or any combination of the foregoing.

[0028] In some embodiments, the cell produces a greater amount of LPL compared to a non-engineered parent cell. In some embodiments, the cell produces a greater amount of LPL compared to a non-engineered parent cell when a phospholipid is provided to the cell. In some embodiments, the cell produces a greater amount of LPL compared to a non-engineered parent cell when a phospholipid is provided to the cell and a transporter moiety is provided to the cell. In some embodiments, the cell produces a greater amount of LPL compared to a non-engineered parent cell as determined by lipidomics. In some embodiments, the cell produces a greater amount of LPL compared to a non-engineered parent cell as determined by quantitative mass spectrometry-based lipidomics. In some embodiments, the LPL is selected from the LPLs shown in Table 1 , Table 2 or Table 3, or any combination thereof.

[0029] In some embodiments, the LPL is selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and lysophosphatidylserine (LPS), or any combination thereof. In some embodiments, the cell is further engineered to increase production of monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), or saturated fatty acids (SFA), or any combination thereof, compared to a non-engineered parent cell. In some embodiments, the PUFA is selected from; an omega-3 fatty acid, an omega-6 fatty acid, and an omega-9 fatty acid, or a combination thereof. In some embodiments, the fatty acid is selected from linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), or any combination thereof. In some embodiments, the PUFA is 008933087selected from linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), or any combination thereof.

[0030] In some embodiments the cell is further engineered to increase production of phospholipids compared to a non-engineered parent cell. In some embodiments, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid comprising a MUFA or PUFA covalently bound at the sn1 position, the sn2 position or the sn1 and sn2 positions of the phospholipid.

[0031] In some embodiments, the cell is engineered to increase production of a phospholipid comprising a PUFA selected from; an omega-3 fatty acid, an omega-6 fatty acid, and an omega-9 fatty acid or a combination thereof. In some embodiments, the fatty acid is selected from oleic acid, linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), and docosapentaenoic acid (DPA), or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid comprising a PUFA selected from; linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), and docosapentaenoic acid (DPA), or any combination thereof.

[0032] In some embodiments, the cell is engineered to increase production of a phospholipid comprising a MUFA selected from hypogeic acid, oleic acid, elaidic acid, gondoic acid, mead acid, erucic acid, nervonic acid and ximenic acid, or any combination thereof.

[0033] In some embodiments, the cell is engineered to reduce beta oxidation of fatty acids, reduce triglyceride (TAG) formation, reduce diacylglycerol (DAG) formation, and / or reduce expression of phospholipase genes. In some embodiments, the cell is engineered to reduce beta oxidation of fatty acids. In some embodiments, the cell is engineered to reduce diacylglycerol (DAG) formation. In some embodiments, the cell is engineered to reduce expression of phospholipase genes. In some embodiments, phospholipase genes are endogenous phospholipase genes.

[0034] In some embodiments, the cell is engineered to increase expression of one or more enzymes of the Kennedy pathway, and / or overexpress lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT), or any combination thereof. In some embodiments, the cell is engineered to increase expression of a gene encoding one or more enzymes of the Kennedy pathway, and / or overexpress lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT), or any combination thereof. In some embodiments, the cell is engineered to increase expression of a heterologous gene encoding one or more enzymes of the Kennedy pathway, and / or overexpress lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT), or any combination thereof.

[0035] In some embodiments, the cell is engineered to reduce expression of any combination of the following, or functional homologues thereof, compared to a non-engineered parent cell; sfk1 , Iem3, pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2.

[0036] 008933087In some embodiments, the cell is engineered to reduce expression of sfk1 and / or Iem3 compared to a non-engineered parent cell. In some embodiments, the cell is engineered to express an enzyme from the TMEM150 protein family, and to reduce expression of endogenous sfk1 and / or Iem3.

[0037] In some embodiments, the cell is engineered to reduce expression of a gene selected from pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof.

[0038] In some embodiments the cell is engineered to redirect lipid flux towards LPL production. Accordingly, in some embodiments, the cell is engineered to reduce expression of a gene selected from; sfk1 , Iem3, pox1 , dga1 , Iro 1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof compared to a non-engineered parent cell. In some embodiments, the engineered cell comprises a deletion of a gene selected from the following genes; sfk1 , Iem3, pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof.

[0039] In some embodiments, the cell is engineered to increase production of fatty acids. For example, in some embodiments, the cell is engineered to increase production of saturated fatty acids, MUFAs or PUFAs, or any combination thereof. In some embodiments, the cell is engineered to increase production of Omega-3, Omega-6, and / or Omega-9 fatty acids. In some embodiments, the cell is engineered to increase production of stearic acid, oleic acid, linoleic acid, linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid, or any combination thereof, compared to a non-engineered parent cell.

[0040] In some embodiments, the cell is engineered to have increased expression of a gene, or functional homologues thereof, selected from elongase 2 (ELO2), delta-9-desaturase (D9D), delta-12-desaturase (D12D), w3-Desaturase (FAD), delta-6-desaturase (D6D), delta-6-elongase (D6E), delta-5-desaturase (D5D), C20 / 22 elongase (ELO2), and delta-5-desaturase (TaFAD4), or any combination thereof.

[0041] In some embodiments, the cell is engineered to have increased expression of a gene, or functional homologues thereof, selected from S. cerevisiae elongase 2 (ScELO2), Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), S. kluyveri w3-Desaturase (skFAD), Ostreococcus tauri delta-6-desaturase (OtD6D), Mortierella alpina delta-6-elongase (MaD6E), Paramecium tetraurelia delta-5-desaturase (Ptet1D5D), Ostreococcus tauri C20 / 22 elongase (OtELO2), and Thraustochytrium sp delta-5-desaturase (TaFAD4), or any combination thereof.

[0042] In some particular embodiments, the cell is engineered to have increased expression of delta-9-desaturase (D9D), delta-12-desaturase (D12D), and w3-desaturase (FAD). In some embodiments, the cell is engineered to have increased expression of Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), and S. kluyveri w3-Desaturase (skFAD). In some embodiments, the cell is engineered to have increased expression of Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), and S. kluyveri w3-Desaturase (skFAD) and is engineered to express TMEM150B.

[0043] 008933087In some embodiments, the cell is a eukaryotic cell, an animal cell, a fungal cell, a bacterial cell, an algal cell, a plant cell, a protist cell, or a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, an insect cell a fungal cell, a bacterial cell, an algal cell, a plant cell, a protist cell, or a prokaryotic cell. In some embodiments, the cell is a fungal cell. In some embodiments, the fungal cell is a yeast cell. In some embodiments, the yeast cell is a Saccharomyces cerevisiae cell. In some embodiments, the yeast cell is a Yarrowia lipolytica cell. In some embodiments, the human cell is from an immortalised cell line. In some embodiments, the human cell is a HeLa cell. In some embodiments, the insect cell is from the Spodoptera genus. In some embodiments the insect cell is a Spodoptera frugiperda cell. In some embodiments the insect cell is an Sf-9 cell.

[0044] In a third aspect, the present disclosure provides a method of producing lysophospholipids comprising culturing a cell according to the second aspect of the invention. In some embodiments, the method comprises culturing the cell of the second aspect of the invention in a culture medium. In some embodiments, the method further comprises isolating an LPL from the culture medium. In some embodiments, the method further comprises a step of harvesting the LPL from the culture medium. In some embodiments the harvesting step comprises contacting the LPL with a transporter moiety. In some embodiments, the method comprises supplementing the culture medium with a source carbon and / or phosphate. In some embodiments, the method comprises providing the culture media with a transporter moiety. In some embodiments, the method of producing lysophospholipids comprising culturing a cell comprises a harvesting step as described herein.

[0045] In a fourth aspect, the present disclosure provides a cell culture comprising any of the cells disclosed herein.

[0046] In a related aspect, the present disclosure also provides compositions comprising LPL. The LPLs within these compositions can be produced following the methods (or through use of the cells) disclosed herein.. The composition may comprise an LPL produced by the methods of the first aspect of the invention or by the cells of the second aspect of the invention.

[0047] In some embodiments, the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% lysophospholipid (LPL) components. In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% total lysophospholipid (LPL) components. In some embodiments, the LPL components are produced by the method or the cell described herein. In some embodiments, the LPL components comprise a fatty acid component, optionally comprising MUFAs and PUFAs. In some embodiments, the LPL components comprise LPC, LPE and / or LPS. In some embodiments, the LPL components comprise one or more MUFAs and / or PUFAs. In some embodiments, the MUFAs or the PUFAs comprise omega-3 or omega-6 fatty acids.

[0048] 008933087In some embodiments, the LPL is selected from LPC, LPE or LPS, or any combination thereof and / or the LPL comprises a MUFA or a PUFA and / or the MUFA or the PUFA is an omega-3 or omega-6 fatty acid. In some embodiments, the fatty acids comprise oleic acid, linoleic acid, linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA) and, docosapentaenoic acid (DPA) or docosahexaenoic acid (DHA), or any combination thereof. In some embodiments, the fatty acid is oleic acid, linoleic acid, linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA) and, docosapentaenoic acid (DPA) or docosahexaenoic acid (DHA), or any combination thereof.

[0049] In some embodiments, the LPL component comprises LPC 22:6 (DHA), LPE 22:6 (DHA), LPS 22:6 (DHA), LPC 18:3 (ALA), LPE 18:3 (ALA), LPS 18:3 (ALA), LPC 20:5 (EPA), LPE 20:5 (EPA), LPS 20:5 (EPA), LPC 20:4 (ETA), LPE 20:4 (ETA) and LPS 20:4 (ETA), or any combination thereof. In some embodiments, the LPL is selected from LPC 22:6 (DHA), LPE 22:6 (DHA), LPS 22:6 (DHA), LPC 18:3 (ALA), LPE 18:3 (ALA), LPS 18:3 (ALA), LPC 20:5 (EPA), LPE 20:5 (EPA), LPS 20:5 (EPA), LPC 20:4 (ETA), LPE 20:4 (ETA) and LPS 20:4 (ETA), or any combination thereof.

[0050] In some embodiments, the LPL component comprises a LPL selected from the LPLs shown in Table 1 , Table 2 or Table 3, or any combination thereof. In some embodiments, the LPL is selected from the LPLs shown in Table 1 , Table 2 or Table 3. In some embodiments, the composition comprises a LPL selected from the LPLs shown in Table 1 , Table 2 or Table 3, or any combination thereof. In some embodiments, the LPL selected from the LPLs shown in Table 1 , Table 2 or Table 3.

[0051] In some embodiments the fatty acid component of the LPL component comprises at least 0.1%, at least 0.3%, at least 0.5%, at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% omega-3 and / or omega-6 fatty acids. For instance, the fatty acid component of the LPL components may comprise at least 0.1%, at least 0.3%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% eicosatetraenoic acid (ETA; 20:4).

[0052] In some embodiments, the composition comprises LPE 20:4. The LPE 20:4 may be present at a concentration of at least 20 nmol / g, at least 30 nmol / g or at least 50 nmol / g.

[0053] The invention enables the production of industrially useful quantities of the compositions disclosed herein. For instance, the composition may have a total mass of 1 g, 10 g, 100 g, 1 kg, 10 kg, 100 kg or 1000 kg. In some embodiments, the composition comprising an LPL is produced by the method or the cell following the methods or through use of the cells disclosed herein

[0054] In some embodiments, the composition comprises a transport moiety which can bind to amphipathic lipids, phospholipids and / or LPLs. In some embodiments, the transport moiety is an albumin protein, alpha-fetoprotein protein, a vitamin D-binding protein, cyclodextrin, a-cyclodextrin, p-cyclodextrin, an apolipoprotein, apolipoprotein A-1 , apolipoprotein B, apolipoprotein D, apolipoprotein E, apolipoprotein F, apolipoprotein M, or any combination thereof. In some embodiments the transporter moiety is albumin. In some embodiments, the transporter moiety is cyclodextrin, for example a-cyclodextrin, and / or p-cyclodextrin.

[0055] 008933087In a further aspect, the disclosure also provides an industrial product comprising any of the compositions described herein. In some embodiments, the industrial product is a pharmaceutical composition, a cosmetic composition, a nutraceutical, or a dietary supplement.

[0056] In a further aspect, the disclosure also provides an emulsifier comprising any of the compositions described herein. In another aspect, the present disclosure provides a use for any of the compositions described herein as an emulsifier. In some embodiments, the emulsifier is a food product emulsifier, a cosmetic emulsifier and / or a pharmaceutical emulsifier.

[0057] In another aspect, the present disclosure provides a use for any of the compositions provided herein as a pharmaceutical excipient.

[0058] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0059] Summary of the Figures

[0060] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0061] Figure 1. Structural modelling of intramembrane lipase activity of TMEM150 family proteins. AlphaFold2 structural model of human TMEM150A with a phosphatidylcholine molecule.

[0062] Figure 2. Images showing localization of human TMEM150A, TMEM150B, TMEM150C, DRAM1 , DRAM2, and yeast Sfk1 proteins in mammalian cells. Myc6His-tagged or FLAG-tagged proteins were overexpressed in HeLa cells and stained with anti-Myc antibody 9E10 or anti-DYKDDDDK [SEQ ID NO:8] antibody (for DRAM1).

[0063] Figure 3A to 3D. Immunoblots and charts showing TMEM150A, TMEM150B, and Sfk1 have phospholipase activity. (A) Lipase assay with NBD-PC 18:1 / 12:0 substrate added to membrane suspensions of HeLa cells overexpressing TMEM150A or TMEM150B or Sfk1 wildtype (WT) or point mutants of the catalytic serine residue. Substrates were added to a working concentration of 4 pM. Reactions were allowed to proceed for 15 minutes (TM EM 150 A) or 60 minutes (TMEM150B and Sfk1) at 37°C without detergent before termination by Folch extraction, and resulting organic extracts were resolved by thin layer chromatography. Incubation of NBD-PC with TMEM150A / TMEM150B / Sfk1 WT membrane suspensions, but not mutant membrane suspensions, produced NBD-LPC consistent with phospholipase A1 (PLA1) activity mediating removal of the sn1 fatty acid. NBD-FA was not a product, indicating TMEM150A is not a phospholipase B / lysophospholipase (PLB). (B) Time course of NBD-LPC evolution by Ni-NTA-purified TMEM150A WT protein or S67A mutant in DDM / CHS detergent. (C) Cofactor dependence of Ni-NTA-purified TMEM150A protein in DDM / CHS detergent. (D) Effects of fatty acid (FA)-free BSA and free fatty acid on activity of Ni-NTA-purified TMEM150A protein in DDM / CHS detergent. Band volumes of the NBD-LPC band were quantified in Biorad Imagelab and plotted in

[0064] 008933087Graphpad Prism using 2-way ANOVA with Dunnett’s test. Fatty-acid-free BSA stimulated activity while pre-incubation with certain free fatty acids appeared to reduce activity.

[0065] Figure 4A and 4B. Lipidomic analysis data showing structure-activity relationship of TMEM150A lipase activity. Ni-NTA purified TMEM150A WT or S67A mutant protein was incubated with various unlabelled substrates in DDM / CHS detergent. Products were quantified by LC-MS / MS lipidomics analysis. (A) Effect of varying the headgroup and bond linkage. TMEM150A showed specificity for PC and PE headgroups, cleaving mainly at acyl (ester) linkages. Shading indicates substrate preference as determined by the amount of lysophosholipid product produced, from not preferred (white) to preferred (gray). (B) Effect of varying the acyl chains. TMEM150A was able to cleave both acyl chains but showed preference for cleaving saturated acyl chains, thereby releasing LPCs comprising unsaturated fatty acids. Shading indicates substrate preference as determined by the amount of LPC product produced, from not preferred (white) to preferred (gray). Statistics are 2-way ANOVA with Tukey’s test, comparing levels of LPC products in reactions incubated with TMEM150A WT protein versus S67A mutant. Abbreviations: ****, p<0.0001; ns, not significant.

[0066] Figure 5A to 5G. Charts showing overexpression of TMEM150 family members increased cellular levels of LPLs. Human (A) TMEM150A, (B) TMEM150B, (C) TMEM150C, (D) DRAM1, (E) DRAM2, (F) PGAP2, or (G) yeast Sfk1 — and mutants thereof — were overexpressed in HeLa cells. The next day, cell pellets were collected and analysed by lipidomics. Expression of TMEM150 family members including yeast Sfk1 increased cellular levels of LPC and LPE lipids, consistent with phospholipase activity. Upper panels show volcano plots comparing levels of lipids in WT-overexpressing cells versus their respective mutant-overexpressing cells. Lower panels show absolute amount of lysophospholipid species in HeLa cell pellets overexpressing TMEM150-family WT proteins versus mutant proteins. Statistics are 2-way ANOVA with Bonferroni correction comparing the WT to the mutant samples.

[0067] Figure 6A to 6D. Charts showing overexpression of TMEM150A and Sfk1 increased levels of LPLs in the conditioned medium. Human (A) TMEM150A, (B) TMEM150B, (C) TMEM150C, or (D) yeast Sfk1 — and mutants thereof — were overexpressed in HeLa cells, this time in the presence of 0.5% (w / v) fatty-acid-free bovine serum albumin (BSA) in serum-free medium. The next day, conditioned medium and cell pellets were collected and lipidomics measurements were performed. Upper panels show volcano plots comparing levels of lipids in conditioned medium and cell pellets from WT-overexpressing cells versus their respective mutant-overexpressing cells. Lower panels show absolute amount of lysophospholipid species in the conditioned medium from HeLa cells overexpressing TMEM150-family WT proteins versus mutant proteins. Statistics are 2-way ANOVA with Sidak’s test comparing the WT to the mutant samples. These results indicate that TMEM150A and Sfk1 were able to generate LPC and LPE at the plasma membrane of mammalian cells that can be secreted into media.

[0068] Figure 7. Diagram showing proposed TMEM150A relationship with albumin. TMEM150A is a membrane-embedded lipase (the first of such to be identified) with active site open to the extracellular leaflet of the plasma membrane. Circulating albumin associates with LPLs produced via the enzymatic activity of TMEM150 family proteins.

[0069] 008933087Figure 8A to 8C. Graphs showing TMEM150A purification and lipase activity. (A) FPLC profile showing the protein peak for purification of Tmem150a-3C-mEGFP-Flag-HIS6 from baculovirus infected HEK29S GnTI- cells with detergent LMNG. (B) Coomassie blue gel stain of Tmem150a-3C-mEGFP-Flag-HIS6 in different elution fractions from FPLC purification in (A). (C) Lipase activities of purified Tmem150a-3C-mEGFP-Flag-HIS6. A 50pl reaction volume containing 5uM NBD-PC 18:1 / 12:0 and indicated concentrations of Tmem150a-GFP protein in assay buffer were prepared and incubated at 37°C for 1 hour. Reactions were terminated and lipids were extracted with Folch method. Lipids were separated by TLC analysis. Typhoon biomolecular scanner was used to detect the fluorescence LPC product.

[0070] Figure 9. Chart showing that Tmem150a knockout mice have significantly reduced plasma LPCs.

[0071] Volcano plot showing targeted lipidomic analysis of 246 lipid species in 26 lipid classes revealed that LPCs were almost exclusively (with the exception of PI 36:3 and PI 36:4) and significantly reduced in Tmem150a knockout (KO) relative to WT controls, supporting the conclusion that Tmem150a is the long sought after lipase responsible for the generation of plasma LPC in vivo (n = 7 mice per genotype).

[0072] Figure 10. Lysolipid production in Spodoptera frugiperda (Sf9) cells. Sf9 cells were infected with baculovirus harbouring Tmem150b-3C-mEGFP-Flag-HIS6 (TMEM150B) orTmem150b (S43A)-3C-mEGFP-Flag-HIS6 (TMEM150B (S43A)) for 72 hours at 28 °C. Lipids harvested from conditioned media was subjected to lipidomic analysis.

[0073] Figure 11. Diagram showing workflow for production of S. cerevisiae strains with increased production of LPLs.

[0074] Figure 12. Charts showing lipidomic analysis of conditioned media from S. cerevisiae strains engineered to secrete of LPC / LPE. The indicated yeast strains were grown according to the workflow in Figure 11. Units are shown in nM.

[0075] Figure 13. Charts showing lipidomic analysis of conditioned media from S. cere visiae strains having genetic backgrounds indicated, with or without 0.5% fatty acid free BSA.

[0076] Figure 14. Diagram showing the lipid metabolic network to redirect the lipid flux toward phospholipid production in S. cerevisiae.

[0077] Figure 15. Heterologous gene expression in S. cerevisiae for production of LPCs. S. cerevisiae codon usage optimized heterologous genes from Homo sapiens Transmembrane protein 150A or 150B (HsTMEM150a, HsTMEM150B) or S. cerevisiae Suppressor of Four Kinase 1 (ScSFKI), S. cerevisiae elongase 2 (ScELO2) (Yu et al., 2017), Mortierella alpina delta-9-desaturase (MaD9D) (Tavares et al., 2011), Mortierella alpina delta-12-desaturase (MaD12D) (Tavares et al., 2011), S. kluyveri w3-Desaturase (skFAD) (Tavares et al., 2011), Ostreococcus tauri delta-6-desaturase (OtD6D) (Tavares et al., 2011), Mortierella alpina delta-6-elongase(MaD6E) (Tavares et al., 2011), Paramecium tetraurelia delta-5-desaturase (Ptet1D5D) (Tavares et al., 2011), Ostreococcus tauri 020 / 22 elongase (OtELO2) (Meyer et al., 2004), and Thraustochytrium sp delta-5-desaturase (TaFAD4) (Qiu et al., 2001), with a strong constitutive promoter can be integrated into the yeast genome using integration constructs from

[0078] 008933087EasyClone2.0 system. All the antibiotic selectable markers that are flanked by loxP sites will be recycled with marker rescue mediated by CreA site-specific recombinase (Stovicek etal., 2015).

[0079] Figure 16. Lipidomic analysis of conditioned media from S. cerevisiae engineered to secrete LPC / LPE-18:2 and LPC / LPE-18:3.

[0080] Figure 17. Alpha- and beta-cyclodextrin can capture LPC / LPE secreted into media from S. cerevisiae strains engineered to secrete of LPC / LPE.

[0081] Detailed Description of the Invention

[0082] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0083] The present invention is based on the identification that enzymes in the TMEM150 protein family can be used in methods of producing lysophospholipids, either in isolation, or in cellular systems.

[0084] Lysophospholipids (LPLs) produced either through use of the methods disclosed herein, or use of the cells disclosed herein, can comprise, for example, essential omega-3 and omega-6 monounsaturated (MUFA) and / or polyunsaturated fatty acids (PUFA). One aspect of the invention relates to methods of producing LPLs using cells, as defined herein.

[0085] LPLs include lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPS). LPCs are biologically significant because they are the physiologically relevant form of omega-3 fatty acids which can cross the blood-brain barrier and supply the brain with omega-3 fatty acids essential for brain development and maintain neurological health. Lysolipid transporter Mfsd2a is known to be responsible for importing LPCs from the plasma and across the blood brain barrier.

[0086]

[0087] In a first aspect, the present invention provides a method of producing lysophospholipids.

[0088] Lysophospholipids (LPLs), also regarded as hydrolyzed lipids, carry either a single alkyl or acyl chain. LPLs can be generated from phospholipids, from which one acyl chain has been removed, leaving a single acyl group. The two main categories of LPLs are characterized based on their backbone of glycerol (lysoglycerophospholipids) ora sphingosine (lysosphingolipids). LPLs are composed of a hydrophobic carbon chain and a hydrophilic phosphate head group attached to a glycerol or sphingosine backbone. LPLs, having only a single hydrophobic carbon chain, display different properties compared with the phospholipids or sphingolipids from which they are derived. In cells, LPLs are intermediate precursors for biosynthesis of other lipids in the cells. Therefore, their intracellular concentrations are low. In contrast, LPLs are highly abundant in extracellular environment such as plasma and interstitial fluids. In the extracellular milieu, LPLs can bind to protein carriers.

[0089] 008933087LPLs derived from phospholipids with a glycerol backbone can retain an acyl chain or alkyl chain at the sn1 or sn2 position of the glycerol backbone. The sn3 position is occupied by the phosphate group which can be esterified to either an alcohol or an amino-alcohol, thereby determining the species of the LPL (see Formula 1). Depending on the sn position of the acyl chain, the LPL can be either a 1-LPL isomer (1-lyso-2-acyl-phospholipid) or a 2-LPL isomer (1-acyl-2-lyso phospholipid).

[0090] 1

[0091] Sn1 CH2O

[0092] 2

[0093] Sn2 CHO - R

[0094] O

[0095] l l 3

[0096] Sn3 CH2O - P - O - R

[0097]

[0098] 0“

[0099] Formula 1

[0100] LPLs have been associated with a range of both normal and pathological processes such as carcinogenesis, neurogenesis, immunity, vascular development and regulation of metabolic diseases. The characteristics of LPLs can depend on the properties of the acyl-chains which they comprise, for example, chain length and degree of unsaturation. Consequently, LPLs comprise a diverse range of molecules with a range of different species. Notably, LPLs are known to be able bind to and activate cognate G-protein-coupled receptors (GPCRs) specific to each LPL type.

[0101] In the blood plasma, LPLs are mainly bound to albumin. Concentration of plasma LPLs considerably varies in certain diseases and as such can be used as diagnostic biomarkers. LPLs can be used as surfactants and emulsifiers in food techniques and cosmetics, and can also be used in drug delivery. The LPLs produced according to the current invention may be for use as a surfactant or an emulsifier, for use in a drug delivery system, or for use in therapy as a drug carrier.

[0102] LPLs can be lysoglycerophospholipids or lysosphingolipids. Unless expressly stated otherwise, the term “lysophospholipid” or “LPL” as used herein to refer to “lysoglycerophospholipid”.

[0103] LPLs can be produced via hydrolysis of one of the two ester bonds of a phospholipid, liberating an acyl chain and generating the LPL. This can also be described as enzyme-catalyzed partial deacylation of phospholipids. Hydrolysis of the ester bond can be achieved by phospholipase enzymes. There are four major classes of phospholipases which are distinguished by the type of reaction which they catalyze. Phospholipase A are relevant to LPLs because they cleave either the sn-1 acyl chain (Phospholipase Ai or PLAi), or the sn-1 acyl chain (Phospholipase A2 or PLA2) of phospholipids, resulting in a fatty acid and LPL. The acyl chain which remains on the LPL can be a saturated fatty acid (SFA), a mono-unsaturated

[0104] 008933087fatty acid (MUFA), or a polyunsaturated fatty acid (PUFA) depending on the fatty acids present in the phospholipid substrate, and whether the phospholipase catalysing the hydrolysis of the ester bond has a positional preference for sn-1 or sn-2, or a preference for acyl chain lengths or degree of unsaturation of an acyl chain.

[0105] Glycerophospholipids are a common class of amphipathic phospholipids that comprise a glycerol backbone with two fatty acyl molecules esterified at the sn-1 and sn-2 positions and a head group linked by a phosphate group at the sn-3 position, sn refers to stereospecific numbering. The sn-2 position refers to the second carbon in the glycerol backbone, which is between the carbon at sn-1 and sn-3. This positional number also applied to LPLs. Phospholipids often contain MUFAs or PUFAs such as arachidonic or docosahexaenoic acids in the sn-2 position, whereas the sn-1 position is normally occupied by saturated fatty acids. Unless expressly stated otherwise the term “phospholipid” is used herein to refer to glycerophospholipids.

[0106] The hydrophilic head of the phospholipid determines the type of phospholipid. The head group may comprise choline, ethanolamine, serine, inositol, or glycerol. The most common phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). The length and saturation of the hydrophobic fatty acyl side chains further influences the properties of the phospholipids.

[0107] LPL as a term also includes ether-linked LPLs (LPL-O), for example LPC-O. LPL-0 comprise of a glycerol backbone linked to a fatty alcohol via an ether bond. LPLs can also be in the type of a vinyl ether-linked phosphatidylcholine (LPL-P), for example, LPC-P. It will be understood that term “LPL” as used herein may refer to any particular LPL type including LPL-0 and LPL-P and it may refer to a mixture of these LPL types. In some embodiments, the LPL may comprise LPL-O. In some embodiments, the LPL may comprise LPL-P. In some embodiments, the LPL may comprise LPL-0 and LPL-P. In some embodiments, the LPL does not comprise LPL-O. In some embodiments, the LPL does not comprise LPL-P. In some embodiments, the LPL does not comprise LPL-0 or LPL-P.

[0108] In mammalian cells, phospholipids are essential components of bilayer membranes, forming the plasma membrane, nuclear envelope, mitochondria, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes, lysosomes, and endosomes. The major phospholipid classes in mammalian cell membranes are phosphatidylcholine (PC) and phosphatidylethanolamine (PE), while phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidate (PA), phosphatidylglycerol (PG), and sphingomyelin (SM) are minor components. PS and PE are enriched in the inner leaflet of the plasma membrane, whereas PC and SM are predominantly located on the outer leaflet.

[0109] The LPL that can be generated from phosphatidylcholine (PC) by hydrolysis of an acyl group is lysophosphatidylcholine (LPC). The LPL that can be generated from phosphatidylethanolamine (PE) by hydrolysis of an acyl group is lysophosphatidylethanolamine (LPE). The LPL that can be generated from phosphatidylserine (PS) by hydrolysis of an acyl group is lysophosphatidylserine (LPS).

[0110] The corresponding LPL for phosphatidylcholine (PC) is lysophosphatidylcholine (LPC). The corresponding LPL for phosphatidylethanolamine (PE) is lysophosphatidylethanolamine (LPE). The 008933087corresponding LPL for or phosphatidylserine (PS) is lysophosphatidylserine (LPS). The term corresponding in this context refers to the product of a phospholipase mediated hydrolysis reaction using the specified phospholipid as a substrate.

[0111] It is envisaged that the methods and cells of the invention may be used to produce any one or more of LPC, LPE, and LPS. In some embodiments, the method of the invention may comprise bringing a mixture of PC and PE into contact with an enzyme of the invention to produce a mixture of LPC and LPE. In some embodiments, the method of the invention may comprise bringing a mixture of PC and PS into contact with an enzyme of the invention to produce a mixture of LPC and LPS. In yet further embodiments, the method of the invention may comprise bringing a mixture of PC, PE and PS into contact with an enzyme of the invention to produce a mixture of LPC, LPE and LPS. Likewise, in some embodiments, the cell of the invention may be engineered to produce an increased amount of any one or more of LPC, LPE and LPS compared to a non-engineered parent cell.

[0112]

[0113] TMEM150

[0114] In a first aspect, the present invention provides a method of producing an LPL, the method comprising providing an enzyme from the TMEM150 protein family. That is, the method of producing an LPL comprises the use of an enzyme from the TMEM150 protein family.

[0115] In the context of the present invention, TMEM150 protein family enzymes are used as biocatalysts for the production of useful biochemicals. In particular, the TMEM150 family of phospholipase enzymes can convert phospholipids into LPLs and a free fatty acid. The TMEM150 protein family can refer to the TMEM150 family of phospholipase enzymes.

[0116] The Transmembrane Protein 150 (TMEM150) family may also be known as the damage-regulated autophagy modulator (DRAM) family. TMEM150 family proteins possess 6 transmembrane domains with both termini positioned within the cytoplasm. The TMEM150 protein family includes TMEM150A, TMEM150B, TMEM150C, Sfk1 , DRAM1 and DRAM2. DRAM2 is also known as TMEM77. The present application demonstrates that members of the TMEM150 family share the same 3D structural arrangement and functional activity.

[0117] In this specification “TMEM150 family protein” refers to a TMEM150 protein from or derived from any species, in particular human (Homo sapiens) and yeast (e.g. Saccharomyces cerevisiae) and includes isoforms, fragments, variants or homologues of a TMEM150 protein from any species. Homologues include orthologues. The term homologue and homolog may be used interchangeably.

[0118] Transmembrane Protein 150A (TMEM150A) is a member of the TMEM150 family of proteins and is identified by UniProtKB Q86TG1. TMEM150A is also be known as Tentonin 1 (TTN1) or TM6P1. To date, TMEM150A is known to interact with the PIPI4Kllla- EFR3 complex at the plasma membrane and positively regulates PI and P2 synthesis (Chung J, etal. EMBO Rep. 2015;16(3):312-20). In this specification, TMEM150A refers to TMEM150A from any species and includes TMEM150A isoforms, fragments, variants or homologues from any species.

[0119] 008933087Transmembrane Protein 150B (TMEM150B) is a member of the TMEM150 family of proteins and is identified by UniProtKB A6NC51. TMEM150B is also be known as TTN2, TMEM224 or DRAM3.

[0120] TMEM150B belongs to the DRAM (damage-regulated autophagy modulator) family of membranespanning proteins. To date, TMEM150B is thought to promote cell viability under glucose deprivation by prompting autophagy (Mrschtik, M. etal. Cell Death Differ. 22, 1714-1726 (2015)). In this specification, TMEM150B refers to TMEM150B from any species and includes TMEM150B isoforms, fragments, variants or homologues from any species.

[0121] Transmembrane Protein 150C (TMEM150C) is a member of the TMEM150 family of proteins and is identified by UniProtKB B9EJG8. TMEM150C is also be known as TTN3. To date, TMEM150C is thought to be a component of a mechanosensitve ion channel that is activated by mechanical stimuli in various cell types and confers slowly adapting, mechanically activated currents in dorsal root ganglion neurons (Pak et al., Cell Rep. 2024;43(6):114334). In this specification, TMEM150C refers to TMEM150C from any species and includes TMEM150C isoforms, fragments, variants or homologues from any species. DNA damage regulated autophagy modulator 1 (DRAM1) is a member of the TMEM150 family of proteins and is identified by UniProtKB Q8N682. DRAM1 is also be known as DRAM. To date, DRAM1 is thought to be a lysosomal membrane protein that is required for the induction of autophagy (Crighton etal. Cell.

[0122] 2006;126(1):121-134). In this specification, DRAM1 refers to DRAM1 from any species and includes DRAM1 isoforms, fragments, variants or homologues from any species.

[0123] DNA damage regulated autophagy modulator 2 (DRAM2) is a member of the TMEM150 family of proteins and is identified by UniProtKB Q6UX65. DRAM2 is also be known as CORD21 , PRO180, TMEM77 or VVWFQ154. Like DRAM1 , DRAM2 is also thought to be a lysosomal membrane protein that is required for the induction of autophagy (Park et al.. Biochem Biophys Res Commun. 2009;390(4):1340-1344). In this specification, DRAM2 refers to DRAM2 from any species and includes DRAM2 isoforms, fragments, variants or homologues from any species.

[0124] Suppressor of four kinase protein 1 (SFK1) is a Saccharomyces cerevisiae protein and a member of the TMEM150 family of proteins. SFK1 is identified by UniProtKB P35735. To date, SFK1 is thought to repress the flip / flop movement of phospholipids across plasma membrane, as well as regulating phospholipid asymmetry in the plasma membrane (Mioka etal., Mol Biol Cell. 2018;29(10):1203-1218). In this specification, SFK1 refers to SFK1 from any species and includes SFK1 isoforms, fragments, variants or homologues from any species.

[0125] The inventors have found that, in addition to their previously recognised roles, enzymes from the TMEM150 protein family have phospholipase activity and can produce LPLs. That is, enzymes from the TMEM150 protein family are phospholipases. Enzymes in the TMEM150 protein family can hydrolyse the ester bond at position sn-1 or position sn-2 of a phospholipid, thereby removing the acyl moiety at either the sn-1 or sn-2 position and generating a free fatty acid and an LPL.

[0126] In the present invention, methods of producing lysophospholipids utilising the enzymatic activity of proteins in the TMEM150 family are disclosed. In a first aspect of the present invention, the method of producing a lysophospholipid (LPL) comprises providing an enzyme from the TMEM150 protein family 008933087and contacting the enzyme with a phospholipid to allow the enzyme to hydrolyse a fatty acid from the phospholipid, thereby producing the LPL.

[0127] In some embodiments, the enzyme from the TMEM150 protein family is TMEM150A, TMEM150B, TMEM150C, DRAM1 , DRAM2 or Sfk1 , or a homologue, or a variant thereof. That is, in some embodiments of the present invention, the enzyme from the TMEM150 protein family is TMEM150A, TMEM150B, TMEM150C, DRAM1 , DRAM2 or Sfk1 , or a homologue, or a variant thereof. Members of the TMEM150 family are closely related and share a high degree of sequence and structural similarity. Fragments, variants, isoforms and homologues of a TMEM150 protein may optionally be characterized by the ability to catalyze the hydrolysis of an ester bond linking an acyl chain to the glycerol backbone of a phospholipid, to produce a fatty acid and a lysophospholipid.

[0128] A “homologue” generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. For example, human TMEM150A (UniProtKB -Q86TG1), and yeast Saccharomyces cerevisiae Sfk1 (UniProtKB - P35735) are homologues of one another. A “functional homologue” generally refers to a homologue of the reference protein which retains, to some degree, one or more of the reference proteins biological activities. In the context of the present invention, a functional homologue of a TMEM150 family member refers to a homologue of the TMEM150 family member which retains, to some degree, the phospholipase activity.

[0129] A “fragment” generally refers to a fraction of the reference protein. A “functional fragment” generally refers to a fraction of the reference protein which retains one or more of the reference proteins biological activities. In the context of the present invention, a functional fragment of a TMEM150 family member refers to a fragment of the TMEM150 family member which retains the phospholipase activity.

[0130] A “variant” generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein. An “isoform” generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein.

[0131] As used herein, a “fragment”, “variant” or “homologue” of a protein may optionally be characterized as having at least 50%, preferably one of 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein. Homologues and variants of a polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. A “fragment” of a reference protein 008933087may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0132] Fragments, variants, isoforms and homologues of a reference protein may be characterised by the ability to perform a function performed by the reference protein. For example, homologous proteins belonging to the TMEM150 protein family may be classified by their ability to perform a function similar to TMEM150A, TMEM150B, TMEM150C, SFK1 , DRAM1 or DRAM2. A homologous protein is also known as a homologue.

[0133] In some embodiments of the invention, the enzyme from the TMEM150 protein family is wild-type or a mutant of TMEM150A. In some embodiments, the enzyme from the TMEM150 protein family is TMEM150A or a homologue thereof. In some embodiments, the enzyme from the TMEM150 protein family is TMEM150A or a functional homologue thereof. In some embodiments, TMEM150A is derived from human TMEM150A (UniProtKB: Q86TG1) represented by the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, TMEM150A comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1. In some embodiments, TMEM150A comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0134] In some embodiments of the invention, the enzyme from the TMEM150 protein family is wild-type or a mutant of TMEM150B. In some embodiments, the enzyme from the TMEM150 protein family is TMEM150B or a homologue thereof. In some embodiments, the enzyme from the TMEM150 protein family is TMEM150B or a functional homologue thereof. In some embodiments, TMEM150B is derived from human TMEM150B (UniProtKB: A6NC51) represented by the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, TMEM150B comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2. In some embodiments, TMEM150B comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0135] In some embodiments of the invention, the enzyme from the TMEM150 protein family is wild-type or a mutant of TMEM150C. In some embodiments, the enzyme from the TMEM150 protein family is TMEM150C or a homologue thereof. In some embodiments, the enzyme from the TMEM150 protein family is TMEM150C or a functional homologue thereof. In some embodiments, TMEM150C is derived from human TMEM150C (UniProtKB: B9EJG8) represented by the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, TMEM150C comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:3. In some embodiments, TMEM150C comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3.

[0136] In some embodiments of the invention, the enzyme from the TMEM150 protein family is wild-type or a mutant of DRAM1. In some embodiments, the enzyme from the TMEM150 protein family is DRAM1 or a 008933087homologue thereof. In some embodiments, the enzyme from the TMEM150 protein family is DRAM1 or a functional homologue thereof. In some embodiments, DRAM1 is derived from human DRAM1 (UniProtKB: Q8N682) represented by the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, DRAM1 comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:4. In some embodiments, DRAM1 comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.

[0137] In some embodiments of the invention, the enzyme from the TMEM150 protein family is wild-type or a mutant of DRAM2. In some embodiments, the enzyme from the TMEM150 protein family is DRAM2 or a homologue thereof. In some embodiments, the enzyme from the TMEM150 protein family is DRAM2 or a functional homologue thereof. In some embodiments, DRAM2 is derived from human DRAM2 (UniProtKB: Q6UX65) represented by the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, DRAM1 comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:5. In some embodiments, DRAM1 comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0138] In some embodiments of the invention, the enzyme from the TMEM150 protein family is wild-type or a mutant of SFK1. In some embodiments, the enzyme from the TMEM150 protein family is SFK1 or a homologue thereof. In some embodiments, the enzyme from the TMEM150 protein family is SFK1 or a functional homologue thereof. In some embodiments, SFK1 is derived from yeast SFK1. In some embodiments, SFK1 is derived from Saccharomyces cerevisiae SFK1 (UniProtKB: P35735) represented by the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, SFK1 comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:6. In some embodiments, SFK1 comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:6.

[0139] The catalytic triad of TMEM150 family proteins is conserved across TMEM150 family proteins including TMEM150A, TMEM150B, TMEM150C, DRAM1, DRAM2 and SFK1. The skilled person would be able to identify functionally equivalent members of the TMEM150 family using standard techniques in the art. In some embodiments, the TMEM150 family protein comprises the catalytic triad sequence of a wild-type TMEM150 family protein. In some embodiments, the TMEM150 family protein catalytic triad comprises a serine, a histidine and glutamate amino acid.

[0140] In some embodiments, the TMEM150 family protein comprises the catalytic triad sequence of wild-type TMEM150A (SEQ ID NO: 1). With reference to SEQ ID NO: 1 , the wild-type TMEM150A catalytic triad is composed of residues serine 67 (S67, Ser67), histidine 140 (H140, His140), and glutamate 211 (E211 , Glu211). In some embodiments, the enzyme in the TMEM150 protein family, or homologue thereof, comprises a catalytic triad of Ser67, His140, and Glu211 , wherein the residue numbering is relative to SEQ ID NO: 1.

[0141] 008933087In some embodiments, the TMEM150 family protein comprises the catalytic triad sequence of wild-type TMEM150B (SEQ ID NO: 2). With reference to SEQ ID NO: 2, the TMEM150B catalytic triad comprises residue serine 43 (S43, Ser43), histidine 116 (H116, His116), and glutamate 185 (E185, Glu185). In some embodiments, the enzyme in the TMEM150 protein family, or homologue thereof comprises a catalytic triad comprising Ser43, His116, and Glu185, wherein the residue numbering is relative to SEQ ID NO:2.

[0142] The present invention provides several examples of TMEM150 family proteins which may be provided in methods of producing LPLs. However, the present disclosure further provides methodology to identify further TMEM150 family proteins with phospholipase activity that may be used in the disclosed methods. Additional members of the TMEM150 family will be readily identifiable to the skilled person using their common general knowledge. For example, further TMEM150 family members may be identified in diverse species through use of tools such as sequence alignment algorithms like BLAST (Basic Local Alignment Search Tool; https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to compare the amino acid sequences of proteins from different organisms, where a high degree of sequence similarity indicates that the proteins are likely related and share a common evolutionary origin and function.

[0143] Enzymes suitable for use in the methods disclosed herein may be validated through use of known phospholipase activity assays. For example, through use of NBD-PC, which is PC molecule with an NBD fluorophore linked to the sn2 fatty acid as detailed in Example 1.2 and used in Example 3. Phospholipase activity of candidate enzymes can be evaluated by setting up NBD-PC digests with any candidate enzyme, and running the results on a Thin Layer Chromatography (TLC) plate to assess whether the NBD fluorophore remains incorporated in a resulting LPL.

[0144]

[0145] of TMEM150

[0146] The methods provided in the present disclosure comprise providing an enzyme form the TMEM150 protein family. The TMEM150 family protein may be derived from a cell expressing or overexpressing a TMEM150 family protein. The TMEM150 family protein may be isolated, solubilised, and / or purified, from a cell expressing, or overexpressing the TMEM150 family protein.

[0147] The TMEM150 family protein according to the present invention may be prepared according to methods for recombinant protein production known to the skilled person. Molecular biology techniques suitable for recombinant production are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, which is herein incorporated by reference in its entirety.

[0148] Expression may be from a nucleic acid sequence and / or an expression vector, e.g. a nucleic acid sequence or expression vector according to the present invention. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from an expression vector according to the invention. Expression may be from a cell according to the present invention. Any cell suitable for the expression of polypeptides may be used.

[0149] 008933087Production may involve culture or fermentation of cell modified or engineered to express the relevant polypeptide(s), for example baculovirus infected HEK29S GnTI- cells. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted or released proteins can be collected by partitioning culture media / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted or expressed peptide or polypeptide. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

[0150] Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

[0151] Following culturing the cells that express the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated from the culture media that contains the secreted polypeptide(s) of interest by centrifugation. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by Bonification, rapid freeze-thaw or osmotic lysis. It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or nutrient medium, which may contain other protein and non-protein components.

[0152] One approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins. Other methods for separating protein components include ion exchange chromatography and size chromatography. These may be used as an alternative to precipitation, or may be performed subsequently to precipitation.

[0153] Once the polypeptide(s) of interest have been isolated from the culture it may be desired or necessary to concentrate the peptide or polypeptide. A number of methods for concentrating proteins are known in the art, such as ultrafiltration and lyophilisation.

[0154] It will be appreciated that the TMEM150 family proteins according to the present invention may be provided as components of larger polypeptides or polypeptide complexes. For example, the polypeptides described herein may be provided as fusion polypeptides. In some embodiments the polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing or purification,

[0155] 008933087e.g. His, (e.g. 6XHis), Myc GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus. For example, Tmem150a-3C-mEGFP-Flag-HIS6.

[0156] The methods provided in the present invention may comprise providing an enzyme situated, or partially situated, within a membrane structure. That is, the method may comprise providing an enzyme in the TMEM150 protein family that is held within a membrane structure. By way of illustration, an enzyme in the TMEM150 protein family that is located in a membrane structure may be from an isolated membrane preparation from HeLa cells that express or overexpress a TMEM150 family protein. Isolated or solubilised membrane fractions may be prepared following the methodology provided in Example 1.1 or Example 1.4.

[0157] TMEM150 family proteins may be provided in a solution, produced through use of known detergents to disintegrate the lipid membrane structure and create detergent micelles which incorporate membrane lipids and the TMEM150 family protein. In these structures, the lipid “tails” orientate to the hydrophobic interior of the detergent micellar structures. The degree of dissociation between lipids and proteins may depend on the solubilised protein and the detergent used.

[0158] TMEM150 family proteins can be purified in the presence of detergent by applying any of the existing protein purification techniques available for soluble proteins. A successful solubilization protocol extracts TMEM150 family proteins at a high yield and may result in stable protein-detergent complexes (or protein-lipid-detergent complexes) where the protein retains its active conformation.

[0159] Purification of TMEM150 family proteins may also be performed using Fast protein liquid chromatography (FPLC) purification methods known in the art. Following FPLC, peak fractions can be isolated and pooled to provide purified TMEM150 family proteins (for details see Example 1.4 of the present application). Isolated TMEM150 family proteins can be used in in-vitro phospholipase assays to produce LPLs.

[0160] Relatedly, in some embodiments of the present invention, the method comprises providing an enzyme situated or partially situated in a membrane. In some embodiments, the membrane forms part of, or previously formed part of, a cell, a liposome or proteoliposome, a lipid nanoparticle, or a micelle. In some embodiments, the membrane is a fragment of a cell membrane, a liposome or proteoliposome, a lipid nanoparticle, or a micelle. In some embodiments the cell membrane is a plasma membrane. In some embodiments the micelle is a detergent micelle. In some embodiments, the method of producing LPLs is an in-vitro method.

[0161] Omega-3 and Omega-6 fatty acids

[0162] Phospholipids, and specifically glycerophospholipids, comprise two acyl chains, which can also be referred to as fatty acids. Phospholipids can comprise saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) or poly unsaturated fatty acids (PUFAs), or a combination of any two of SFA, MUFA and PUFA. Thus, in some embodiments of the present invention, the phospholipid which contacts the enzyme can comprise SFA, MUFA, or PUFA, or a combination of any two of SFA, MUFA and PUFA.

[0163] 008933087The term monounsaturated fatty acid (MUFA) refers a subclass of fatty acid characterised by having one double bond in the fatty acid chain, with all of the remaining carbon atoms being single-bonded. The term polyunsaturated fatty acids (PUFA) refer to a subclass of fatty acids characterised by a carbon backbone with two or more carbon-carbon double bonds. MUFA include hypogeic acid, oleic acid, elaidic acid, gondoic acid, mead acid, erucic acid, nervonic acid and ximenic acid. PUFA include, linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The term saturated fatty acid (SFA) refers to a subclass of fatty acid which contain only single bonds between the carbon atoms.

[0164] Omega-3 (w-3), Omega-6 (w-6) and Omega-9 (w-9) represent 3 major classes of MUFAs and PUFAs. Omega-3 fatty acids (omega-3s) have a carbon-carbon double bond located three carbons from the methyl end (i.e. the w-carbon) of the alkyl chain. Alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are the best characterised omega-3 fatty acid acids. ALA contains 18 carbon atoms, whereas EPA and DHA are considered long-chain (LC) omega-3s because EPA contains 20 carbons and DHA contains 22 carbons.

[0165] Omega-6 fatty acids (omega-6s) have a carbon-carbon double bond that is six carbons away from the methyl end of the fatty acid chain. Linoleic acid (C18:2n-6) and arachidonic acid (C20:4n-6) are two of the major and best characterised omega-6s.

[0166] Omega-9 fatty acids (omega-9s) have a carbon-carbon double bond that is nine carbons away from the methyl end of the fatty acid chain. Oleic acid is a well characterised omega-9 fatty acid that can be incorporated into LPLs. LPC-oleate (18:1) is a major omega-9 containing LPL.

[0167] Delta nomenclature may be used to identify omega fatty acids. In delta nomenclature, the first number indicates the number of carbons in the carbon chain, and the second number indicates the number of double bonds. The third number, which can be optional, indicates the number of carbons from the carboxylic acid end to the first carbon in the double bond. This nomenclature also applies to identifying the acyl chain within an LPL, with the first number referencing the number of carbons in the acyl chain, and the second number referencing the number of double bonds. For example, LPC 18:2 denotes lysophosphatidylcholine comprising an acyl chain with 18 carbons and 2 double bonds (linoleic acid). Omega-3 fatty acids include Hexadecatrienoic acid (HTA) (16:3 n— 3), a-Linolenic acid (ALA) (18:3 n— 3), Stearidonic acid (SDA) (18:4 n— 3), Eicosatrienoic acid (ETE) (20:3 n— 3), Eicosatetraenoic acid (ETA) (20:4 n— 3), Eicosapentaenoic acid (EPA) (20:5 n— 3), Heneicosapentaenoic acid (HPA) (21 :5 n— 3), Docosapentaenoic acid (DPA) (22:5 n-3), Clupanodonic acid (22:5 n— 3), Docosahexaenoic acid (DHA) (22:6 n-3), Tetracosapentaenoic acid (24:5 n-3), Tetracosahexaenoic acid (Nisinic acid) (24:6 n-3). The methods and cells of the present invention may be used to produce LPLs comprising any of the foregoing Omega-3 fatty acids

[0168] Omega-6 fatty acids include Linoleic acid (LA) (C18:2n-6), Gamma-linolenic acid (GLA) (18:3 n— 6), Calendic acid (18:3 n— 6), Eicosadienoic acid (20:2 n— 6), Dihomo-gamma-linolenic acid (DGLA) (20:3 n— 6) , Arachidonic acid (AA, ARA) (20:4 n— 6) , Docosadienoic acid (22:2 n— 6), Adrenic acid (22:4 n— 6),

[0169] 008933087Docosapentaenoic acid (Osbond acid) (22:5 n-6), Tetracosatetraenoic acid (24:4 n-6), and Tetracosapentaenoic acid (24:5 n-6). The methods and cells of the present invention may be used to produce LPLs comprising any of the foregoing Omega-6 fatty acids.

[0170] Omega-9 fatty acids include hypogeic acid 16:1 (n-9), oleic acid 18:1 (n-9) and trans isomer elaidic acid 18:1 (n-9), gondoic acid 20:1 (n-9), mead acid 20:3 (n-9), erucic acid 22:1 (n-9), nervonic acid 24:1 (n-9), ximenic acid26:1 (n-9). The methods and cells of the present invention may be used to produce an LPL comprising any of the foregoing Omega-9 fatty acids.

[0171] In some embodiments, the methods and cells of the present invention may be used to produce any one or more of the foregoing Omega-3, Omega-6 and Omega-9 fatty acids, or any combination thereof.

[0172]

[0173] The present invention provides a method of producing a lysophospholipid (LPL), the method comprising providing an enzyme from the TMEM150 protein family and contacting the enzyme with a phospholipid to allow the enzyme to hydrolyse a fatty acid from the phospholipid, thereby producing the LPL. That is, in the disclosed methods the phospholipid is a precursor for the lysophospholipid. Thus, the species of the phospholipid directly relates to the species of lysophospholipid produced by the method.

[0174] In some embodiments, a method of producing a LPC is provided. In some embodiments, a method of producing a LPE is provided. In some embodiments, a method of producing a LPS is provided.

[0175] Contacting the enzyme with a phospholipid should be understood to mean bringing a phospholipid into close enough proximity to the enzyme to allow the enzyme to hydrolyse an ester bond of the phospholipid. The ester bond can be at either the sn1 or the sn2 position of the glycerol backbone.

[0176] In some embodiments, the phospholipid of the method is a glycerophospholipid. In some embodiments, the phospholipid comprises a SFA, a MUFA and / or a PUFA, or any combination thereof. In some embodiments, the phospholipid comprises a SFA. In some embodiments, the phospholipid comprises a MUFA. In some embodiments, the phospholipid comprises a PUFA.

[0177] The phospholipids which find use in the present invention comprise fatty acids covalently bound at the sn1 and sn2 positions. The fatty acids may be independently selected from a SFA, MUFA and a PUFA. In some embodiments, the phospholipid comprises SFA covalently bound at the sn2 position. In some embodiments, the phospholipid comprises a SFA covalently bound at the sn1 position. In some embodiments, the phospholipid comprises a MUFA covalently bound at the sn2 position. In some embodiments, the phospholipid comprises a MUFA covalently bound at the sn1 position. In some embodiments, the phospholipid comprises a PUFA covalently bound at the sn2 position. In some embodiments, the phospholipid comprises a PUFA covalently bound at the sn1 position. In some embodiments, the enzyme hydrolyses a SFA, a MUFA or a PUFA from the sn2 position of the phospholipid. In some embodiments, the enzyme hydrolyses a SFA, a MUFA or a PUFA from the sn1 position of the phospholipid. Preferably, the enzyme hydrolyses a SFA, MUFA, or a PUFA from the sn1 position of the phospholipid.

[0178] 008933087In some embodiments, the MUFA is selected from an omega-3 fatty acid, or an omega-6 fatty acid. In some embodiments, the PUFA is selected from an omega-3 fatty acid or an omega-6 fatty acid. In some embodiments, the phospholipid comprises a MUFA which is an omega-3 MUFA. In some embodiments, the phospholipid comprises a PUFA which is an omega-3 PUFA. In some embodiments, the phospholipid comprises a MUFA which is an omega-6 MUFA. In some embodiments, the phospholipid comprises a PUFA which is an omega-6 PUFA.

[0179] In some embodiments, the phospholipid comprises hexadecatrienoic acid (HTA), linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), tetracosapentaenoic acid or tetracosahexaenoic, or any combination thereof. In some embodiments, the phospholipid comprises linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA), or any combination thereof. In some embodiments, the phospholipid comprises docosahexaenoic acid (DHA). In some embodiments, the phospholipid comprises linoleic acid (LA), gamma-linolenic acid (GLA), calendic acid, eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA, ARA), docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid or tetracosapentaenoic acid. In some embodiments the omaga-3 fatty acid is selected from hexadecatrienoic acid (HTA), linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), tetracosapentaenoic acid or tetracosahexaenoic. In some embodiments, the omaga-3 fatty acid is selected from linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA). In some embodiments the omaga-3 fatty acid is docosahexaenoic acid (DHA).

[0180] In some embodiments the omaga-6 fatty acid is selected from linoleic acid (LA), gamma-linolenic acid (GLA), calendic acid, eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA, ARA), docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid or tetracosapentaenoic acid.

[0181] In some embodiments, the phospholipid is phosphatidylcholine (PC). In some embodiments, the PC comprises a MUFA or PUFA. In some embodiments, the PC comprise a MUFA. In some embodiments, the PC comprises a PUFA.

[0182] In some embodiments, the PC comprises a MUFA which is an omega-3 MUFA. In some embodiments, the PC comprises a PUFA which is an omega-3 PUFA. In some embodiments, the PC comprises a MUFA which is an omega-6 MUFA. In some embodiments, the PC comprises a PUFA which is an omega-6 PUFA.

[0183] In some embodiments, the PC comprises hexadecatrienoic acid (HTA), linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA),

[0184] 008933087tetracosapentaenoic acid or tetracosahexaenoic, or any combination thereof. In some embodiments, the PC comprises linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA), or a combination of any two. In some embodiments, the PC comprises docosahexaenoic acid (DHA).

[0185] In some embodiments, the PC comprises an omega-6 MUFA or an omega-6 PUFA. In some embodiments, the PC comprises an omega-6 MUFA. In some embodiments, the PC comprises an omega-6 PUFA. In some embodiments, the PC comprises linoleic acid (LA), gamma-linolenic acid (GLA), calendic acid, eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA, ARA), docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid or tetracosapentaenoic acid, or a combination of any two of the foregoing.

[0186] In some embodiments, the phospholipid is phosphatidylethanolamine (PE). In some embodiments, the PE comprises MUFAs or PUFAs. In some embodiments, the PE comprises MUFAs. In some embodiments, the PE comprises PUFAs. In some embodiments, the PE comprises a MUFA which is an omega-3 MUFA. In some embodiments, the PE comprises a PUFA which is an omega-3 PUFA. In some embodiments, the PE comprises a MUFA which is an omega-6 MUFA. In some embodiments, the PE comprises a PUFA which is an omega-6 PUFA. In some embodiments, the PE comprises hexadecatrienoic acid (HTA), linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), tetracosapentaenoic acid or tetracosahexaenoic, or any combination thereof. In some embodiments, the PE comprises linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA) a combination of any two of the foregoing fatty acids. In some embodiments, the PE comprises docosahexaenoic acid (DHA).

[0187] In some embodiments, the PE comprises an omega-6 PUFA. In some embodiments, the PE comprises linoleic acid (LA), gamma-linolenic acid (GLA), calendic acid, eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA, ARA), docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid or tetracosapentaenoic acid, a combination of any two of the foregoing fatty acids.

[0188] In some embodiments, the phospholipid is phosphatidylserine (PS). In some embodiments, the PS comprises MUFAs or PUFAs. In some embodiments, the PS comprises MUFAs. In some embodiments, the PS comprises PUFAs. In some embodiments, the PS comprises a MUFA which is an omega-3 MUFA. In some embodiments, the PS comprises a PUFA which is an omega-3 PUFA. In some embodiments, the PS comprises a MUFA which is an omega-6 MUFA. In some embodiments, the PS comprises a PUFA which is an omega-6 PUFA. In some embodiments, the PS comprises hexadecatrienoic acid (HTA), linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), tetracosapentaenoic acid or tetracosahexaenoic, or any combination thereof. In some embodiments, the PS comprises linolenic acid

[0189] 008933087(ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA) a combination of any two of the foregoing fatty acids.

[0190] In some embodiments, the PS comprises docosahexaenoic acid (DHA). In some embodiments, the PS comprises linoleic acid (LA), gamma-linolenic acid (GLA), calendic acid, eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA, ARA), docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid or tetracosapentaenoic acid or a combination of any two of the foregoing fatty acids.

[0191] It will be appreciated that the methods and cells of the invention may be used to produce multiple species of LPL derived from multiple species of phospholipids in a single reaction or cell culture, respectively. That is to say, the methods and cells may be used to produce one or more species of LPC, LPE, and LPS discussed supra.

[0192] In some embodiments the PUFA is selected from Hexadecatrienoic acid (HTA), a-Linolenic acid (ALA), Stearidonic acid (SDA), Eicosatrienoic acid (ETE), Eicosatetraenoic acid (ETA), Eicosapentaenoic acid (EPA), Heneicosapentaenoic acid (HPA), Docosapentaenoic acid (DPA) (Clupanodonic acid), Docosahexaenoic acid (DHA), Tetracosapentaenoic acid, Tetracosahexaenoic acid (Nisinic acid), Linoleic acid (LA), Gamma-linolenic acid (GLA), Calendic acid, Eicosadienoic acid, Dihomo-gamma-linolenic acid (DGLA), Arachidonic acid (AA, ARA), Docosadienoic acid, Adrenic acid, Osbond acid, Tetracosatetraenoic acid, or Tetracosapentaenoic acid.

[0193] In some embodiments the PUFA is an omega-3 fatty acid selected from Hexadecatrienoic acid (HTA), a-Linolenic acid (ALA), Stearidonic acid (SDA), Eicosatrienoic acid (ETE), Eicosatetraenoic acid (ETA), Eicosapentaenoic acid (EPA), Heneicosapentaenoic acid (HPA), Docosapentaenoic acid (DPA), Clupanodonic acid, Docosahexaenoic acid (DHA), Tetracosapentaenoic acid, Tetracosahexaenoic acid (Nisinic acid).

[0194] In some embodiments the PUFA is an omega-6 fatty acid selected from Linoleic acid (LA), Gammalinolenic acid (GLA), Calendic acid, Eicosadienoic acid, Dihomo-gamma-linolenic acid (DGLA), Arachidonic acid (AA, ARA), Docosadienoic acid, Adrenic acid, Osbond acid, Tetracosatetraenoic acid, or Tetracosapentaenoic acid.

[0195] In some embodiments the PUFA is selected from, linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), docosapentaenoic acid (DPA), or any combination thereof.

[0196] LPL species

[0197] The methods and cells provided in aspects of the present invention can be used to produce a range of LPLs. LPLs include lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPS). The roles of LPC, LPE and LPS in health and diseases are reviewed in Tan et al., Prog Lipid Res. (2020) 80:101068, which is hereby incorporated by reference in its entirety.

[0198] 008933087Lysophosphatidylcholines (LPCs, lysoPCs, lysolecithins), are a class of lysophospholipids which are derived from phosphatidylcholines (PCs). LPCs can be generated by the cleavage of an acyl side chain from phosphatidylcholine (PC) via the action of a phospholipase A. PC is a major component of biological membranes found in animal and plant cells. LPCs activate multiple signalling pathways that are involved in oxidative stress and inflammatory responses. LPCs can comprise a range of PUFA and MUFA, giving rise to multiple species of LPC. The main types of LPC include LPC16:0, LPC18:1 , LPC20:4, and LPC22:6. These LPC constitute more than 90% of total LPC level in plasma. Thus, essential fatty acids can move around the body as a unit of a LPC, for example as LPC22:4 and LPC22:6. Notably, LPC-DHA is the physiologically relevant form of the essential fatty acid DHA because LPC-DHA can cross the blood brain barrier, a process facilitated by MFSD2A. Aberrant (i.e. reduced) levels of LPCs are associated with several human diseases including cardio-vascular disease and brain diseases. The liver is thought to be the source of plasma LPCs, but the major molecular mechanism by which LPC is secreted from hepatocytes was previously unknown.

[0199] Lysophosphatidylethanolamines (LPEs) are a class of lysophospholipids which are derived from phosphatidylethanolamine (PEs). LPEs can be generated by the cleavage of an acyl side chain from PE via the action of phospholipase A. PE is present in cell membranes. In blood, LPE is the second highest lysoglycerophospholipids. Like LPC, albumin is also a carrier for LPE in blood. However, the cell types and tissues that produce circulating LPE still remains uncharacterized. Several studies demonstrated the involvement of LPE in various cellular activities, particularly differentiation and migration of PC-12 neuronal cells, MDA-MB-231 breast cancer cells and SK-OV3 ovarian cancer cells. LPE transporters expressed in the plasma membrane likely import circulating LPE species. Notably, Mfsd2a also accepts LPE as a ligand. PE species containing essential fatty acids such as docosahexaenoic acid (DHA) are abundant in brain. LPE is involved in various cellular activities, particularly differentiation and migration of PC-12 neuronal cells, MDA-MB-231 breast cancer cells and SK-OV3 ovarian cancer cells.

[0200] Lysophosphatidylserine (Lyso-PS or LPS) are a class of lysophospholipids which are derived from phosphatidylserine (PS). LPS can be generated by the cleavage of an acyl side chain from PS via the action of phospholipase A1 or A2. LPS can be found in various sites including thymus, peripheral lymphoid tissues, central nervous system (CNS) and colon, and is also detected in human plasma. LPS is thought to be involved in the immune response and contribute to histamine release, stimulation of mast cell degranulation, promotion of cell clearance by macrophages and suppression of T cell proliferation. LPS is also thought to be important in the inflammatory response.

[0201] The present invention provides methods of producing a lysophospholipid (LPL). In some embodiments, the LPL comprises a MUFA or PUFA. In some embodiments, the LPL comprises a MUFA. In some embodiments, the LPL comprises a PUFA. In some embodiments the LPL is an LPL-0 or an LPL-P. The fatty acid chain not hydrolysed from the glycerol backbone of a phospholipid during LPL synthesis remains part of the synthesised LPL. Phospholipase A1 enzymes preferentially hydrolyse phospholipids at the sn1 position, liberating the fatty acid at position sn1. Thus, for example, a PC having an omega-3 PUFA at position sn2 hydrolysed by a phospholipase A1 enzyme will produce an LPC comprising an

[0202] 008933087omega-3 PUFA at position sn2. In some embodiments, the LPL comprises a PUFA, MUFA, or SFA at position sn2. In some embodiments, the LPC comprises a PUFA, MUFA, or SFA at position sn2. In some embodiments, the LPE comprises a PUFA, MUFA, or SFA at position sn2. In some embodiments, the LPS comprises a PUFA, MUFA, or SFA at position sn2.

[0203] In some embodiments the enzyme from the TMEM150 protein family preferentially hydrolyses the fatty acid from the sn2 position of a phospholipid. In some embodiments enzyme from the TMEM150 protein family preferentially hydrolyses the fatty acid from the sn1 position of a phospholipid. In some embodiments, the enzyme from the TMEM150 protein family hydrolyses the fatty acid from the sn1 and sn2 position of a phospholipid. In some embodiments the enzyme from the TMEM150 protein family hydrolyses the fatty acid from the sn2 position of a phospholipid. In some embodiments enzyme from the TMEM150 protein family hydrolyses the fatty acid from the sn1 position of a phospholipid.

[0204] In some embodiments, the PUFA or MUFA is an omega-3 fatty acid. In some embodiments, the PUFA or MUFA is an omega-6 fatty acid.

[0205] In some embodiments, the omega-3 fatty acid is hexadecatrienoic acid (HTA), linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), tetracosapentaenoic acid or tetracosahexaenoic, or any combination thereof. In some embodiments, the omega-3 fatty acid is linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA), or any combination thereof. In some embodiments, the omega-3 fatty acid is docosahexaenoic acid (DHA). In some embodiments, the omega-6 fatty acid is linoleic acid (LA), gamma-linolenic acid (GLA), calendic acid, eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA, ARA), docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid or tetracosapentaenoic acid, or any combination thereof.

[0206] In some embodiments the LPL comprises a fatty acid with a chain length longer than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 carbons. In some embodiments the LPL comprises a fatty acid with a chain length longer than 17 carbons. In some embodiments the LPL comprises a fatty acid with a chain length longer than 21 carbons. In some embodiments the LPL comprises a fatty acid with a chain length longer than 22 carbons. In some embodiments the LPL comprises a fatty acid with a chain length shorter than 23 carbons. In some embodiments the LPL comprises a fatty acid with a chain length shorter than 19 carbons. In some embodiments the LPL comprises a fatty acid with a chain length between, and including, 16 to 22 carbons.

[0207] The present invention provides methods to produce LPLs. LPL yields can be measured through methods known in the art. By way of example, highly quantitative mass spectrometry-based lipidomics can be used to analyse LPL yields, as use in Example 1.3 of the present application. Details and discussion of lipidomic analysis suitable for quantifying LPLs are provided in Zullig etal. Anal Bioanal Chem.

[0208] 2020;412(10):2191-2209.

[0209] 008933087Relatedly, in some embodiments of the present invention, production of LPLs is measured using lipidomic analysis. In some embodiments, production of LPLs is measured using quantitative mass spectrometrybased lipidomics.

[0210]

[0211] a TMEM150

[0212] Aspects of the present invention relate to a cell that is engineered to express an enzyme from the TMEM150 protein family. The cells can be recombinant cells. Recombinant cells are cells that are engineered to express recombinant plasmids, genes or proteins. A further aspect of the present invention relates to a microorganism that is engineered to express an enzyme from the TMEM150 protein family. Further aspects of the present invention relate to methods of producing LPLs, wherein cells are utilised in said methods. That is, in some embodiments, the method comprises the use of a cell defined herein. In some embodiments, the cell is provided in isolated form and / or in culture. In some embodiments, cells may be provided in vitro.

[0213] A cell comprising an enzyme according to the present invention may do so through expression from a nucleic acid / expression vector according to the present invention that has been introduced into the cell. Cells contemplated for use with the present invention include prokaryotic and eukaryotic cells. For example, the prokaryotic cell may be a bacteria or archaea, and the eukaryotic cell may be / may be derived from a fungi, protist, insect, or microscopic animal or microscopic plant organism. Cells may be derived from any eukaryotic organism including mammals. Cells may be human cells.

[0214] Host cells are cells that are engineered to express a heterologous gene or protein. Cells that are engineered or modified to express an enzyme from the TMEM150 protein family can be described as the host cell.

[0215] In some embodiments, the cell is a eukaryotic cell, an animal cell, a fungal cell, a plant cell, an algal cell, a protist cell, a prokaryotic cell, or a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a fungal cell.

[0216] In preferred aspects, the microorganism is, or the cell is derived from, a fungus. In some embodiments the cell is a unicellular fungus. Any fungi may be used, such as laboratory strains (such as S. cerevisiae or Yarrowia lipolytica) , or field strains. In some embodiments, Saccharomyces yeast such as S. cerevisiae and cyanobacteria are contemplated for use in the present invention.

[0217] In some embodiments, the fungi may be Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, or Neocallimastigomycota. In some embodiments, the fungi may be Dikarya (including Deuteromycota), such as fungi of the Ascomycota, including Pezizomycotina, Saccharomycotina, and Taphrinomycotina; or Basidiomycota, including Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina. In some embodiments, the fungi may be fungi of the Entomophthoromycotina, Kickxellomycotina, Mucoromycotina, or Zoopagomycotina.

[0218] 008933087In some embodiments, the cell is a yeast cell. In some embodiments, the microorganism is Saccharomyces cerevisiae. In some embodiments, the cell is a Saccharomyces cerevisiae cell. In some embodiments, the microorganism is Yarrowia lipolytica. In some embodiments, the cell is a Yarrowia lipolytica cell.

[0219] Microorganisms and cells commonly used in commercial and industrial processes are contemplated, including cells used for the commercial or industrial production of chemicals, enzymes or other biological molecules.

[0220] In some embodiments the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments the cell is a HeLa cell. In some embodiments, the cell is a HEK 293T cell.

[0221] Insect cell lines are extensively used to produce enzymes peptides, and other recombinant proteins. They can also be used as systems to produce other valuable products. Often this is achieved by transient expression of vectors, such as plasmids, harbouring genes of interest such as enzymes. Insect cell lines can be adapted for high-density suspension culture for large-scale expression. Baculovirus is often utilised as a vector to introduce genes of interest into insect cell lines. Insect cell transformation and culture can be performed by any known means of the art.

[0222] The Spodoptera frugiperda cell line Sf-9 is commonly used for recombinant expression. Sf-9 cells can achieve high product yields and demonstrate good passaging stability compared to other insect cell lines. Sf21 cells are also derived from Spodoptera frugiperda. Other commonly used insect cell lines are derived from the cabbage looper Trichoplusia ni (High Five), and the fruit fly Drosophila melanogaster (S2). Relatedly, in some embodiments the cell is an insect cell. In some embodiments the insect cell is from a cell line derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster, Bombyx mori, or Mamestra brassicae. In some embodiments insect cell is a Sf-9 cell or a Sf21 cell. In some embodiments insect cell is a High Five Cell. In some embodiments, the insect cell is an S2 cell.

[0223] The cell may be modified. The cell may be engineered. Herein, the terms modified and engineered are used interchangeably. The cell may be modified through any known genetic engineering methodology. In some embodiments, the cell expresses a heterologous gene. In some embodiments, the cell is modified or engineered to express a heterologous gene. In some embodiments, the cell is modified or engineered to overexpress a gene. In some embodiments, the cell is modified or engineered to express a heterologous vector. In some embodiments, the cell is modified or engineered to express a heterologous plasmid.

[0224] In some embodiments, the cell is modified or engineered to express a gene which encodes an enzyme from the TMEM150 protein family. In some embodiments, the cell is modified or engineered to express a gene which encodes an enzyme from the TMEM150 protein family described herein. In some embodiments, the enzyme from the TMEM150 protein family is selected from TMEM150B or a homologue thereof; TMEM150A or a homologue thereof; SFK1 or a homologue thereof; TMEM150C or a homologue thereof; DRAM1 or a homologue thereof; or DRAM2 or a homologue thereof.

[0225] 008933087In some embodiments, the enzyme from the TMEM150 protein family comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6. In some embodiments, the enzyme from the TMEM150 protein family comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0226] Cells that are modified or engineered to express an enzyme from the TMEM150 protein family can produce a greater amount of LPL than a non-engineered parent cells. An increase in LPL production may be determined by quantifying LPL production in a modified or engineered cell and comparing it to LPL production in a non-engineered parent cell. LPLs may be isolated from the cells, or the culture media in which cells are grown, or the reaction solution. LPLs measured for this purpose may be LPLs bound to a carrier protein such as albumin. Carrier proteins are also known as transporter moieties. LPL amount may be expressed as a concentration of a sample. LPL amount or concentration can be determined by mass spectrometry-based lipidomic analysis as performed in Example 6 of the present disclosure.

[0227] A ‘parent cell’ as disclosed hereinabove refers to a cell of the same lineage, type or class of cell as an engineered cell, but which lacks one or more of the engineered modifications. A non-engineered parent cell is a cell which was genetically identical to the engineered cell before it was genetically engineered. It will be understood from the context that the parent cell may comprise one or more engineered modifications but will lack at least one engineered modification compared to the engineered cell. For example, in some embodiments, the engineered cell may overexpress an exogenous enzyme of the TMEM150 protein family in and be further engineered to reduce beta oxidation of fatty acids, reduce triglyceride (TAG) formation, reduce diacylglycerol (DAG) formation, and / or reduce expression of phospholipase genes compared to a parent cell. The parent cell in this context may overexpress an exogenous TMEM150 protein but not comprise a further modification to reduce beta oxidation of fatty acids, reduce triglyceride (TAG) formation, reduce diacylglycerol (DAG) formation, and / or reduce expression of phospholipase genes.

[0228] Relatedly, in some embodiments cells engineered to express an enzyme from the TMEM150 protein family show increased production of LPLs relative to a non-engineered parent cell. In some embodiments, the cell produces a greater amount of LPL compared to a non-engineered parent cell. In some embodiments, the cell produces a greater concentration of LPL compared to a non-engineered parent cell.

[0229] In some embodiments, the cell engineered to express an enzyme from the TMEM150 protein family produces a greater amount of LPL compared to a non-engineered parent cell. In some embodiments, the cell engineered to express an enzyme from the TMEM150 protein family produces a greater concentration of LPL compared to a non-engineered parent cell.

[0230] In some embodiments, the cell secretes a greater amount of LPL compared to a non-engineered parent cell. In some embodiments, the cell engineered to express an enzyme from the TMEM150 protein family secretes a greater amount of LPL compared to a non-engineered parent cell.

[0231] 008933087A cell that is engineered to express or overexpress an enzyme from the TMEM150 protein family releases a greater amount of LPL compared to a non-engineered parent cell. In some embodiments, the cell releases a greater amount of LPL compared to a non-engineered parent cell.

[0232] In some embodiments, the cell that is engineered to express or overexpress an enzyme from the TMEM150 protein family produces a greater amount of LPL compared to a non-engineered parent cell as a result of the phospholipase activity of the enzyme. In some embodiments, the enzyme produces LPL. In some embodiments, the enzyme produces LPL from a phospholipid.

[0233] Cells can be grown in a cell culture media. In some embodiments, the cell is grown in a culture media. In some embodiments, the cell is grown in a cell culture media.

[0234] In some embodiments, the culture media in which the cell is cultured comprises a greater amount of LPL compared to the culture media in which a non-engineered parent cell is cultured.

[0235] In some embodiments, the cell releases a greater amount of LPL into the culture media compared to a non-engineered parent cell. In some embodiments, the cell that is engineered to express an enzyme from the TMEM150 protein family releases a greater amount of LPL into the culture media compared to a nonengineered parent cell.

[0236] In some embodiments, a carrier protein liberates LPLs from the cell membrane. In some embodiments, the carrier protein is albumin. In some embodiments, the carrier protein is cyclodextrin. In some embodiments, a transport moiety liberates LPLs from the cell membrane. In some embodiments, the transport moiety is albumin. In some embodiments, the transport moiety is cyclodextrin.

[0237] In some embodiments, the growth media or solution in which the cell is grown comprises a greater concentration of LPLs compared to a growth media or solution in which a non-engineered parent cell was grown.

[0238] Inventors have shown that cells engineered to express an enzyme from the TMEM150 protein family produce a range of LPL species. The LPL species produced can depend on the characteristics of the phospholipid available to the cell for the hydrolyses reaction.

[0239] Accordingly, in some embodiments, the LPL produced by the cell is lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and / or lysophosphatidylserine (LPS) as described herein, or any combination thereof. In some embodiments, the LPL produced by the cell comprises omega-3 fatty acids and / or omega-6 fatty acids as described herein. In some embodiments, the LPLs produced by the cell comprises omega-3 fatty acids and / or omega-6 fatty acids as described herein. In some embodiments, the LPL produced by the cell comprises MUFAs. In some embodiments, the LPL produced or secreted by the cell comprises omega-3 MUFAs and / or omega-6 MUFAs. In some embodiments, the LPLs produced by the cell comprises omega-3 MUFAs and / or omega-6 MUFAs.

[0240] In some embodiments, the LPL produced by the cell comprises PUFAs. In some embodiments, the LPL produced by the cell comprises omega-3 PUFAs and / or omega-6 PUFAs. In some embodiments, the LPLs produced by the cell comprises omega-3 PUFAs and / or omega-6 PUFAs.

[0241] 008933087In some embodiments, the LPL produced by the cell comprises hexadecatrienoic acid (HTA), linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), tetracosapentaenoic acid ortetracosahexaenoic, or any combination thereof. In some embodiments, the LPL produced or secreted by the cell comprises linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA), or any combination thereof. In some embodiments, the LPL comprises docosahexaenoic acid (DHA).

[0242] In some embodiments, the LPL produced by the cell comprises linoleic acid (LA), gamma-linolenic acid (GLA), calendic acid, eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA, ARA), docosadienoic acid, adrenic acid, osbond acid, tetracosatetraenoic acid or tetracosapentaenoic acid, or any combination thereof.

[0243] In some embodiments, the LPL is LPC 14:0, LPC 16:0, LPE 16:0, LPS 16:0, LPC-0 16:0, LPE-P 16:0, LPC 16:1 , LPC-0 18:0, LPE-P 18:0, LPC 18:0, LPE 18:0, LPS 18:0, LPC 18:1 , LPE 18:1 , LPS 18:1 , LPC-O 18:1 , LPC 18:2, LPC 20:4, LPE 20:4, LPS 20:4 and / or LPS 22:6. In some embodiments, the LPL is LPC 14:0, LPC 16:0, LPE 16:0, LPS 16:0, LPC-0 16:0, LPE-P 16:0, LPC 16:1 , LPC-0 18:0, LPE-P 18:0, LPC 18:0, LPE 18:0, LPS 18:0, LPC 18:1 , LPE 18:1 , LPS 18:1 , LPC-0 18:1 , LPC 18:2, LPC 20:4, LPE 20:4, LPS 20:4 and / or LPS 22:6 and the cell is a human cell. In some embodiments, the LPL is LPC 16:0, LPE 18:0, LPS 18:1 , LPC-0 16:0, LPC-P 18:0, LPE-P 16:0, LPC 16:1 , LPE 18:1 , LPC-0 18:1 , LPC 18:0, LPE 20:4 and / or LPC 18:1. In some embodiments, the LPL is LPC 16:0, LPE 18:0, LPS 18:1 , LPC-0 16:0, LPC-P 18:0, LPE-P 16:0, LPC 16:1 , LPE 18:1 , LPC-0 18:1 , LPC 18:0, LPE 20:4 and / or LPC 18:1 , and the cell is a human cell. In some embodiments, the LPL is LPC 14:0, LPC 16:0, LPC 16:1 , LPC 18:1 , LPE 18:1 and / or LPE-P 16:0. In some embodiments, the LPL is LPC 14:0, LPC 16:0, LPC 16:1 , LPC 18:1 , LPE 18:1 and / or LPE-P 16:0, and the cell is a human cell.

[0244] In some embodiments, the LPL is LPC 16:0, LPC 16:1 , LPC 17:0, LPC 18:0, LPC 18:1 , LPC 18:2, LPC 20:1. LPE 18:0, LPE 18:1 , LPE 18:2, LPE 20:4 and / or LPS 18:1. In some embodiments, the LPL is LPC 16:0, LPC 16:1 , LPC 17:0, LPC 18:0, LPC 18:1 , LPC 18:2, LPC 20:1. LPE 18:0, LPE 18:1 , LPE 18:2, LPE 20:4 and / or LPS 18:1 , and the cell is an insect cell.

[0245] In some embodiments, the LPL is LPC 18:1 , LPC 18:0, LPC 16:1 , LPC 16:0, LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16:0. In some embodiments, the LPL is LPC 18:1 , LPC 18:0, LPC 16:1 , LPC 16:0, LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16:0, and the cell is a yeast cell. In some embodiments, the LPL LPC 18:1 , LPC 18:0, LPC 16:1 , LPC 16:0, LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16:0, and the cell is a yeast cell expressing TMEM150B.

[0246] In some embodiments, the LPL is LPC-0 16:0, LPC 20:1 , LPC 18:1 , LPC 16:1 , LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16:0. In some embodiments, the LPL is LPC-0 16:0, LPC 20:1 , LPC 18:1 , LPC 16:1 , LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16, and the cell is a yeast cell. In some embodiments, the LPL LPC 18:1 , LPC 18:0, LPC 16:1 , LPC 16:0, LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16:0, and the cell is a yeast cell expressing TMEM150B.

[0247] 008933087Cell culture

[0248] The present disclosure provides methods of producing LPLs, comprising the provision of a cell engineered to express an enzyme from the TMEM150 protein family. The enzymatic activity of the enzyme in the TMEM150 protein family can convert a phospholipid into an LPL. Cells can be cultured in culture media.

[0249] In some aspects, the present invention provides a method of producing LPLs comprising the use of a cell engineered to express an enzyme from the TMEM150 protein family. In some embodiments, the method of producing lysophospholipids comprises culturing a cell engineered to express an enzyme from the TMEM150 protein family. In some embodiments, the method comprises culturing a cell engineered to express an enzyme from the TMEM150 protein family in a culture medium. In some embodiments, the method comprises supplementing the culture medium with a source carbon and / or phosphate. In some embodiments, the method comprises supplementing the culture medium with a carrier protein such as albumin.

[0250] Specific methods of producing an LPL according to the present disclosure are disclosed within the examples herein. Additionally, the chemical and biochemical components of the method have been discussed in previous sections.

[0251] The method may comprise more than one stage. For example, the method of producing lysophospholipids comprises culturing a cell engineered to express an enzyme from the TMEM150 protein family. The cell culture method may comprise one or more of a fatty acid production stage, a growth stage, and a conversion stage.

[0252] The method may comprise more than one method of growing or utilising a cell. The method may comprise more than one substrate conversion process. The method may comprise fermentation, whole cell biocatalysis, biotransformation, and / or product isolation.

[0253] In some embodiments, the method of producing an LPL comprises whole cell biocatalysis. In some embodiments, the method of producing an LPL is a whole cell biocatalysis method of producing an LPL. Whole cell biocatalysis is reviewed in detail in Lin and Tao (Microbial Cell Factories volume 16, Article number: 106 (2017)). Whole-cell catalysis approaches can broadly be classified into biotransformation (biocatalysis) and fermentation bioprocesses. In fermentations, the products are synthesized from growth substrates via the host cells’ native metabolism and are accompanied in the fermentation broth by metabolic intermediates that make downstream processing complicated. In biotransformations, cell growth (the enzyme manufacturing phase) and production phase (conversion stage) are separated. Substrates are converted to the desired products by resting cells. The key advantages of whole-cell biocatalysis are its abilities to use cheap and abundant raw materials and to catalyse multistep reactions. The method may involve culture or fermentation of a cell. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. LPLs liberated from cell membranes can be collected from the culture media by partitioning culture

[0254] 008933087media / fermentation broth from the cells, extracting the lipid content, and separating LPLs. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

[0255] Microbial cultures, or cultures or microbiological cultures, generally comprise a culture vessel, a growth medium, and at least one microbial cell. In some embodiments, the microbial culture is a lab-scale culture. In some embodiments, the microbial culture is an industrial-scale culture. In some embodiments of the present disclosure, the microbial cell is a yeast cell, such as a Saccharomyces cerevisiae cell, or a derivative of a Saccharomyces cerevisiae cell.

[0256] A microbial medium, microbial growth medium or microbial culture medium is a liquid, semi-solid or solid designed to support the growth and proliferation of microbial cells. Microbial culture media are well known by scientists in the area of cell culture. Microbial cell culture media types and methods are comprehensively reviewed in A Rouf, Varsha Kanojia, HR Naik, Bazilla Naseer and Tahiya Qadri (2017) An overview of microbial cell culture, Journal of Pharmacognosy and Phytochemistry, Vol. 6, Issue 6 p 1923-1928, the contents of which are incorporated by reference.

[0257] Microbial culture media may be liquid (i.e. aqueous), semi-solid (i.e. gelatinous), or completely solid. Semi-solid and solid media may contain agar, gellan gum, or other solidification agents. Liquid media generally do not contain solidification agents.

[0258] The culture media may be any suitable media known to the skilled person. In some embodiments, the culture media minimal yeast media. In some embodiments the, culture media is Synthetic Defined (SD) Yeast Media without Uracil (SD-URA ). SD-URA-includes all necessary components for selective growth of Saccharomyces cerevisiae including various amino acids except uracil. This makes it a selective growth medium for yeast cells harbouring a URA3 gene.

[0259] Microbial culture media may comprise an appropriate source of energy and compounds which regulate the cell cycle. In addition to nutrients, the medium also helps maintain pH and osmolality. In some embodiments, the media can contain dextrose. In some embodiments, the media can contain 2% dextrose. In some embodiments, the media can be supplemented with glucose. In some embodiments, there is no glucose and / or no carbon source present in the media.

[0260] In some embodiments, a fed batch method is utilised. In some embodiments, a fed batch fermentation is utilised. In some embodiments, a precursor of fatty acid synthesis is provided during cell growth. In some embodiments, acetyl-coenzyme A (acetyl-CoA) is provided during cell growth. In some embodiments, a dose a precursor of fatty acid synthesis (or more than one dose) is provided during exponential cell growth. In some embodiments, a dose acetyl-CoA (or more than one dose) is provided during exponential cell growth.

[0261] Culture media may also be supplemented with Bovine Serum Albumin (BSA). BSA can act as a carrier for small molecules such as steroids, fatty acids, and thyroid hormones. In some embodiments, a carrier protein is provided to the cell. In some embodiments, albumin is provided to the cell. In some

[0262] 008933087embodiments albumin is bovine serum albumin (BSA). In some embodiments, growth media is supplemented with BSA. In some embodiments, cell culture media is supplemented with BSA.

[0263] In some embodiments, LPL production occurs within the cell. In some embodiments, LPL biosynthesis occurs within the cell. In some embodiments, conversion of a phospholipid acid to LPL occurs within the cell. In some embodiments, LPL production occurs within a membrane of the cell. In some embodiments, LPL biosynthesis occurs within a membrane of the cell. In some embodiments, conversion of a phospholipid acid to LPL occurs within a membrane of the cell. In some embodiments, LPL production is associated with a membrane of the cell. In some embodiments, LPL biosynthesis associated with a membrane of the cell. In some embodiments, conversion of a phospholipid acid to LPL associated with a membrane of the cell. In some embodiments, the membrane is the plasma membrane.

[0264] In some embodiments, LPL production occurs within the buffer, culture media, or cell lysate. In some embodiments, LPL biosynthesis occurs within the buffer, culture media, or cell lysate. In some embodiments, conversion of phospholipid to LPL occurs within buffer, culture media, or cell lysate.

[0265] LPLs may be harvested from culture medium or reaction solution using filtration. In this context, the term ‘harvested’ refers to collecting the LPLs produced by a cell engineered to express or overexpress a TMEM150 family member protein, and / or collecting LPLs produced by the methods of the invention. For example, collecting the LPLs produced under the reaction conditions discussed above, including collection from culture medium or any solution / surface the method has been performed in / on. Harvesting can involve the addition of an alcohol, such as ethanol, to a reaction or growth media. Harvesting can be completed following the Folch method which was first described in Folch, J. (1942). J. Biol. Chem., 146, 35-44. The Folch method is frequently used for lipid extractions and is based on the partitioning of lipids in a biphasic mixture of chloroform and methanol. Methanol disrupts hydrogen bonds between lipids and protein following addition of an organic solvent such as chloroform. Chloroform methanol and water can be used in a ratio of, for example 8:4:3. Further details of LPL harvesting methods are discussed in Example 1.

[0266] In some embodiments of the present invention the method comprises a step of harvesting the LPL. In some embodiments, the method comprises a subsequent step of harvesting the LPL. In some embodiments, the step of harvesting the LPL comprises homogenizing and / or dissolving the membrane in which the TMEM150 family protein is situated.

[0267] Secretion of LPLs into culture medium or reaction solution can be enhanced by adding solvent to the culture medium or reaction solution. As such, in some embodiments, the step of harvesting the LPL comprises the use of a solvent to increase the secretion of LPLs into the medium. In some embodiments the solvent is an alcohol. In some embodiments, the alcohol is ethanol. In some embodiments the alcohol is added to the reaction solution, the growth medium, or the culture medium. In some embodiments, the

[0268] 008933087step of harvesting involves use of a concentration of alcohol in media of between 1 to 6% alcohol / vol media.

[0269] Albumin or human serum albumin is a carrier protein for LPLs in blood plasma, with six LPC molecules thought to be able to bind albumin at the primary fatty acid binding sites (Wang et al., 2023). Like LPC, albumin is also a carrier for LPE in blood. In plasma, the majority of LPC is bound to albumin.

[0270] In some embodiments, the method of producing LPLs further comprises the step of contacting the LPL with a transporter moiety to non-covalently bind to the LPL. In some embodiments, the method of producing LPLs further comprises the step of contacting the LPL with a carrier protein to non-covalently bind to the LPL. The transporter moiety can be a carrier protein. In some embodiments the transporter moiety can be any moiety capable of binding to amphipathic lipids. In some embodiments the transporter moiety is an albumin protein, alpha-fetoprotein protein, or vitamin D-binding protein, cyclodextrin, an apolipoprotein, apolipoprotein A-1, apolipoprotein B, apolipoprotein D, apolipoprotein E, apolipoprotein F, or apolipoprotein M, or any combination thereof. In some embodiments the transporter moiety is albumin. In some embodiments the transporter moiety is cyclodextrin.

[0271] In some embodiments, the method of producing LPLs comprises providing an albumin, alpha-fetoprotein protein, vitamin D-binding protein, cyclodextrin, an apolipoprotein, apolipoprotein A-1, apolipoprotein B, apolipoprotein D, apolipoprotein E, apolipoprotein F, or apolipoprotein M to a reaction or cell culture. In some embodiments, the method of producing LPLs comprises providing albumin, alpha-fetoprotein protein, or vitamin D-binding protein to the membrane. In some embodiments, the method of producing LPLs comprises providing albumin, alpha-fetoprotein protein, or vitamin D-binding protein to the membrane surface. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is bovine serum albumin (BSA). In some embodiments, the albumin is human serum albumin. In some embodiments of the present invention the method comprises a step of harvesting the LPL. In some embodiments, the harvesting step comprises contacting the LPL with a transporter moiety which can bind to amphipathic lipids. In some embodiments, the transporter moiety non-covalently binds to the LPL. The transporter moiety can be a carrier protein. The transporter moiety can be any moiety capable of binding to amphipathic lipids. In some embodiments the transporter moiety is an albumin protein, alpha-fetoprotein protein, vitamin D-binding protein, cyclodextrin, an apolipoprotein, apolipoprotein A-1, apolipoprotein B, apolipoprotein D, apolipoprotein E, apolipoprotein F, apolipoprotein M, or any combination thereof. In some embodiments the transporter moiety is albumin. In some embodiments the transporter moiety is cyclodextrin.

[0272] In some embodiments, the harvesting step comprises providing albumin, alpha-fetoprotein protein, or vitamin D-binding protein to a reaction or cell culture medium. In some embodiments the harvesting step comprises providing albumin, alpha-fetoprotein protein, or vitamin D-binding protein to the membrane. In some embodiments, the method of producing LPLs comprises providing albumin, alpha-fetoprotein protein, or vitamin D-binding protein to the membrane surface. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is bovine serum albumin (BSA). In some embodiments, the albumin is human serum albumin.

[0273] 008933087Cyclodextrins are a family of cyclic oligosaccharides comprising a macrocyclic ring typically of six to eight glucose monomer subunits joined by a-1 ,4 glycosidic bonds, creating a cone shape. Within each ring is a hydrophobic cavity which allows cyclodextrins to form soluble complexes with lipids and translocate hydrophobic phospholipids into cultured cells (Kainu et al., 2010). In these complexes the cyclodextrin cones sit around the tail groups. Cyclodextrins can also be used as carriers to extract lipids from cells and for the exchange of lipids in membranes. The three major species cyclodextrins are a-cyclodextrin (a-CD), p-cyclodextrin (p-CD) and y-cyclodextrin (y-CD). Cyclodextrin can include any derivatives of a-cyclodextrin, methyl-a-cyclodextrin, p-cyclodextrin, methyl-p-cyclodextrin. In the present invention the inventors utilise the ability of cyclodextrins to complex with phospholipids and LPLs to harvest LPLs from the methods or cells defined herein. That is, cyclodextrins can bind to amphipathic lipids, phospholipids and / or LPLs.

[0274] Relatedly, in some embodiments, the harvesting step comprises the use of a cyclodextrin. In some embodiments, the harvesting step comprises contacting the LPL with a cyclodextrin. In some embodiments, the harvesting step comprises the addition of a cyclodextrin to the reaction solution or culture medium. In some embodiments, the cyclodextrin associates with the LPL. In some embodiments, the cyclodextrin binds to the LPL. In some embodiments, the cyclodextrin solubilises the LPL. In some embodiments, contacting the LPL with the cyclodextrin isolates the LPL from the cell. In some embodiments, the cyclodextrin extracts LPLs from the cell.

[0275] In some embodiments, the cyclodextrin is a-cyclodextrin (a-CD), p-cyclodextrin (p-CD) or y-cyclodextrin (y-CD). In some embodiments, the cyclodextrin is methyl-a-cyclodextrin (a-CD), methyl-p-cyclodextrin (p-CD) or methyl-y-cyclodextrin (y-CD). In some embodiments, the cyclodextrin is a derivative of a-cyclodextrin, methyl-a-cyclodextrin, p-cyclodextrin, methyl-p-cyclodextrin, y-cyclodextrin (y-CD) or methyl-y-cyclodextrin (y-CD). In some embodiments, the cyclodextrin is a-cyclodextrin, methyl-a-cyclodextrin, p-cyclodextrin, methyl-p-cyclodextrin. In some embodiments, the cyclodextrin is a derivative of a-cyclodextrin, methyl-a-cyclodextrin, p-cyclodextrin, methyl-p-cyclodextrin.

[0276] Aspects of the current disclosure relate to methods of producing LPLs comprising culturing a cell (e.g. a S. cerevisiae cell) and isolating or harvesting a LPL from the culture. The LPL may be an LPC, LPE or LPS. As used herein, “isolation of an LPL from the culture” means separation from another portion of the culture e.g. the cells and / or the culture medium. However, “isolation of an LPL from the culture” does not mean that the LPL needs to be removed in a pure form.

[0277] In some embodiments, LPLs of interest are collected from a membrane through the action of a transport moiety, for example albumin, into the media and can be isolated from the media. In this context, the term collected refers to the process of the transport moiety coming into contact with the LPL associated with the membrane or membrane protein (e.g. a TMEM150 phospholipase), forming of a non-covalent bond between the transport moiety and LPL, and the movement of the LPL-transport moiety complex away from the membrane.

[0278] In other embodiments, LPLs of interest are not collected into the media and need to be isolated from the cell fraction.

[0279] 008933087The first step for both of these methods is to separate the cell fraction from the liquid fraction. The liquid fraction is the liquid portion (e.g. culture media) which lies above a sediment formed by the solid cells and below the culture headspace (gaseous fraction). The cell fraction is the solid sediment fraction which forms below the liquid fraction.

[0280] The cell fraction can be separated from the liquid fraction in a number of ways, including filtration, chromatography, evaporation, sedimentation, and centrifugation. Following this crude separation, the LPLs of interest can be separated from their fraction.

[0281] LPLs which are collected into the media by the action of a transport moiety, for example albumin, are present in the liquid fraction. The liquid fraction comprising LPLs can be separated from the cell fraction according to any suitable method known in the art, including those described above. The LPLs present in the liquid fraction can be further purified in a number of ways, including chromatography, distillation, crystallisation, and pervaporation.

[0282] Distillation, e.g. vacuum distillation of a solvent is the process of separating the components or substances from a liquid mixture by using selective boiling and condensation. The process takes advantage of the fact that different compounds have different boiling points. There are a number of different types of distillation that could be used to separate LPLs from media liquid fraction: simple distillation, fractional distillation, vacuum distillation, and azeotropic distillation, to name a few.

[0283] The isolation of secreted LPLs from the growth medium can be performed continuously throughout the growth of the cell.

[0284] To isolate LPLs which may not be collected from cell membranes, the cells may need to be lysed. Cells can be lysed through mechanical homogenization, ultrasonic homogenisation, pressure homogenisation, freeze-thaw treatment, heat treatment, osmotic lysis and chemical lysis. Following this, the LPLs can be isolated through methods known in the art. In some embodiments of the present invention, harvesting LPLs comprises lysis of a membrane and / or a cell. In some embodiments, the harvesting step comprises a step selected from mechanical homogenization, ultrasonic homogenisation, pressure homogenisation, freezethaw treatment, heat treatment, osmotic lysis and chemical lysis, or any combination thereof, of a membrane and / or cell. In some embodiments, a step of harvesting LPLs comprises homogenizing and / or dissolving a membrane.

[0285] Remaining cell mass from the cell fraction can be harvested and used as either fertiliser or in animal feeds, or as feedstock for other biotechnological processes. These products are often high in nutrients, minerals, protein, oil, and / or carbohydrates, and have value as fertiliser or in animal feeds. The remaining cell mass can be either whole cells or the solid fraction of lysed cells.

[0286] In some embodiments, the LPL is purified. As used herein, “purification of a LPL” means that the relative amount of the LPL is increased compared to other components of the extract / product i.e. contaminants are removed from the extract / product. The LPL does not necessarily have to be 100% pure, although it may be in some embodiments.

[0287] 008933087In some embodiments, the LPL is purified after it is isolated from the culture. In some embodiments, the LPL is more than 50% pure after purification. In some embodiments, the LPL is more than one of 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure after purification.

[0288] LPLs may be extracted or harvested from reaction solutions using the Folch method. This involves terminating hydrolysis reactions by adding 200 pL chloroform:methanol (2:1) to form Folch ratio of chloroform:methanol:water (8:4:3) and vigorously vortexing the solution followed by centrifugation. The lower organic phase is then recovered by pipetting and dried under argon. Samples can then be redissolved in chloroform:methanol (3:2) and 13.3% spotted on a TLC plate. Mobile phase for TLC can be chloroform:methanol:water (65:25:4). Methods such as this involve homogenising and dissolving any membranes the TMEM150 family protein may be situated in. Other lipid extraction methods include the methanol / tert-butyl methyl ether (MTBE) (Matyash) method, and the single-phase methanol / 1 -butanol (Alshehry) method.

[0289] In some methods the step of harvesting comprises the Folch method. In some embodiments, the Folch method comprises the use of chloroform methanol and water. In some embodiments, the Folch method comprises the use of chloroform methanol and water in a ratio of around 8:4:3. In some embodiments, the harvesting step comprises the Matyash method of lipid extraction. In some embodiments, the harvesting step comprises the Alshehry method of lipid extraction. In some embodiments, the harvesting step comprises a combination of lipid extraction methods.

[0290] It will be understood that “the LPL” may refer to a particular LPL species or it may refer to a mixture of LPL species as described above.

[0291] In some embodiments, cells of the invention are engineered to produce one or more species of LPL compared to a non-engineered parent cell. In some embodiments, the relative abundance of LPL produced by an engineered cell of the invention as compared to a non-engineered parent cell may be determined using any suitable method known to the skilled person, for example highly quantitative mass spectrometry-based lipidomics.

[0292] The yield, (also referred to as titre, or reaction yield) is the amount of product obtained in a reaction or enzymatic process. The absolute yield can be given as the weight in grams or in moles (molar yield). Yield can also be given as a concentration or the desired product. Cellular yield refers to product within the cellular pool, e.g. LPL amount within the cellular LPL pool. Environmental yield refers to product within the environment in which the method has been completed, e.g. in the culture media or reaction solution. Total yield can refer to the combination of environmental and cellular yields.

[0293] In some embodiments, yield is cellular yield. In some embodiments, yield is environmental yield. In some embodiments, yield is total yield. In some embodiments, LPL is LPC, LPE and / or LPS.

[0294] In some embodiments, yield of LPL is greater than 1 nmol / g, 2 nmol / g, 3 nmol / g, 4 nmol / g, 5 nmol / g, 10 nmol / g, 20 nmol / g, 30 nmol / g, 40 nmol / g, 50 nmol / g, 60 nmol / g, 70 nmol / g, 80 nmol / g, 90 nmol / g, 100 nmol / g, 200 nmol / g, 300 nmol / g, 400 nmol / g, 500 nmol / g, 600 nmol / g, 700 nmol / g, 800 nmol / g, 900 nmol / g, 1000 nmol / g, 1100 nmol / g, 1200 nmol / g, 1300 nmol / g, 1400 nmol / g, 1500 nmol / g, 1600 nmol / g,

[0295] 0089330871700 nmol / g, 1800 nmol / g, 1900 nmol / g, 2000 nmol / g, 2200 nmol / g, 2400 nmol / g, 2600 nmol / g, 2800 nmol / g or 3000 nmol / g.

[0296] In some embodiments, yield of LPL is greater than 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500 nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, 2000 nM, 2200 nM, 2400 nM, 2600 nM, 2800 nM or 3000 nM.

[0297] In some embodiments, yield of LPL is between 50 nM to 3000 nM, 100 nM to 3000 nM, 200 nM to 3000 nM, 300 nM to 3000 nM, 400 nM to 3000 nM, 500 nM to 3000 nM, 600 nM to 3000 nM, 700 nM to 3000 nM, 800 nM to 3000 nM, 900 nM to 3000 nM, 1000 nM to 3000 nM, or 2000 nM to 3000 nM.

[0298] In some embodiments, yield of LPL is between 3000 nM and 15000 nM.

[0299] Genetic modification of cells

[0300] The cells, including microorganisms, according to some aspects of the present disclosure may be genetically modified or engineered to express heterologous genes, which may result in additional enzymatic activity. For example, the cells may be genetically modified or engineered to express an enzyme from the TMEM150 protein family.

[0301] Cells which are genetically modified or engineered to express a heterologous gene may be referred to as recombinant cells. Vectors may be used to introduce heterologous genes into cells. The vector may be an expression vector for expression of the foreign genetic material in the cell. Such vectors may include a promoter and / or a ribosome binding site (RBS) sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, RBS, enhancers and other elements, such as for example polyadenylation signals, which may be necessary and which are positioned in the correct orientation in order to allow for recombinant protein expression.

[0302] The vector may be used to replicate the nucleic acid in a compatible host cell. Therefore, nucleic acids can be produced by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell and growing the host cell under conditions that bring about replication of the vector. Vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the gene sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express the enzymes from a vector according to the invention. Suitable vectors include plasmids, binary vectors, viral vectors, cosmids, and artificial chromosomes (e.g. yeast artificial chromosomes).

[0303] A construct or vector comprising a nucleic acid as described above need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

[0304] 008933087Constructs and vectors may further comprise selectable genetic markers consisting of genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, chloramphenicol, ampicillin, etc.

[0305] Those skilled in the art can construct vectors and design protocols for recombinant gene expression, for example in a microbial cell. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook et al, 2001, Cold Spring Harbor Laboratory Press and Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992.

[0306] Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a polypeptide from a vector according to the invention. In some embodiments, the vector may be a plasmid, phage, cosmid, MAC, virus, etc.

[0307] Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al., 2001 , Molecular Cloning: a laboratory manual, 3rd edition, Cold Harbour Laboratory Press.

[0308] The term “operably linked” may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of the nucleotide sequence. The resulting transcript may then be translated into a desired peptide or polypeptide. The promoter may be a GAL promoter.

[0309] In some embodiments, the vector may comprise an element for facilitating translation of encoded protein from mRNA transcribed from the construct. For example, the construct may comprise a ribosomal binding site (RBS) sequence upstream of the start codon. In some embodiments, RBS sequences may be designed to provide for different levels of expression of the encoded proteins.

[0310] In some embodiments, the vector may encode one or more regulatory elements for modulating expression of the encoded protein(s). In some embodiments, the response element is an element that causes upregulation of gene or protein expression in response to treatment with a particular agent. For example, the agent may induce transcription of DNA encoding the protein(s) from a vector including a response element for the agent. Other induction agent / response element combinations are known in the art.

[0311] In some embodiments, the vector may encode one or more response elements for constitutive expression of the encoded protein(s), such that no induction is necessary. In some embodiments the vector may comprise a transcription terminator sequence downstream of the sequences encoding to the protein or proteins of interest. In some embodiments the terminator may be a T7 terminator sequence. In some

[0312] 008933087embodiments the vector may comprise a sequence encoding a detectable marker in-frame with the sequence encoding the protein of interest to facilitate detection of expression of the protein, and / or purification or isolation of the protein (e.g. a His, (e.g. 6XHis (SEQ ID NO:7), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus).

[0313] The nucleic acids / expression vectors can be introduced into a cell by any suitable means, which are well known to the skilled person. In some embodiments the nucleic acids / expression vectors are introduced into a cell by transformation, transduction, conjugation, transfection or electroporation.

[0314] Vectors containing hybrid promoters may be preferred. This may contain native yeast or other promoters that have been engineered to convert them to inducible or constitutive promoters. Hybrid vectors may contain randomised variable region sequences that confer different expression levels of recombinant proteins.

[0315] The TDH3 or the GPD promoter may be preferred. Constitutive promoters are defined as promoters active in vivo in all circumstances, and, on the other hand, inducible promoters are switched ON and OFF by transcription factors depending on the in vivo conditions. Constitutive promoters with different strength such as ADH1 (alcohol dehydrogenase 1) promoter, PGK1 (Phosphoglycerate kinase) promoter and other promoters can be used to drive the expression of a heterologous gene. Inducible promoters include copper inducible promoter (CUP1), or galactose inducible promoter (GAL1 , GAL10). GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae

[0316] A vector, or multiple vectors, can be used to introduce one or more heterologous genes into cells. In some embodiments of the present invention, cells may be modified with a heterologous pathway for the production of phospholipids and / or LPLs. In some embodiments, the vector is a plasmid. In some embodiments, the vector is an integrative plasmid.

[0317] Engineered host cells

[0318] Some organisms that can be utilised in the production of LPLs may not naturally produce PLs with chain lengths longer than 18 carbons, which are the substrates of the TMEM150 family proteins described in this invention. In such organisms, the host cell may be engineered to produce longer chain omega-3 and omega-6 fatty acids. That is, to enhance or promote production of LPLs with longer acyl chains, the metabolic pathways of host cells can be modified or engineered to produce fatty acids with a chain length greater than 18 carbons. Fatty acids provide substrate for phospholipid synthesis and subsequent lysophospholipid production by enzymes in the TMEM150 protein family.

[0319] In some embodiments, the cell is modified or engineered to increase production of a fatty acid or a combination of fatty acids compared to a non-engineered parent cell. In some embodiments, the cell is modified or engineered to increase production of an omega-3 or omega-6 fatty acid or a combination of an omega-3 or omega-6 fatty acids compared to a non-engineered parent cell. In some embodiments, the cell is modified or engineered to increase production of an omega-3 fatty acid or a combination of an 008933087omega-3 fatty acids compared to a non-engineered parent cell. In some embodiments, the cell is modified or engineered to increase production of an omega-6 fatty acid or a combination of an omega-6 fatty acids compared to a non-engineered parent cell.

[0320] In some embodiments, the cell is modified or engineered to increase production of a fatty acid or a combination of fatty acids selected from stearic acid, oleic acid, linoleic acid, linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid, or any combination thereof, compared to a non-engineered parent cell.

[0321] In some embodiments, the cell is modified or engineered to express a gene which encodes an enzyme which increases the concentration of long chain unsaturated fatty acids and / or long chain saturated fatty acids.

[0322] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes S. cerevisiae elongase 2 (ScELO2). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes ScELO2 described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an ScELO2 homologue. In some embodiments, the ScELO2 homologue comprises an amino acid sequence with at least 40% sequence identity to ScELO2. In some embodiments, the ScELO2 homologue comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of ScELO2

[0323] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes Mortierella alpina delta-9-desaturase (MaD9D). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes MaD9D described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an MaD9D homologue. In some embodiments, the MaD9D homologue comprises an amino acid sequence with at least 40% sequence identity to MaD9D. In some embodiments, the homologue MaD9D comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of MaD9D.

[0324] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes Mortierella alpina delta-12-desaturase (MaD12D). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes MaD12D described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an MaD12D homologue. In some embodiments, the MaD12D homologue comprises an amino acid sequence with at least 40% sequence identity to MaD12D. In some embodiments, the MaD12D homologue comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of MaD12D.

[0325] In some embodiments, the cell is modified or engineered to express a gene which encodes S. kluyveri w3-Desaturase (skFAD). In some embodiments, the cell is modified or engineered to express or overexpress a gene which skFAD described herein. In some embodiments, the cell is modified or 008933087engineered to express or overexpress a gene which encodes an skFAD homologue. In some embodiments, the skFAD homologue comprises an amino acid sequence with at least 40% sequence identity to skFAD. In some embodiments, the skFAD homologue comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of skFAD.

[0326] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes Ostreococcus tauri delta-6-desaturase (OtD6D). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes OtD6D described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an OtD6D homologue. In some embodiments, the OtD6D homologue comprises an amino acid sequence with at least 40% sequence identity to OtD6D. In some embodiments, the OtD6D homologue comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of OtD6D.

[0327] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes Mortierella alpina delta-6-elongase (MaD6E). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes MaD6E described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an MaD6E homologue. In some embodiments, the MaD6E homologue comprises an amino acid sequence with at least 40% sequence identity to MaD6E. In some embodiments, the MaD6E homologue comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of MaD6E.

[0328] In some embodiments, the cell is modified or engineered to express or overexpress a gene which Paramecium tetraurelia delta-5-desaturase (Ptet1D5D). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes Ptet1D5D described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an Ptet1D5D homologue. In some embodiments, the Ptet1D5D homologue comprises an amino acid sequence with at least 40% sequence identity to Ptetl D5D. In some embodiments, the Ptetl D5D homologue comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of Ptetl D5D.

[0329] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes Ostreococcus tauri C20 / 22 elongase (OtELO2). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes OtELO2 described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an OtELO2 homologue. In some embodiments, the OtELO2 homologue comprises an amino acid sequence with at least 40% sequence identity to OtELO2. In some embodiments, the OtELO2 homologue enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of OtELO2.

[0330] 008933087In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes Thraustochytrium sp delta-5-desaturase (TaFAD4). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes TaFAD4 described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an TaFAD4 homologue. In some embodiments, the TaFAD4 homologue comprises an amino acid sequence with at least 40% sequence identity to TaFAD4. In some embodiments, the TaFAD4 comprises homologue, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of TaFAD4. Increasing cellular production of a phospholipid, or a phospholipid precursor, in a cell described herein has the downstream consequence of increasing the production of the corresponding LPL following hydrolysis of the LP by the TMEM150 family member protein.

[0331] In some embodiments, the cell is modified or engineered to express a gene which encodes an enzyme in a branch of a Kennedy pathways (CDP-choline and CDP-ethanolamine pathways). The CDP-choline pathway is the major pathway by which mammalian cells synthesise phosphatidylcholine (PC) for incorporation into membranes or lipid-derived signalling molecules. The CDP-ethanolamine pathway is responsible for the biosynthesis of the phospholipid phosphatidylethanolamine (PE). Enzymes of the Kennedy pathway CDP-choline pathway include choline kinase (CK), CTP:phosphocholine cytidylyltransferase (CCT) and Choline / ethanolamine phosphotransferase (CEPT). Enzymes of the Kenedy pathway CDP-ethanolamine pathway include ethanolamine kinase (EK), phosphoethanolamine cytidylyltransferase (ECT) and ethanolaminephosphotransferase (ETP).

[0332] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an enzyme in a Kennedy pathway. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an enzyme in a Kennedy pathway described herein. In some embodiments, the enzyme in the Kennedy pathway is one of CK, CCT, CPT, EK, ECT or ETP. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes one of CK, CCT, CPT, EK, ECT or ETP. In some embodiments, the cell is modified or engineered to express or overexpress one or more genes which encodes for one or more of CK, CCT, CPT, EK, ECT or ETP.

[0333] In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT). In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT) described herein. In some embodiments, the cell is modified or engineered to express or overexpress a gene which encodes an LPCAT homeolog. In some embodiments, the LPCAT homeolog comprises an amino acid sequence with at least 40% sequence identity to mammalian LPCAT In some embodiments, the LPCAT homeolog comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of to mammalian LPCAT.

[0334] 008933087In some embodiments, the cell is engineered to increase production of a phospholipid by reducing beta oxidation of fatty acids, reducing triglyceride (TAG) formation, reducing diacylglycerol (DAG) formation, or reducing expression of phospholipase genes, or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid by modulating expression of one or more enzymes of the Kennedy pathway, or lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT), or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid by adjusting expression of one or more enzymes of the Kennedy pathway, or lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT), or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid by increasing expression of one or more enzymes of the Kennedy pathway, or lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT), or any combination thereof.

[0335] In some embodiments, the cell is engineered to increase production of a phospholipid by modulating the expression of a gene, or functional homologues thereof, selected from sfk1 , Iem3, pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid by adjusting the expression of a gene, or functional homologues thereof, selected from; sfk1 , Iem3, pox1 , dga1 , Iro 1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid by reducing expression of a gene, or functional homologues thereof, selected from; sfk1 , Iem3, pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid by reducing expression of a gene, or functional homologues thereof, selected from; sfk1 , Iem3, pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof. In some embodiments, the cell is engineered to redirect lipid flux towards phospholipid production by: (i) increasing expression of one or more enzymes of the Kennedy pathway; (ii) overexpressing lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT) (iii) reducing expression of a gene or functional homologues thereof, selected from; sfk1 , Iem3, pox1 , dga1 , Iro 1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof. In some embodiments, the cell is engineered to redirect lipid flux towards phospholipid production by a combination of (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii).

[0336] In some embodiments, the cell is engineered to redirect lipid flux towards phospholipid production by: (i) adjusting expression of one or more enzymes of the Kennedy pathway, and / or overexpressing lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT), or any combination thereof; and / or

[0337] (ii) adjusting expression of any combination of the following, or functional homologues thereof, compared to a non-engineered parent cell; sfk1 , Iem3, pox1 , dga1 , Iro 1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2.

[0338] In some embodiments, the cell is engineered to increase production of a phospholipid comprising an unsaturated fatty acid. In some embodiments, the cell is engineered to increase production of a phospholipid comprising an unsaturated fatty acid selected from stearic acid, oleic acid, linoleic acid, 008933087linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid, or any combination thereof, compared to a non-engineered parent cell.

[0339] Accordingly, in some embodiments, the cell is engineered to adjust the expression of a gene, or a functional homologue thereof, selected from; sfk1 , Iem3, pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2 are1 , are2, faa1 , faa2, or any combination thereof.

[0340] In some embodiments, the cell is engineered to increase production of a phospholipid comprising an unsaturated fatty acid by increasing expression of a gene, or functional homologues thereof, selected from elongase 2 (ELO2), delta-9-desaturase (D9D), delta-12-desaturase (D12D), w3-Desaturase (FAD), delta-6-desaturase (D6D), delta-6-elongase (D6E), delta-5-desaturase (D5D), C20 / 22 elongase (ELO2), and delta-5-desaturase (TaFAD4), or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid comprising a unsaturated fatty acid by increasing expression of a gene, or functional homologues thereof, selected from elongase 2 (ELO2), delta-9-desaturase (D9D), delta-12-desaturase (D12D), w3-Desaturase (FAD), delta-6-desaturase (D6D), delta-6-elongase (D6E), delta-5-desaturase (D5D), C20 / 22 elongase (ELO2), and delta-5-desaturase (TaFAD4), or any combination thereof, compared to a non-engineered parent cell.

[0341] In some embodiments, the cell is engineered to increase production of a phospholipid comprising an unsaturated fatty acid by increasing expression of a gene, or functional homologues thereof, selected from; S. cerevisiae elongase 2 (ScELO2), Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), S. kluyveri w3-Desaturase (skFAD), Ostreococcus tauri delta-6-desaturase (OtD6D), Mortierella alpina delta-6-elongase (MaD6E), Paramecium tetraurelia delta-5-desaturase (Ptet1D5D), Ostreococcus tauri C20 / 22 elongase (OtELO2), and Thraustochytrium sp delta-5-desaturase (TaFAD4), or any combination thereof. In some embodiments, the cell is engineered to increase production of a phospholipid comprising an unsaturated fatty acid by increasing expression of a gene, or functional homologues thereof, selected from; S. cerevisiae elongase 2 (ScELO2), Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), S. kluyveri w3-Desaturase (skFAD), Ostreococcus tauri delta-6-desaturase (OtD6D), Mortierella alpina delta-6-elongase (MaD6E), Paramecium tetraurelia delta-5-desaturase (Ptet1D5D), Ostreococcus tauri C20 / 22 elongase (OtELO2), and Thraustochytrium sp delta-5-desaturase (TaFAD4), or any combination thereof, compared to a non-engineered parent cell.

[0342] In some embodiments, the cell is engineered to increase production of a phospholipid comprising an unsaturated fatty acid by adjusting expression of a gene, or functional homologues thereof, selected from S. cerevisiae elongase 2 (ScELO2), Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), S. kluyveri w3-Desaturase (skFAD), Ostreococcus tauri delta-6-desaturase (OtD6D), Mortierella alpina delta-6-elongase (MaD6E), Paramecium tetraurelia delta-5-desaturase (Ptet1D5D), Ostreococcus tauri C20 / 22 elongase (OtELO2), and Thraustochytrium sp delta-5-desaturase (TaFAD4), or any combination thereof.

[0343] In some embodiments of the present invention, the cell is modified or engineered to express a plasmid comprising a gene encoding an enzyme from the TMEM150 protein family. In some embodiments, the

[0344] 008933087cell is modified or engineered to express a plasmid comprising a gene encoding an enzyme from the TMEM150 protein family under the control of a constitutive promoter. In some embodiments, the cell is modified or engineered to express a plasmid comprising a gene encoding an enzyme from the TMEM150 protein family under the control of an inducible promoter. In some embodiments, the cell is modified or engineered to express a plasmid comprising a gene encoding an enzyme from the TMEM150 protein family under the control of a TDH3, TEF1 Pcyd , GPD, ADH1 , PGK1 , CUP1 , GAL1 , or GAL10 promoter. In some embodiments, the cell is modified or engineered to express one or more plasmids comprising a gene encoding an enzyme from the Kennedy pathway, for example, one of CK, CCT, CPT, EK, ECT, ETP or any combination thereof. In some embodiments, the cell is modified or engineered to express one or more plasmids comprising a gene encoding an enzyme from the Kennedy pathway, for example, one of CK, CCT, CPT, EK, ECT, ETP, or any combination thereof, under the control of a constitutive promoter. In some embodiments, the cell is modified or engineered to express one or more plasmids comprising a gene encoding an enzyme form the Kennedy pathway, for example, one of CK, CCT, CPT, EK, ECT, ETP, or any combination thereof, under the control of an inducible promoter. In some embodiments, the cell is modified or engineered to express one or more plasmids comprising a gene encoding an enzyme form the Kennedy pathway, for example, one of CK, CCT, CPT, EK, ECT, ETP, or any combination thereof, under a TDH3, TEF1 Pcyd , GPD, ADH1 , PGK1 , CUP1 , GAL1 , and / or GAL10 promoter.

[0345] In some embodiments, the cell is modified or engineered to express a plasmid comprising a gene encoding an enzyme from the TMEM150 protein family and a gene encoding an enzyme selected from the Kennedy pathways, LPCAT, ScELO2, MaD9D, MaD12D, skFAD, OtD6D, MaD6E, Ptet1D5D, OtELO2, and TaFAD4, or any combination thereof under the control of a constitutive promoter. In some embodiments, the cell is modified or engineered to express a plasmid comprising a gene encoding an enzyme from the TMEM150 protein family and a gene encoding an enzyme from the Kennedy pathways LPCAT, ScELO2, MaD9D, MaD12D, skFAD, OtD6D, MaD6E, Ptet1D5D, OtELO2, and TaFAD4 or any combination thereof under the control of an inducible promoter. In some embodiments, the cell is modified or engineered to express a plasmid comprising a gene encoding an enzyme from the Kennedy pathway. In some embodiments, the gene, or functional homologue, is selected from LPCAT, ScELO2, MaD9D, MaD12D, skFAD, OtD6D, MaD6E, Ptet1D5D, OtELO2, and TaFAD4, under the control of a promoter selected from a TDH3, TEF1 Pcyd , GPD, ADH1 , PGK1 , CUP1 , GAL1 , and GAL10 promoter. In some embodiments, the cell is transformed or engineered with a plasmid or an integrative plasmid comprising Saccharomyces cerevisiae codon optimized heterologous genes. In some embodiments, the plasmid or integrative plasmid comprises Saccharomyces cerevisiae codon optimized heterologous genes operably linked to a URA selectable marker, a LEU selectable marker, and / or a TRP selectable marker. In some embodiments, the plasmid or integrative plasmid comprises Saccharomyces cerevisiae codon optimized heterologous genes operably linked to a gene encoding a URA selectable marker, a LEU selectable marker, and / or a TRP selectable marker.

[0346] 008933087In some embodiments, the plasmid or integrative plasmid comprises pRSII-GAP-Sfk1-myc-6HIS-tADH1-URA, pRSII-GAP-yEhTmem150a-myc-6HIS-tADH1-URA, or pRSII-GAP-yEhTmem150b-myc-6HIS-tADH1-URA or any combination thereof. In some embodiments, the plasmid or integrative plasmid is selected from pRSII-GAP-Sfk1-myc-6HIS-tADH1-URA, pRSII-GAP-yEhTmem150a-myc-6HIS-tADH1-URA, or pRSII-GAP-yEhTmem150b-myc-6HIS-tADH1-URA or any combination thereof.

[0347] In some embodiments, the plasmid or integrative plasmid comprises YIPIac128-TDH3-skFAD3-tADH, YIPIac204-TDH3-MaD12D-tADH, or YIPIac128-TDH3-MaD9D-tADH, or any combination thereof. In some embodiments, the plasmid or integrative plasmid is selected from YIPIac128-TDH3-skFAD3-tADH, YIPIac204-TDH3-MaD12D-tADH, or YIPIac128-TDH3-MaD9D-tADH, or any combination thereof.

[0348] The cell may be further modified or engineered to redirect lipid flux toward phospholipid synthesis to increase fatty acid turnover and secretion. Lipid flux can be redirected towards phospholipid synthesis by silencing or down-regulating, or decreasing expression of fatty acid activation genes (the first step in p-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes. Methods to achieve this are known in the art and are discussed in Ferreira et al., Proc Natl Acad Sci U S A.

[0349] 2018;115(6):1262-1267, which is herein incorporated by reference in its entirety.

[0350] The cell may be modified or engineered through any known gene silencing methodology. For example, the cell may be modified or engineered through methodologies involving, siRNA, miRNA, shRNA, TALEN, CRISPR and / or RNAi.

[0351] Small RNA molecules may be employed to regulate gene expression. These include targeted degradation of mRNAs by small interfering RNAs (siRNAs), post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs) and targeted transcriptional gene silencing.

[0352] A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has also been demonstrated. Double-stranded RNA (dsRNA)-dependent post transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.

[0353] In the art, these RNA sequences are termed "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending on their origin. Both types of sequence may be used to down-regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNAs are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro-interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.

[0354] 008933087Methods of RNAi are known in the art, and are described in: Hannon, G.J. 2002. RNA interference. Nature. 418:244-51.

[0355] Highly specific methods reducing or removing endogenous gene expression are now possible through the use of gene editing techniques, such as site-specific nuclease (SSN) systems (e.g., CRISPR, TALEN, meganuclease, and Zinc Finger Nuclease systems).

[0356] In some embodiments the methods employ targeted nucleic acid editing using SSNs. Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequence(s) of interest. DSBs may be repaired by either error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively, DSBs may be repaired by highly homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB.

[0357] SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR-associated (CRISPR / Cas) systems.

[0358] ZFN systems are reviewed e.g. in Umov etal., Nat Rev Genet. (2010) 11 (9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a Fokl endonuclease domain). The DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence. TALEN systems are reviewed e.g. in Mahfouz etal., Plant Biotechnol J. (2014) 12(8):1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g. a Fokl endonuclease domain). TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: “HD” binds to C, “Nl” binds to A, “NG” binds to T and “NN” or “NK” binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501.).

[0359] CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) systems are prokaryotic adaptive immune system that bind and cleave foreign nucleic acids. The most frequently used type II CRISPR system is composed of two components: Cas9 nuclease and a guide RNA (gRNA), e.g., an artificial single guide RNA (sgRNA), a fusion of a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). When the SpCas9-sgRNA complex recognizes an NGG (N = A, T, C, or G) protospacer-adjacent motif (PAM) sequence, the spacer of the sgRNA pairs with the target DNA strand to form an “R-loop” structure. Subsequently, the Cas9 nuclease cleaves the DNA strands and produces a blunt-end DSB 3 bp upstream of the PAM into the protospacer. CRISPR-Cas gene editing tools are flexible, highly efficient, and inexpensive and have been widely applied. Recently, various Cas orthologs and variants with useful additional properties have been identified and harnessed for use in gene editing.

[0360] 008933087Moreover, novel tools for precise gene modification, such as base editors (BEs) and prime editors (PEs), have greatly expanded the applications of gene editing and have been leveraged for use in a variety of fields of research. Methods of CRISPR-Cas gene editing are reviewed in Liu et al. Mol Cell. 2022 Jan 20;82(2):333-347. doi: 10.1016 / j.molcel.2021.12.002, and Kantor et al. Int J Mol Sci. 2020 Aug 28;21 (17):6240. doi: 10.3390 / ijms21176240.

[0361] In some embodiments, the cell has been modified or engineered to reduce expression of or knock-out an endogenous gene. In some embodiments, the cell has been cured of an endogenous plasmid.

[0362] In some embodiments, the cell has been modified or engineered to remove / knock-out an endogenous gene using any known methodology including siRNA, miRNA, shRNA, SSN systems, ZFN, TALEN, CRISPR and / or RNAi. In some embodiments, the cell has been engineered to reduce expression of an endogenous gene or a combination of endogenous genes compared to a non-engineered parent cell gene using any known methodology including siRNA, miRNA, shRNA, SSN systems, ZFN, TALEN, CRISPR and / or RNAi. In some embodiments, the cell has been engineered to reduce expression of an of functional homologues of an endogenous gene, ora combination of functional homologues of an endogenous gene, compared to a non-engineered parent cell using any known methodology including siRNA, miRNA, shRNA, SSN systems, ZFN, TALEN, CRISPR and / or RNAi.

[0363] Lem3 is a protein that acts as a subunit of yeast phospholipid flippases which acts to flip plasma membrane phospholipids from the inner membrane leaflet to the outer membrane leaflet of the plasma membrane.

[0364] In some embodiments, the cell has been engineered or modified to reduce expression of an endogenous gene or a combination of endogenous genes compared to a non-engineered parent cell. In some embodiments, the cell has been engineered to reduce expression of an of functional homologues of an endogenous gene, ora combination of functional homologues of an endogenous gene, compared to a non-engineered parent cell. In some embodiments, the cell has been modified or engineered to reduce expression of or knock-out an endogenous gene or a combination of endogenous genes selected from skf1 and / or Iem3 or any combination thereof. In some embodiments, the cell has been engineered to reduce expression of a functional homologues or homologue of an endogenous gene, or a combination of functional homologues of an endogenous gene, compared to a non-engineered parent cell. In some embodiments, the cell has been engineered to reduce expression of an endogenous gene or a combination of endogenous genes any combination of the following, or functional homologues thereof, compared to a non-engineered parent cell; skf1 and / or Iem3. In some embodiments, the endogenous genes or functional homologues are selected from skf1 and / or Iem3 or any combination thereof. In some embodiments, the cell has been engineered to reduce expression of skf1 and / or Iem3 or any combination thereof.

[0365] In some embodiments, deletion of skf1 and / or Iem3 is achieved by transforming the cell with a deletion cassette comprising lem3D::KANMX6 and sfk1::Hph1MX6. In some embodiments, knock-down of skf1 and / or Iem3 is achieved by transforming the cell with a deletion cassette comprising lem3D::KANMX6

[0366] 008933087and / or sfk1 ::Hph1MX6. In some embodiments, a plasmid comprises the deletion cassette comprising lem3D::KANMX6 and / or sfk1 ::Hph1MX6.

[0367] To redirect lipid flux towards phospholipid production in yeast, pox1 (fatty-acyl coenzyme A oxidase 1), dga1 (diacylglycerol acyltransferase 1), Iro1 (lecithin cholesterol acyl transferase 1), pah1 (phosphatidic acid phosphohydrolase 1), Ipp1 (lipid phosphate phosphatase 1), dpp1 (diacylglycerol pyrophosphate phosphatase 1), plb1 (phospholipase B1) and / or plb2 (phospholipase B2), are1 (Acyl-coenzyme A: cholesterol acyl transferase-Related Enzyme ), are2 (Acyl-coenzyme A: cholesterol acyl transferase- Related Enzyme 2), faa1 (Fatty Acid Activation 1), faa1 (Fatty Acid Activation 2) can be deleted (see Ferreira et al., Proc Natl Acad Sci U S A. 2018;115(6):1262-1267).

[0368] In some embodiments of the present invention, the cell is modified or engineered to reduce expression or knock-out an endogenous gene selected from pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 plb2, are1 , are2, faa1 and / or, faa2, or any combination thereof. In some embodiments, the cell has been engineered to reduce expression of an endogenous gene or a combination of endogenous genes compared to a nonengineered parent cell. In some embodiments, the cell has been engineered to reduce expression of an endogenous gene or a combination of endogenous genes any combination of the following, or functional homologues thereof, compared to a non-engineered parent cell; pox1 , dga1 , Iro1 , pah1 , Ipp1 , dpp1 , plb1 , plb2, are1 , are2, faa1 and / or faa2. In some embodiments, the endogenous genes or functional homologues are selected from pox1 , dga1 , Iro 1 , pah1 , Ipp1 , dpp1 , plb1 plb2, are1 , are2, faa1 and / or faa2 or any combination thereof.

[0369]

[0370] LPL

[0371] The LPLs produced following the methods of the present invention, or through use of the cells of the present invention, can be utilised for a range of purposes. For example, in nutraceuticals, dietary supplements, emulsifiers, pharmaceutical compositions, and cosmetic compositions. Emulsifiers comprising the LPLs of the present invention may be used in, for example, food products, cosmetics and pharmaceuticals. Food products can be for human or animal consumption.

[0372] Dietary supplements derived from fish oil can have proportions of omega-3 fatty acids such as DHA and EPA as low as 10%. Despite the most biologically available forms of omega-3 and omega-6 being LPL- Omega-3 / 6, omega-3 supplements derived from fish oils and krill are in the form of triglycerides or phospholipids respectively, and do not comprise significant proportions of biologically available LPLs. The present application provides compositions that are enriched for the LPLs described in the present application. Certain compositions can be enriched in LPLs comprising essential omega-3 and omega-6 fatty acids, such as DHA and EPA.

[0373] Accordingly, in a further aspect, the present disclosure provides a composition comprising LPLs. In some embodiments, the LPLs are produced from any of the methods described herein and / or produced by any of the cells described herein. In some embodiments, the composition is enriched for LPLs. In this context,

[0374] 008933087a composition enriched for LPLs refers to composition comprising a greater proportion of LPLs than a composition from a naturally occurring source, for example from fish or krill.

[0375] In some embodiments the LPL is lysophosphatidylcholine (LPC) lysophosphatidylethanolamine (LPE) or lysophosphatidylserine (LPS). In some embodiments the LPL comprises a MUFA or a PUFA. In some embodiments the LPL comprises an omega-3 or an omega-6 fatty acid.

[0376] In some embodiments, the omaga-3 fatty acid comprises hexadecatrienoic acid, a-Linolenic acid (ALA), Stearidonic acid (SDA), Eicosatrienoic acid (ETE), Eicosatetraenoic acid (ETA), Eicosapentaenoic acid (EPA), Heneicosapentaenoic acid (HPA), Docosapentaenoic acid (DPA) (Clupanodonic acid), Docosahexaenoic acid (DHA), Tetracosapentaenoic acid, Tetracosahexaenoic acid (Nisinic acid). In some embodiments, the omega-6 fatty comprises linoleic acid (LA), gamma-linolenic acid (GLA), Calendic acid, Eicosadienoic acid, Dihomo-gamma-linolenic acid (DGLA), Arachidonic acid (AA, ARA), Docosadienoic acid, Adrenic acid, Osbond acid, Tetracosatetraenoic acid, and Tetracosapentaenoic acid.

[0377] In some embodiments, the LPL is selected from the LPL species shown in Table 1 , for example LPC 16:3.

[0378] Table 1 : LPL species comprising omega-3 and omega-6 fatty acids

[0379] LPC LPE LPS

[0380] Omega-3 Hexadecatrienoic acid LPC 16:3 LPE 16:3 LPS 16:3

[0381] (16:3)

[0382] a-Linolenic acid (ALA) LPC 18:3 LPE 18:3 LPS 18:3 (18:3)

[0383] Stearidonic acid (SDA) LPC 18:4 LPE 18:4 LPS 18:4 (18:4)

[0384] Eicosatrienoic acid LPC 20:3 LPE 20:3 LPS 20:3 (ETE) (20:3)

[0385] Eicosatetraenoic acid LPC 20:4 LPE 20:4 LPS 20:4 (ETA) (20:4)

[0386] Eicosapentaenoic acid LPC 20:5 LPE 20:5 LPS 20:5 (EPA) (20:5)

[0387] Heneicosapentaenoic LPC 21:5 LPE 21:5 LPS 21:5 acid (HPA) (22:5)

[0388] Docosapentaenoic LPC 22:5 LPE 22:5 LPS 22:5 acid (clupanodonic)

[0389] (DPA) (22:5)

[0390] Docosahexaenoic acid LPC 22:6 LPE 22:6 LPS 22:6 (DHA) (22:6)

[0391] Tetracosapentaenoic LPC 24:5 LPE 24:5 LPS 24:5 acid (24:5)

[0392] Tetracosahexaenoic LPC 24:6 LPE 24:6 LPS 24:6 acid (Nisinic acid)

[0393] (24:6)

[0394] Omega-6 Linoleic acid (LA) LPC 18:2 LPE 18:2 LPS 18:2

[0395] (18:2)

[0396] gamma-linolenic acid LPC 18:3 (GLA) LPE 18:3 (GLA) LPS 18:3 (GLA) (GLA) (18:3)

[0397] Calendic acid (18:3) LPC 18:3 LPE 18:3 LPS 18:3 Eicosadienoic acid LPC 20:2 LPE 20:2 LPS 20:2 (20:2)

[0398] Dihomo-gamma- LPC 20:3 LPE 20:3 LPS 20:3 linolenic acid (DGLA)

[0399]

[0400] (20:3)

[0401] 008933087Arachidonic acid (AA, LPC 20:4 LPE 20:4 LPS 20:4 ARA) (20:4)

[0402] Docosadienoic acid LPC 22:2 LPE 22:2 LPS 22:2 (22:2)

[0403] Adrenic acid (22:4) LPC 22:4 LPE 22:4 LPS 22:4 Osbond acid (22:5) LPC 22:5 LPE 22:5 LPS 22:5 Tetracosatetraenoic LPC 24:4 LPE 24:4 LPS 24:4 acid (24:4)

[0404] Tetracosapentaenoic LPC 25:5 LPE 25:5 LPS 25:5

[0405]

[0406] acid (24:5)

[0407] In some embodiments, the LPL is selected from the LPL species shown in Table 2, for example LPC 18:1.

[0408] Table 2: LPL species

[0409] Fatty LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P Acid

[0410] 14:0 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 14:0 14:0 14:0 14:0 14:0 14:0 14:0 14:0 14:0 15:0 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 15:0 15:0 15:0 15:0 15:0 15:0 15:0 15:0 15:0 16:0 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:1 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 16:1 16:1 16:1 16:1 16:1 16:1 16:1 16:1 16:1 17:0 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 17:0 17:0 17:0 17:0 17:0 17:0 17:0 17:0 17:0 18:0 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 18:0 18:0 18:0 18:0 18:0 18:0 18:0 18:0 18:0 18:1 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 18:1 18:1 18:1 18:1 18:1 18:1 18:1 18:1 18:1 18:2 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 18:2 18:2 18:2 18:2 18:2 18:2 18:2 18:2 18:2 18:3 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 18:3 18:3 18:3 18:3 18:3 18:3 18:3 18:3 18:3 20:1 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 20:1 20:1 20:1 20:1 20:1 20:1 20:1 20:1 20:1 20:3 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 20:3 20:3 20:3 20:3 20:3 20:3 20:3 20:3 20:3 20:4 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 20:4 20:4 20:4 20:4 20:4 20:4 20:4 20:4 20:4 20:5 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 20:5 20:5 20:5 20:5 20:5 20:5 20:5 20:5 20:5 22:5 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P 22:5 22:5 22:5 22:5 22:5 22:5 22:5 22:5 22:5 22:6 LPC LPE LPS LPC-0 LPE-0 LPS-0 LPC-P LPE-P LPS-P

[0411]

[0412] 22:6 22:6 22:6 22:6 22:6 22:6 22:6 22:6 22:6 In some embodiments, the LPL is selected from the LPL species shown in Table 3, for example LPC 18:1. In some embodiments, the LPL is selected from any combination of LPL species shown in Table 3.

[0413] 008933087Table 3: LPL species

[0414] LPC LPE LPS LPC-0 LPC-P LPE-P LPC 16:0 LPE 18:0 LPS18:0 LPC-0 16:0 LPC-P 18:0 LPE-P 16:0 LPC 16:1 LPE 18:1 LPS 18:1 LPC-0 18:1

[0415] LPC 18:0 LPE 20:4

[0416] LPC 18:1 LPE 18:2

[0417] LPC 17:0 LPE 16:0

[0418] LPC 18:2

[0419] LPC 20:1

[0420]

[0421] LPC 14:0

[0422] In some embodiments, the LPL is selected from the LPL species shown in Table 1 , Table 2 and / or Table 3, or any combination thereof.

[0423] In some embodiments, the LPL is selected from LPC 14:0, LPC 16:0, LPE 16:0, LPS 16:0, LPC-0 16:0, LPE-P 16:0, LPC 16:1 , LPC-0 18:0, LPE-P 18:0, LPC 18:0, LPE 18:0, LPS 18:0, LPC 18:1 , LPE 18:1 , LPS 18:1 , LPC-0 18:1 , LPC 18:2, LPC 20:4, LPE 20:4, LPS 20:4 and / or LPS 22:6. In some embodiments, the LPL is selected from LPC 16:0, LPE 18:0, LPS 18:1 , LPC-0 16:0, LPC-P 18:0, LPE-P 16:0, LPC 16:1 , LPE 18:1 , LPC-0 18:1 , LPC 18:0, LPE 20:4 and / or LPC 18:1. In some embodiments, the LPL is selected from LPC 14:0, LPC 16:0, LPC 16:1 , LPC 18:1 , LPE 18:1 and / or LPE-P 16:0.

[0424] In some embodiments, the LPL is selected from LPC 16:0, LPC 16:1 , LPC 17:0, LPC 18:0, LPC 18:1 , LPC 18:2, LPC 20:1. LPE 18:0, LPE 18:1 , LPE 18:2, LPE 20:4 and / or LPS 18:1.

[0425] In some embodiments, the LPL is selected from LPC 18:1 , LPC 18:0, LPC 16:1 , LPC 16:0, LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16:0.

[0426] In some embodiments, the LPL is selected from LPC-0 16:0, LPC 20:1 , LPC 18:1 , LPC 16:1 , LPE 18:1 , LPE 16:0, LPS 18:1 and / or LPS 16:0.

[0427] In some embodiments, the LPL is selected from LPC 22:6 (DHA), LPE 22:6 (DHA), LPS 22:6 (DHA), LPC 18:3 (ALA), LPE 18:3 (ALA), LPS 18:3 (ALA), LPC 20:5 (EPA), LPE 20:5 (EPA), LPS 20:5 (EPA), LPC 20:4 (ETA), LPE 20:4 (ETA) and / or LPS 20:4 (ETA).

[0428] In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% LPL. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% LPL.

[0429] In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% of a mixture of LPL. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of a mixture of LPL.

[0430] In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least

[0431] 00893308760%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of an LPL. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of an LPL. In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% of an LPL species. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of an LPL species.

[0432] In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% omega-3 or omega-6 fatty acids. In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% an omega-3 or and omega-6 fatty acid. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% omega-3 or omega-6 fatty acids. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% an omega-3 or an omega-6 fatty acid.

[0433] In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% of any one of the LPLs of Table 1 , Table 2 or Table 3. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of any one of the LPLs of Table 1 , Table 2 or Table 3.

[0434] In some embodiments the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% of a combination of any of the LPLs of Table 1 , Table 2 or Table 3. In some embodiments the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of a combination of any one of the LPLs of Table 1 , Table 2 or Table 3.

[0435] In some embodiments, the composition comprises at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% LPL. In some embodiments the LPL is selected from LPC-DHA, LPE-DHA and LPS-DHA. In some embodiments, the composition comprises at least 12%, at least 13%, at least 14%, at

[0436] 008933087least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% LPL and the LPL is selected from LPC-DHA, LPE-DHA and LPS-DHA.

[0437] In some embodiments, the composition comprises at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% LPL. In some embodiments the LPL is selected from LPC-EPA, LPE-EPA and LPS-EPA, or any combination thereof. In some embodiments, the composition comprises at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% LPL and the LPL is selected from LPC-EPA, LPE-EPA and LPS-EPA, or any combination thereof.

[0438] In some embodiments the LPL is selected from LPC-ALA, LPE-ALA and LPS-ALA, or any combination thereof. In some embodiments, the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% LPL and the LPL is selected from LPC-ALA, LPE-ALA and LPS-ALA, or any combination thereof.

[0439] In some embodiments the LPL is selected from LPC-ETA, LPE-ETA and LPS-ETA, or any combination thereof. In some embodiments, the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% LPL and the LPL is selected from LPC-ETA, LPE-ETA and LPS-ETA, or any combination thereof.

[0440] In some embodiments, the composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% of a mixture of LPL and the LPLs are selected from LPC 22:6 (DHA), LPE 22:6 (DHA), LPS 22:6 (DHA), LPC 18:3 (ALA), LPE 18:3 (ALA), LPS 18:3 (ALA), LPC 20:5 (EPA), LPE 20:5 (EPA), LPS 20:5 (EPA), LPC 20:4 (ETA), LPE 20:4 (ETA) and LPS 20:4 (ETA). In some embodiments, the composition comprises more than one LPL. In some embodiments, the mixture comprises one or more LPLs. In some embodiments, the mixture comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 LPL species.

[0441] In some embodiments, the composition comprises a transporter moiety. In some embodiments, the composition comprises a transport moiety which can bind to amphipathic lipids, phospholipids and / or LPLs. In some embodiments, the transport moiety is an albumin protein, alpha-fetoprotein protein, a vitamin D-binding protein, cyclodextrin, an apolipoprotein, apolipoprotein A-1, apolipoprotein B,

[0442] 008933087apolipoprotein D, apolipoprotein E, apolipoprotein F, or apolipoprotein M, or any combination thereof. In some embodiments, the transport moiety is albumin protein or cyclodextrin.

[0443] In a further aspect, the disclosure also provides an emulsifier comprising any of the compositions described herein. In another aspect, the present disclosure provides a use for any of the compositions described herein as an emulsifier. In some embodiments, the emulsifier is a food product emulsifier, a cosmetic emulsifier and / or a pharmaceutical emulsifier.

[0444] In a further aspect, the disclosure also provides an industrial product comprising any of the compositions described herein. In some embodiments, the industrial product is a pharmaceutical composition, a cosmetic composition, a nutraceutical, or a dietary supplement.

[0445] In addition to the defined LPL component, pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. The term can refer to molecular entities and compositions approved by a regulatory agency of the US federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans. Those with skill in the art are familiar with pharmaceutical carriers such as diluents, binders, lubricants and disintegrants and methods of compounding pharmaceutical compositions using such carriers.

[0446] The compositions provided in the present disclosure also find use as pharmaceutical excipients.

[0447] Pharmaceutical excipients are important components of pharmaceutical compositions and can be solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the pharmaceutical compositions will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredients of the pharmaceutical. Thus, the skilled person will appreciate that pharmaceutical excipients and compositions are provided wherein there is no incompatibility between any of the components of the dosage form. In a related aspect aspect, the present disclosure provides a use for any of the compositions provided herein as a pharmaceutical excipient.

[0448] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” whether or not explicitly indicated.

[0449] Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should

[0450] 008933087at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0451] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass all sub ranges subsumed therein. For example, a range of “1 to 10” includes any and all sub ranges between (and including) the minimum value of 1 and the maximum value of 10, that is, any and all sub ranges having a minimum value of equal to or greater than 1 and a maximum value of equal to or less than 10, e.g., 5.5 to 10.

[0452] Sequence identity

[0453] To identify gene or protein homologues pairwise and multiple sequence alignment can be used. Pairwise and multiple sequence alignment for the purpose of determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0454] Sequences

[0455] SEQ Description Sequence

[0456] ID NO:

[0457] 1 TMEM150A Homo MTAWILLPVSLSAFSITGIWTVYAMAVMNHHVCPVENWSYNESCPP sapiens DPAEQGGPKTCCTLDDVPLISKCGSYPPESCLFSLIGNMGAFMVALI UniProtKB Q86TG1 CLLRYGQLLEQSRHSWVNTTALITGCTNAAGLLVVGNFQVDHARSL HYVGAGVAFPAGLLFVCLHCALSYQGATAPLDLAVAYLRSVLAVIAFI TLVLSGVFFVHESSQLQHGAALCEWVCVIDILIFYGTFSYEFGAVSSD TLVAALQPTPGRACKSSGSSSTSTHLNCAPESIAMI

[0458] 2 TMEM150B Homo MWGYLSLMPVFLAVWAISGVWIVFAIAVTNRTVDLSKGFPYISICGSF sapiens PPQSCIFSQVLNMGAALAAWICIVRYHQLRDWGVRRWPNQLILWTG UniProtKB A6NC51 LLCALGTSVVGNFQEKNQRPTHLAGAFLAFILGNVYFWLQLLLWRLK RLPQPGAAWIGPLRLGLCSVCTILIVAMIVLHACSLRSVSAACEWVVA MLLFALFGLLAVDFSALESCTLCVQPWPSLSPPPASPISLPVQL

[0459]

[0460] 0089330873 TMEM150C Homo MDGKKCSVWMFLPLVFTLFTSAGLWIVYFIAVEDDKILPLNSAERKPG sapiens VKHAPYISIAGDDPPASCVFSQVMNMAAFLALWAVLRFIQLKPKVLN UniProtKB B9EJG8 PWLNISGLVALCLASFGMTLLGNFQLTNDEEIHNVGTSLTFGFGTLTC WIQAALTLKVNIKNEGRRVGIPRVILSASITLCVVLYFILMAQSIHMYAA RVQWGLVMCFLSYFGTFAVEFRHYRYEIVCSEYQENFLSFSESLSEA SEYQTDQV

[0461] 4 DRAM1 Homo MLCFLRGMAFVPFLLVTWSSAAFIISYWAVLSGHVNPFLPYISDTGT sapiens UniProtKB TPPESGIFGFMINFSAFLGAATMYTRYKIVQKQNQTCYFSTPVFNLVS Q8N682 LVLGLVGCFGMGIVANFQELAVPVVHDGGALLAFVCGVVYTLLQSIIS YKSCPQWNSLSTCHIRMVISAVSCAAVIPMIVCASLISITKLEWNPREK DYVYHWSAICEWTVAFGFIFYFLTFIQDFQSVTLRISTEINGDI

[0462] 5 DRAM 2 Homo MWWFQQGLSFLPSALVIWTSAAFIFSYITAVTLHHIDPALPYISDTGTV sapiens UniProtKB APEKCLFGAMLNIAAVLCIATIYVRYKQVHALSPEENVIIKLNKAGLVL Q6UX65 GILSCLGLSIVANFQKTTLFAAHVSGAVLTFGMGSLYMFVQTILSYQM QPKIHGKQVFWIRLLLVIWCGVSALSMLTCSSVLHSGNFGTDLEQKL HWNPEDKGYVLHMITTAAEWSMSFSFFGFFLTYIRDFQKISLRVEAN LHGLTLYDTAPCPINNERTRLLSRDI

[0463] 6 SFK1 MIQFKSPGNWLFIVPWIAFIPWYGMLIAMLICWASQGHPIYWFMHSE Saccharomyces QFPVYISDIGATNLRPLFISCAGWQGLGYVITVALEFFQRSGYLPFQL cerevisiae KKKDPSISDSTSYAEKLHSGKYLMPPYYTKDERNLIFAAFVLGSIGEL UniProtKB P35735 ALLFSSIFSTALYHRVHIAMVSVFVVFMFLSTCCLIAEYFLMGRHYASV HPLASPHFNPQSSEKSFNQDYNTVDELPWYKWKGHVWNKFTISATL KVIWLTLAVVWAICFGAINDRSKSACFEWLLAFWFGIIFMILSADFYLG GRYRQSRYFNHVESFSGYYKYDKALGLYHSEDVLPSDDNAGVIATE TASSNIYNNSSSNESIQVW

[0464] 7 6-His Tag HHHHHH

[0465]

[0466] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0467] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0468] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0469] 008933087Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0470] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0471] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0472] Examples

[0473] Example 1: Materials and Methods

[0474] 1.1 Lipase assay of membrane fractions to assess phospholipase activity

[0475] To evaluate the phospholipase activity of TM EM 150 family proteins, HeLa cells in two 6-wells were transfected with 2.5 pg of plasmid / well encoding either TMEM150 family proteins or mutants of the putative catalytic residue. pcDNA was transfected as a negative control. The next day, cells were washed with PBS, scraped, and cell pellets were resuspended in 400 pL lysis buffer (10 mM Na HEPES, 250 mM sucrose, 1 mM MgCI2, 1.5 mM MgOAc, 1 mM CaCI2) and lysed by passage through a 26-gauge needle 50 times. Lysates were centrifuged at 800xg and supernatants were ultracentrifuged at 100000xg for 45 min. Membrane pellets were resuspended in 100 pL of TBS pH 7.4 and unused membranes were frozen in aliquots. Digests were set up in triplicate, each using 10 pL membrane suspension and 40 pL of 5 pM NBD-PC 18:1 / 12:0 (Avanti 810133C, 10 mM stock in 100% ethanol) in reaction buffer (TBS plus 1 mM MgCI2 and 1 mM CaCI2) for 15 min or 60 min at 37°C with gentle agitation. Reactions were terminated by adding 200 pL chloroform:methanol (2:1) to form Folch ratio of chloroform:methanol:water (8:4:3), vortexed vigorously for 5 min followed by centrifugation at 4000xg for 10 min. The lower organic phase was recovered by pipetting and dried under argon. Samples were redissolved in chloroform:methanol (3:2) and 13.3% was spotted on a TLC plate. Mobile phase was chloroform:methanol:water (65:25:4).

[0476] 1.2 Lipase assay of Ni-NTA purified TMEM150A protein

[0477] Assays were performed on Ni-NTA-purified protein from HEK293S GnTI- cells carrying wildtype TMEM150A or its S67A mutant (both C-term Myc6His-tagged) knocked into the AAVS1 locus (mixed clone, Puromycin-selected). Cells were seeded into stir flask at 0.5 million cells / mL and allowed to grow in 240 mL of Freestyle medium + 2% FBS + Penstrep to ~3 million cells / mL, upon which cell pellets were 008933087harvested and frozen in 4 aliquots. Cell pellets from 1 aliquot (60 mL culture) were resuspended in 4 mL lysis buffer (20 mM Na HEPES, 150 mM NaCI, pH 7.5 containing EDTA-free protease inhibitor cocktail and Pierce universal nuclease) and lysed by an IKA T25 homogenizer (10k rpm, 1 min). Detergents were then added from 5x stocks in TBS to a working concentration of 40 mM (2%) DDM + 3.3 mM (0.2%) CHS. Proteins were extracted by rotating for 2 hours in the cold room. Thereafter, extracts were centrifuged at 8000xg for 30 minutes to remove insoluble debris, and supernatants were added to 0.2 mL of preequilibrated Ni-NTA resin and protein was bound by rotating for 2 hours in the cold room in the presence of 20 mM imidazole. Mixtures were poured into gravity columns in the cold room and washed with 20 CV (4 mL) of wash buffer, namely 20 mM Na HEPES, 200 mM NaCI, 60 mM imidazole, pH 7.5 with 2 mM (0.1%) DDM and 165 pM (0.01%) CHS forTMEM150A. Proteins were eluted by 5 min incubation in 20 mM Na HEPES, 200 mM NaCI, 300 mM imidazole, pH 7.5 with 1 mM (0.05%) DDM and 82.5 pM (0.005%) CHS for TMEM150A. Eluates were collected in two fractions, fraction E1 with 1.0 mL (5 CV) and fraction E2 with 0.5 mL (2.5 CV). Upon total protein quantification by absorbance at 280 nm, only E1 was used. Ni-NTA eluates were snap frozen with 10% glycerol and stored at -80°C.

[0478] For the time-course assay, NBD-PC digests were set up using frozen protein aliquots (thawed in a beaker of room temperature water) at 37°C with agitation. Reactions were initiated by adding 10 pL protein into 9 replicate tubes each containing 40 pL of 5 pM NBD-PC 18:1 / 12:0 (Avanti 810133C, 10 mM stock in 100% ethanol) in reaction buffer at pH 7.4 (TBS, 1 mM DDM, and 82.5 pM CHS). 10 pL of Ni-NTA elution buffer plus 10% glycerol was added for the no enzyme control. 200 pL chloroform:methanol (2:1) was added immediately to the 0 min samples. Thereafter, tubes were withdrawn at the indicated timepoints and reactions were terminated by adding 200 pL chloroform: methanol (2:1) to form Folch ratio of chloroform:methanol:water (8:4:3), vortexed vigorously for 1 min followed by centrifugation at 4000xg for 5 min. The lower organic phase was recovered by pipetting, dried under argon, and stored at -30°C in a staggered fashion as soon as the samples were ready. The next day, samples were redissolved in chloroform:methanol (3:2) and 8.3% was spotted on a TLC plate. Mobile phase was chloroform:methanol:water (65:25:4).

[0479] To assess the effect of divalent cations on TMEM150A activity, NBD-PC digests were set up in triplicate, each using 10 pL of protein (frozen protein was thawed in a beaker of room temperature water) and 40 pL of 5 pM NBD-PC 18:1 / 12:0 (Avanti 810133C, 10 mM stock in 100% ethanol) in reaction buffer at pH 7.4 (TBS, 1 mM DDM, and 82.5 pM CHS) plus the relevant cofactors (from 500 mM stocks in deionized water) or EDTA+EGTA (100 mM stocks adjusted to neutral pH with NaOH) for 15 minutes at 37°C with gentle agitation. Reactions were terminated by adding 200 pL chloroform:methanol (2:1) to form Folch ratio of chloroform:methanol:water (8:4:3), vortexed vigorously for 3 min followed by centrifugation at 4000xg for 10 min. The lower organic phase was recovered by pipetting and dried under argon. Samples were redissolved in chloroform:methanol (3:2) and 16.7% was spotted on a TLC plate. Mobile phase was chloroform:methanol:water (65:25:4).

[0480] To assess the effect of FA-free BSA and free fatty acids on TMEM150A activity, protein was preincubated for 30 minutes at 37°C with 100 pM of either Na palmitate or Na oleate or Na DHA (all from 10 mM stocks in 12% w / v FA-free BSA) or an equivalent volume of 12% FA-free BSA as negative control.

[0481] 008933087NBD-PC digests were then set up in triplicate, each using 10 pL of pre-incubated protein (~1.5 pg) and 40 pL of 5 pM NBD-PC 18:1 / 12:0 (Avanti 810133C, 10 mM stock in 100% ethanol) in reaction buffer at pH 7.4 (TBS, 1 mM DDM, and 82.5 pM CHS, with or without 0.5% FA-free BSA) for 60 minutes at 37°C with gentle agitation. Reactions were terminated by adding 200 pL chloroform:methanol (2:1) to form Folch ratio of chloroform:methanol:water (8:4:3), vortexed vigorously for 3 min followed by centrifugation at 4000xg for 5 min. The lower organic phase was recovered by pipetting and dried under argon. Samples were redissolved in chloroform:methanol (3:2) and 16.7% was spotted on a TLC plate. Mobile phase was chloroform:methanol:water (65:25:4).

[0482] For structure activity relationship assays, digests were set up in triplicate, each using 10 pL of WT or S67A mutant protein (frozen protein was thawed in a beaker of room temperature water) and 40 pL of 10 pM unlabeled substrates (from 10 mM stocks in 100% ethanol) in reaction buffer (TBS pH 7.4, 1 mM DDM, and 82.5 pM CHS) for 1 hour at 37°C with gentle agitation. Reactions were terminated by adding 200 pL chloroform:methanol (2:1) to form Folch ratio of chloroform:methanol:water (8:4:3), vortexed vigorously for 3 min followed by centrifugation at 4000xg for 10 min. The lower organic phase was recovered by pipetting and dried under argon. Dried organic phases were analyzed by QTOF mass spectrometry.

[0483] 1.3 Lipidomics analyses

[0484] HeLa cells:

[0485] HeLa cells in 12-wells were transfected in triplicate with 1 pg of plasmid / well encoding either TMEM150 family proteins or mutants of the putative catalytic residue. pcDNA was transfected as a negative control. For whole cell samples, transfected cells were washed once with PBS the next day and then scraped in 1 mL PBS. A small aliquot was withdrawn to measure protein concentration (BCA assay) for normalization, and the remaining cell suspension was spun down and frozen as cell pellets.

[0486] For conditioned medium samples, transfected cells were washed with PBS the next day and then incubated in serum-free DMEM containing 0.5% (w / v) fatty acid-free BSA overnight. The next day, the conditioned medium was collected, centrifuged at 3000xg for 5 minutes to remove cell debris, and supernatants were frozen. The remaining cells were washed once with PBS and then scraped in 1 mL PBS. A small aliquot was withdrawn to measure protein concentration (BCA assay) for normalization of both conditioned medium and cell pellet samples, and the remaining cell suspension was spun down and frozen as cell pellets. Frozen cell pellets and conditioned medium samples were submitted for lipidomic measurements.

[0487] Yeast:

[0488] 1 ml of yeast condition medium was mixed with 1.1 mL of chloroform spiked with internal standards (ISTD) and 2.2 mL of methanol. The standards included acylcarnitine 16:0 D3, cholesterol-D7, cholesterol ester 18:0 D6, dihydroceramide d18:0 / 08:0, ceramide d18:1 / 12:0, deoxyceramide m18:1 / 12:0, diacylglycerol 15:0 / 15:0, GM3 d18:1 / 18:0 D3, monohexosylceramide d18:1 / 12:0, dihexosylceramide d18:1 / 12:0, trihexosylceramide d18:1 / 18:0 D3, lysophosphatidylcholine 13:0,

[0489] 008933087lysophosphatidylethanolamine 14:0, phosphatidylcholine 13:0 / 13:0, phosphatidylethanolamine 17:0 / 17:0, phosphatidylglycerol 17:0 / 17:0, phosphatidylinositol 12:0 / 13:0, phosphatidylserine 17:0 / 17:0, sphingomyelin d18:1 / 12:0, sphingosine d17:1 and triacylglycerol 12:0 / 12:0 / 12:0.

[0490] The mixture was vortexed for 10 seconds and shaken for 5 mins. A further 1.1 mL of chloroform wase added and the mixture vortexed for a further 10 seconds and mixed for 5 minutes. Water (1.1 mL) was added to each sample, the mixture was vortexed for 10 seconds, shaken for 5 mins and then centrifuged at 3,000 xg for 10 mins at 4°C. An equal volume from the lower fraction was collected for all the samples and dried. Extracts were resuspended in 100 pL of butanol:methanol (1 :1 , v / v) and vortexed for 30 seconds before analysis by LC-MS / MS. A pooled lipid extract was used as a quality control (QC) sample and injected every five samples. Data were normalized to original volume of each medium sample.

[0491] LC-MS / MS analysis: LC-MS / MS analysis was performed on an Agilent UHPLC 1290 Infinity II liquid chromatography system connected to an Agilent QqQ 6495C. An Agilent Zorbax RRHD Eclipse Plus C18 column (2.1 x 50 mm, 1.8 pm) was used for the RPLC separation. The mobile phases A (60% water and 40% acetonitrile with 10 mmol / L ammonium formate) and B (10% acetonitrile and 90% isopropanol with 10 mmol / L ammonium formate) were used for the chromatographic analysis.

[0492] The following gradient was applied: 0-2 min, 20-60% B; 2-12 min, 60-100% B; 12-14 min, 100% B; 14.01-15.8 min, 20% B to equilibrate the column. The oven temperature was maintained at 40°C. Flow rate was set at 0.4 mL / min and the sample injection volume was 2 pL. The positive ionization spray voltage and nozzle voltage were set at 3,000 V and 1 ,000 V, respectively. The drying gas and sheath gas temperatures were both maintained at 250°C. The drying gas and sheath gas flow rates were 14 L / min and 11 L / min, respectively. The nebulizer nitrogen gas flow rate was set at 35 psi. The iFunnel high and low pressure RF were 150 V and 60 V, respectively.

[0493] Lipidomic Data Analysis: The acquired MS data were analyzed using Agilent MassHunter software version 10.1. For each analyte, signal to noise ratios (S / N) were calculated using the raw peak areas corresponding to the analytes in QC samples and processed blanks. Lipids associated with S / N <10, CV > 20% in the QC samples and did not show a linear behaviour (R2 < 0.8) in dilution curves were excluded from further analysis. Internal standards were used to normalize the raw peak areas for the corresponding lipid class (one internal standard per class) and relative concentrations were further normalized to the protein concentration in the original sample.

[0494] 1.4 TMEM150 protein expression, purification and sample preparation for in-vitro assays Homo sapiens TMEM150A was cloned into a modified BacMam expression vector (Goehring etal., 2014) with a C-terminal GFP-6his tag and a 3C protease cleavage site. The baculovirus was generated using sf9 insect cells (Expression Systems #94-001 S) following the published protocol (Invitrogen LifeTechnologies). Recombinant TMEM150A protein was expressed in HEK293S cells (ATCC #CRL-3022) grown in 293 Freestyle medium (Life Technologies) supplemented with 2% FBS. Cells were infected with baculovirus at a density of 2.5 * 106cells per ml. After 12 hours’ incubation at 37°C, 10 mM sodium butyrate was added to the culture, and the cells were moved to 30°C for an additional 48 hours before harvesting. All protein purification steps were carried out at 4°C. Cell pellets were homogenized in 0089330874x cell volume low-salt buffer (10 mM HEPES pH 7.5, 10 mM KCI, 10 mM MgCb, 0.5 mM PMSF, complete EDTA-free protease inhibitor cocktail, 10 pg ml-1DNase I and 8 pg ml-1RNase)) with cell homogenizer at 9000rpm for 3 mins on ice. Membrane fractions were isolated by ultracentrifugation at 100,000 g (35000rpm) in a type 70 Ti rotor (Beckman Coulter) for 30mins at 4 °C. Membrane pellet were then homogenized and washed once with 4x cell vol of high-salt buffer (10 mM HEPES pH 7.5, 10 mM KCI, 10 mM MgCh, 1 M NaCI, 0.5 mM PMSF, complete protease inhibitor cocktail, 10 pg ml-1DNase I and 8 pg ml-1RNase), and centrifuged as described above. The membrane pellet was resuspended and washed with storage buffer comprising 20 mM HEPES pH 7.5, 200 mM NaCI and 0.5 mM PMSF and complete protease inhibitor cocktail and ultracentrifuge. To solubilize the membrane fraction, the pellet was homogenized and resuspended in 4x the cell pellet weight of solubilization buffer (20 mM HEPES pH 7.5, 200 mM NaCI, 0.5 mM PMSF, complete protease inhibitor cocktail, 10 pg ml-1DNase I and 8 pg ml-1RNase) for 3 mins and followed by addition of LMNG to a final concentration of 1% (w / v) detergent and incubated at 4 °C for 1 h with gentle agitation. Insoluble material was removed by ultracentrifugation at 100000 g in a type 70 Ti rotor (Beckman Coulter) at 4 °C for 30 min. The supernatant was then placed in a Falcon tube containing pre-equilibrated Ni2+-NTA resin (Qiagen) (wash Nickel beads 3 times with 1ml storage buffer) in the presence of 20 mM imidazole and incubated at 4 °C for 1 hour with gentle rotation. The resin was washed with 10 column volumes of buffer comprising 20 mM HEPES pH 7.5, 200 mM NaCI, 60 mM imidazole 0.01% LMNG). Bound protein was first eluted with 0.5ml buffer followed by second elution with 0.25ml consisting of 20 mM HEPES pH 7.5, 200 mM NaCI, 300 mM imidazole 0.005% LMNG. Eluate was concentrated with Microcon-10kDa Centrifugal Filter to 0.5ml and insoluble protein aggregates were removed with centrifugation at 4°C for 20mins. Samples were then subjected to FPLC fractionation. The peak fraction was pooled and concentrated.

[0495] 1.5 In-vitro lipase activity assay

[0496] Lipase activities of Tmem150a-3C-mEGFP-Flag-HIS6 purified from baculovirus infected HEK29S GnTI-cells. A 50ul PLA reaction containing 5uM NBD-PC 18:1 / 12:0 (dissolved in absolute ethanol) and respective concentration of Tmem150a-GFP protein (in 10ul 0.005% LMNG) in PLA assay buffer (5mM CaCI2 , 1 mM EDTA, 100mM NaCI, 75mM Tris-HCI pH9.0) were prepared and incubated at 37°C for 1 hour. Reaction was terminated and lipids were extracted with 0.2ml folch solution (chloroform: methanol, 2:1). Organic phase (0.15ml) were dried down with agon gas. Lipids were dissolved in 70pl chlorofornrmethanol (3:2). 10% of the lipids were spotted into TLC plate and run with solvent system chloroform: methanol: water (65:25:4). Typhoon scanner was used to detect the fluorescence signal.

[0497] 1.6 Generating a mouse TMEM150A KO

[0498] TMEM150A knock-out mouse line was generated using the CRISPR / Cas9 method. Briefly, two gRNAs were designed in the exon 5 region of the Tmem150a gene. These two gRNAs were used with the intention of creating a fragment deletion in the flanking region. Cas9 mRNA and gRNAs were co-injected into one-cell mouse embryos. A total of 332 embryos were injected, out of which 227 were implanted into 11 pseudo-pregnant females. From the implantation, 9 pups were born, 5 of which contained a fragment deletion, and one contained a small indel mutation, as determined by PCR and T7 endonuclease I assay.

[0499] 008933087After sequencing analysis, two founders were selected for germline transmission breeding with wild-type mice. One founder contained an 89-bp deletion (Del89) mutation, and the other contained two indel mutations (8-bp and 6-bp, respectively, at the two gRNA sites) that collectively caused a 14-bp reading frame shift deletion (Dell 4). The genotype of F1 heterozygotes was confirmed by sequencing.

[0500] Genomic DNA extracted from tail clippings was amplified using Tmem150GT1 / GT2. For Del89 mutant, the PCR product was directly resolved on 2% agarose gel. For Dell 4 mutant, half volume of the PCR product were used for T7 Endonuclease I assay, and then resolved using 2% agarose gel with reference of the other half of undigested PCR products.

[0501] 1.7 Spodoptera frugiperda (Sf9J cells engineered to express TMEM150B

[0502] Sf9 cells were resuspend in Sf-900™ II SFM (Gibco) to final concentration of 1x106cells / ml. Cells were then infected with baculovirus harbouring Tmem150b-3C-mEGFP-Flag-HIS6 orTmem150b (S43A)-3C-mEGFP-Flag-HIS6 at 1 :100 medium ratio and grown at 28°C, at 100rpm, for 24 hours. Cells were then removed by centrifugation and conditioned culture medium was harvested and filtered through a 0.22pm syringe filter. Lipids were extracted from 1ml conditioned medium and subjected to lipidomic analysis. Graphs showing concentration of LPC, LPE, and LPS, respectively.

[0503] 1.8 S. cerevisiae strains and plasmids

[0504] A combination of standard molecular biology and molecular genetics techniques such of PCR-based deletion of endogenous genes were used to construct deletion mutants. The plasmids for deletion cassette were obtained from European Saccharomyces cerevisiae Archive for functional analysis (Euroscarf). For cells harbouring respective episomal plasmids, gene of interest (yeast codon usage optimized) was cloned into yeast 2-micron vector, pRSII with TDH3 promoter and ADH terminator, and URA as selectable marker.

[0505] Wild-type W303 yeast was transformed with deletion cassette, lem3D::KANMX6 and sfk1 ::Hph1 MX6 to generate lem3D sfk1D strains in which Iem3 and sfk1 expression is knocked down. 2-micron plasmid that carries either Tmeml 50a, Tmem150b, or Sfk1 was transformed into cells harboring lem3D sfk1D cells to create individual strains. Yeast transformation was performed as described previously (Taxis and Knop 2006). The strains generated are shown in Table 4.

[0506] Transformed cells were grown in yeast extract peptone (YP) or synthetic defined URA-media (SD-URA) supplemented with 2% dextrose at 24°C prior to experimentation (for growth after transformation and prior to lipid analysis).

[0507] 1.9 S. cerevisiae culture for LPC production

[0508] To evaluate the effect of TMEM150 family proteins and the corresponding catalytic triad mutants on LP production, S. cerevisiae cells (shown in Table 4) were grown overnight in SD-URA- media supplemented with 2% dextrose overnight at 24 °C, at 130rpm. Overnight culture was inoculated to final, 2x106cells / ml, into SD-URA-media supplemented with 2% dextrose and 0.5% fatty acid free BSA. Culture was grown at 24 °C, at 130rpm, for 24 hours. Cells were removed by centrifugation and conditioned culture medium was harvested and filtered through a 0.22pm syringe filter.

[0509] 008933087To further analyse the effect of endogenous yeast lipase skf1 and accessory component Iem3 on LP production, S. cerevisiae cells with background skf1 and / or Iem3 knocked-down were engineered to overexpress TMEM150B and grown in conditions with or without BSA. Cells were grown overnight in SD-URA' media supplemented with 2% dextrose overnight at 24°C, at 130rpm. Overnight culture was inoculated to final, 1x107cells / ml, into SD-URA-+2% glucose supplemented with or without 0.5% fatty acid free BSA. Cultures were grown at 24°C, at 130rpm, for 24 hours. Cells were then removed by centrifugation and conditioned culture medium was harvested and filtered through a 0.22pm syringe filter. Lipids were extracted from 1ml conditioned medium and subjected to lipidomic analysis. Graphs show concentrations of LPC, LPE, and LPS.).

[0510]

[0511] To identify the enzyme responsible for generating LPC and other LPLs present in blood plasma, the inventors focussed their search for an enzyme which: (i) is not secreted but instead resides in the plasma membrane; (ii) could hydrolyse phosphatidylcholine (PC) located in the outer leaflet of the plasma membrane; (iii) is a phospholipase A1 (PLAI)-type enzyme, and (iv) is expressed in the liver.

[0512] To date, there are no known PLA1 enzymes or other families of lipases (e.g. PNPLA, ABHD) that fulfils these criteria and so the inventors focused on a group of proteins designated as TMEM for transmembrane protein. Proteins with the TMEM prefix do not form a single protein family but are structurally diverse proteins that are often uncharacterized or poorly characterized. Within the broad TMEM class of proteins are smaller subfamilies which can show structural and functional similarities. Among TMEM proteins, a number of lipid flippases / scramblases (TMEM30A, TMEM16, TMEM41B) and lipid enzymes (TMEM189) have been identified. Analysis of the TMEM family revealed TMEM150A as a candidate transmembrane enzyme because it localises to the plasma membrane and is expressed in the liver. Whether TMEM150A was a lipase was unknown. TMEM150A is in the TMEM150 protein family. Candidate enzyme TMEM150A was modelled using AphaFold2 software to provide insight into TMEM150A structure and function. The resulting model is shown in Figure 1 and revealed a conserved central cavity in TMEM150A lined by a potential catalytic triad composed of residues Ser67, His140, and Glu211. Notably, Ser67 is located at the beginning of an alpha helix, allowing activation of the serine nucleophile by helical dipole. In this catalytic triad, the serine nucleophile may be activated by deprotonation by His with the extra electron pair stabilized by the adjacent acid residue (Asp / Glu). Thus, Ser67 is predicted to be the nucleophilic residue that mediates attack on the fatty acid ester bond of a PC molecule. Structural analysis of the other TMEM150 family members indicates TMEM150B and yeast Sfk1 have this conserved putative catalytic triad, while the acidic residue is substituted by glutamine in TMEM150C. Remarkably, the central cavity exhibits a lateral opening to the outer leaflet of the membrane, suggesting that TMEM150A would act exclusively on outer leaflet phospholipids of the plasma membrane. Computational docking of a palmitoyl-oleoyl-phosphatidylcholine (POPC) revealed that the saturated acyl tail docked within the central cavity with the monounsaturated acyl tail hanging outside the protein. The putative enzymatic triad was located just below the glycerol backbone, placing 008933087the putative Ser67 nucleophile in proximity to the sn1 ester bond of POPC. Computational analysis indicates there are molecular determinants of substrate selectivity for PC and phosphatidylethanolamine (PE) over the other lipids phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidylinositol (PI).

[0513] Accordingly, TMEM150A satisfied criteria (i), (ii) and (iv). Moreover, TMEM150A, TMEM150B and TMEM150C all have catalytic triads configured to accept a phospholipid. To establish whether TMEM150A and other members of the TMEM150 protein family had phospholipase activity enzyme required further experimental verification.

[0514] Example 3: TMEM150A is a PC lipase aeneratina LPC

[0515] The cellular locations of TMEM150A, TMEM150B, TMEM150C and Sfk1 were imaged and can be seen in Figure 2. This confirmed that TMEM150A, TMEM150C and Sfk1 are expressed at the PM, while TMEM150B is intracellular and DRAM1 and DRAM2 are localized to lysosomes.

[0516] To test whether TMEM150A has phospholipases A1 (PLA1) activity to generate LPLs including LPC, an enzyme assay using isolated membrane preparations from HeLa cells that overexpress TMEM150A was used. Individual mutants in the catalytic dyad Ser67Ala and His140Ala were generated to serve as negative controls in this assay. The assay set-up involved adding NBD-PC — a PC molecule with an NBD fluorophore linked to the sn2 fatty acid — into the membrane preps and incubating samples for 2 hours. Reactions were stopped by organic extraction and products were resolved on TLC plates and scanned using a fluorescent scanner.

[0517] Addition of WT TMEM150A membrane suspensions, but not Ser67Ala, His140Ala, or pcDNA, resulted in the production of NBD-LPC but not NBD-fatty acid (Figure 3A). These data show that TMEM150A preferentially hydrolyses the fatty acid at the sn1 position from NBD-PC to generate NBD-LPC molecules with MUFAs / PUFAs at the sn2 position, demonstrating PLA1 activity. The same was observed for TMEM150B and yeast Sfk1 (Figure 3A).

[0518] A time course of TMEM150A activity with Ni-NTA purified protein in detergent, with continuous evolution of NBD-LPC up to 2 hours incubation at 37°C was also characterised (Figure 3B). This shows TMEM150A can continually produce LPLs including LPC. Cofactor dependence of Ni-NTA-purified TMEM150A was also evaluated. The results show that TMEM150A is not dependent on divalent cations (Mg2+ or Ca2+) but is inhibited by Zn2+ (Figure 3C). Similarly, the effects of fatty acid (FA)-free BSA and free fatty acid on activity of Ni-NTA-purified TMEM150A protein were also assessed. Addition of fattyacid free bovine serum albumin (BSA) stimulates TMEM150A activity, while pre-incubation with unsaturated free fatty acids reduces TMEM150A activity (Figure 3D).

[0519] To rule out the possibility that the NBD fluorophore linked to the sn2 position sterically affects its entry into the enzyme active site, highly quantitative mass spectrometry-based lipidomics assay was used. The experimental set-up is similar as described above, but with the use of unlabelled PCs having different headgroups or acyl chains without fluorophores. This experiment clearly demonstrated that TMEM150A 008933087has high specificity for the acyl linkages of PC and PE lipids (Figure 4A), preferentially hydrolyzing the sn1 position, particularly if the sn2 position contains an unsaturated fatty acid (Figure 4B; see boxes with darker shading).

[0520] The preference of TMEM150A for generating LPCs with sn2 PUFAs is in line with similar LPC species commonly found in human plasma. To test whether the LPCs generated by TMEM150A alter the cellular phospholipid pool, changes in cellular phospholipids using lipidomics in HeLa cells expressing TMEM150A, TMEM150B, TMEM150C, DRAM1, DRAM2, and Sfk1 were quantified. Volcano plots of changes relative to controls indicated that TMEM150A increased cellular LPCs and LPEs (Figure 5A to Figure 5G). TMEM150B and Sfk1 increased LPCs, LPEs, LPSs, lyso-ether PC, and lyso-plasmalogens (Figure 5B and Figure 5G). DRAM2 overexpression increased LPCs and LPE species, while DRAM1 overexpression resulted in changes to saturated lyso-ether PC and lyso-plasmalogens (Figure 5D and Figure 5E). These data show that TMEM150 family proteins have similar phospholipase activity.

[0521] Specifically, TMEM150 family proteins can generate a range of LPLs, with specificity for generating LPCs and LPEs.

[0522] Whether TMEM150A, TMEM150B, TMEM150C, and Sfk1 overexpression can lead to albumin-inducible efflux of LPCs was then tested. Using the same cell-based experimental set-up described above, cells overexpressing these TMEM150 constructs, and negative control mutants were incubated in media overnight containing albumin without serum. This conditioned media was used for lipidomic analysis. As shown in the volcano plots, overexpression of TMEM150A, TMEM150C and Sfk1 results in large efflux of LPCs into the conditioned medium (Figure 6A to Figure 6D), while TMEM150B overexpression did not increase levels of lysophospholipids comprising MUFA and PUFA in conditioned medium in this assay to the same degree as TMEM150A, TMEM150C and Sfk1, consistent with its intracellular location.

[0523] Accordingly, TMEM150A is a lipase that generates LPCs with preference for generating LPC-PUFA and LPC-MUFA. Moreover, TMEM150A generates LPC species that resemble the composition of the outer leaflet of cellular membranes. TMEM150A expression in cells also results in efflux of LPCs to extracellular albumin. These data show that TMEM150A is a new class of lipase in terms of both enzymatic function and structure. The TMEM150A - LPL - albumin relationship is summarised in Figure 7.

[0524] The ability of TMEM150 family proteins to generate LPL activity is completely unexpected because prior to the present disclosure they were not known to have phospholipase activity and certainly not phospholipase A1 activity specifically.

[0525]

[0526] 4: TMEM150 demonstrate Hi in-vitro

[0527] To demonstrate the intrinsic activity of TMEM150 family proteins as PLA1 lipases, TMEM150a protein from baculovirus infected HEK29S GnTI- cells was purified (Figure 8A and Figure 8B). The FPLC purified protein was then used in an in-vitro lipase assay. Similar to the TMEM150a membrane extracts from cell overexpressing Tmem150a, the purified TMEM150a cleaves NBD-PC 18:1 / 12:0 efficiently to produce NBD-LPC (Figure 8C). This data shows that TMEM150a is a PLA1 lipase that digests PC to

[0528] 008933087form LPC, and demonstrates that purified TMEM150 family proteins can be used in-vitro to generate LPLs.

[0529] Example 5: TMEM150a is essential for maintaining plasma LPC-PUFAs

[0530] To further characterise the activity of TM EM 150A, a Tmem150a mouse KO line using CRISPR / Cas9 was generated. Lipidomic analysis of the mutant found that LPC-PUFA were significantly reduced in adult KO mice relative to controls (Figure 9). This demonstrates that TMEM150a is the major LPC-PUFA generating enzyme in mammals.

[0531] Example 6: Exogenous expression of TMEM150 family proteins increases LPL production in insect cells

[0532] To evaluate whether TMEM150 family proteins can be engineered into non-human cellular system to create an efficient production method for LPLs, TMEM150B or the corresponding catalytic triad mutant were introduced into Spodoptera frugiperda (Sf9) cells. Lipidomic analysis of the conditioned growth media of the engineered cells shows that introduction of functional TMEM150B results in an increase in a variety LPL species (Figure 10). Of note, TMEM150B increased production of LPLs comprising unsaturated fatty acids with chain length of 20, for example LPE 20:4 which comprises omega-3 fatty acid ETA. These data demonstrate that exogenous expression of TMEM150 family proteins can increase production of LPLs in a non-human cellular system, specifically in insect cells. The data also show TMEM150 family proteins can produce LPLs comprising omega-3 fatty acids.

[0533] Example 7: Precision fermentation to produce LPC-PUFAs.

[0534] The inventors next looked to exploit the unique enzymatic activity of TMEM150 family proteins for precision fermentation with the aim of engineering an efficient production system for LPLs.

[0535] To create such a system, transgenic S. cerevisiae strains were engineered to knock down endogenous expression of Iem3 and endogenous sfk1 and overexpress TMEM150 family proteins following the workflow shown in Figure 11.

[0536] 008933087sfk1A lem3A MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 TMEM150A bar1A sfk1::hphMX6 lem3::KANMX4 pRSII-TDH3-yEhTmem150a- myc-6HIS-tADH1-URA

[0537] sfk1A lem3A MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 TMEM150A-S67A bar1A sfk1::hphMX6 lem3::KANMX4 pRSII-TDH3-yEhTmem150a- S67A-myc-6HIS-tADH1-URA

[0538] sfk1A lem3A MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 TMEM150B bar1A sfk1::hphMX6 lem3::KANMX4 pRSII-TDH3-yEhTmem150b- myc-6HIS-tADH1-URA

[0539] sfk1A lem3A MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 TMEM150B-S43A bar1A sfk1::hphMX6 lem3::KANMX4 pRSII-TDH3-yEhTmem150b- S43A-myc-6HIS-tADH1-URA

[0540] sfk1A lem3A SFK1 MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15

[0541] bar1A sfk1::hphMX6 lem3::KANMX4 pRSII-TDH3-ySFK1-myc- 6HIS-tADH1-URA

[0542] sfk1A lem3A SFK1- MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 S54A bar1A sfk1::hphMX6 lem3::KANMX4 pRSII-TDH3-ySFK1-S54A- myc-6HIS-tADH1-URA

[0543] MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 sfk1A lem3A vector

[0544] bar1A sfk1::hphMX6 lem3::KANMX4 pRSII-GAP-MCS-tADH-URA control

[0545] (MCS refers to multiple cloning sites)

[0546] lem3A TMEM150A MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15

[0547] bar1A lem3::KANMX4 pRSII-GAP-yEhTmem150b-myc-6HIS- tADH1-URA

[0548] MaD9D MaD12D MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15

[0549] bar1A TDH3-MaD9D-tADH::LEU2 TDH3-MaD12D-tADH::TRP1 lem3::KANMX4 sfk1::hphMX6 pRSII-GAP-MCS-tADH1-URA MaD9d MaD12D MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 TM EM 150b bar1A TDH3-MaD9D-tADH::LEU2 TDH3-MaD12D-tADH::TRP1 lem3::KANMX4 sfk1::hphMX6 pRSII-GAP-yEhTmem150b-myc- 6HIS-tADH1-URA

[0550] skFAD3 MaD12D MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15

[0551] bar1A TDH3-skFAD3-tADH::LEU2 TDH3-MaD12D-tADH::TRP1 lem3::KANMX4 sfk1::hphMX6 pRSII-GAP-MCS-tADH1-URA

[0552]

[0553] 008933087skFAD3 MaD12D MAT a leu2-3, 112 trp1-1 ura3-1 can1-100 ade2-1 his3-11, 15 TM EM 150b bar1A TDH3-skFAD3-tADH::LEU2 TDH3-MaD12D-tADH::TRP1 lem3::KANMX4 sfk1::hphMX6 pRSII-GAP-yEhTmem150b-myc- 6HIS-tADH1-URA

[0554]

[0555] The resulting yeast strains overexpress TMEM150A, TMEM150B and SFK1 , or the equivalent protein with a mutated catalytic region (Table 4). Each strain was cultured in liquid media for 24 h before lipids from the media were extracted. Lipidomic analysis revealed that each TMEM150 family protein produced a greater concentration of lysophospholipids than the respective TMEM150 mutant (Figure 12), demonstrating the importance of the catalytic triad to lipase TMEM150 activity. Moreover, the strain expressing TMEM150B secreted the most LPC / LPE primarily in the form of LPC-18:1 and LPE-18:1 (Figure 12). The effectiveness of TMEM150B to generate unsaturated LPLs in the yeast system was surprising because TMEM150B did not perform as well as TMEM150A at producing unsaturated LPLs in the HeLa cell assays (Figure 6B). These results mean the strain expressing TMEM150B can secrete approximately 3g / Litre of culture of each LPC-18:1 and LPE-18:1. When scaled using industrial fermenters this will result in high production levels in a sustainable system. LPC-18:1 and LPE-18:1 are already commodity lipids used in food, cosmetics and other industries.

[0556] To further analyse the effect of endogenous yeast lipase skf1 and accessory component Iem3 on LPLs production, S. cerevisiae cells with background skf1 and / or Iem3 knocked down were engineered to overexpress TMEM150B. To analyse lysophospholipid production, yeast cells were supplemented either with or without 0.5% fatty acid free BSA. Lipids from the media were extracted and analysed. The results in Figure 13 demonstrate that TMEM150B mediated production of LPC, LPE and LPS is enhanced when native Lem3 and SFK1 are deleted from the strain expressing TMEM150B (i.e. sfkldel Iem3del Tmem150b+0.5% BSA). That is, deleting native Lem3 and SFK1 had an additive effect on TMEM150B mediated production of LPC-18:1 and LPE-8:1. The inventors concluded that deletion of Iem3 from the host strain prevented phospholipid flipping back to the inner leaflet and therefore provided more substrate to TMEM150B for hydrolysis and efflux from the cell into media. Accordingly, deletion of Iem3 increases flux of LPC out of the cell and prevents flipping of the LPC back into the cell, thus creating a more efficient LPL production system.

[0557] Taken together, the results demonstrate that TMEM150B increases expression of LPLs including LPC, LPE and LPS. Moreover, LPL production is enhanced when endogenous Iem3 and sfk1 are deleted, and when the media is supplemented with BSA.This shows that TMEM150 protein family members can be engineered into biological systems to create an efficient production method for LPLs.

[0558] Example 8: S. cerevisiae strains with enhanced lysophospholipid production

[0559] The knock down of endogenous genes skf1 and Iem3 demonstrate that manipulation of host networks can result in enhanced productions of lysophospholipid (Figure 14). The system can be enhanced further by manipulating additional components of endogenous lipid synthesis pathways. For example, the lipid 008933087metabolic network of S. cerevisiae can be genetically altered to redirect the lipid flux toward phospholipid production. This will result in S. cerevisiae strains capable of producing lysophospholipid pools with varying profiles. It has been demonstrated that deletion of genes involved in beta oxidation, triglyceride (TAG) formation, phosphatidate phosphatase genes, and phospholipase genes, resulted in significant increases of phospholipids in yeast cell as demonstrated in Ferreira et al., Proc Natl Acad Sci USA 115, 1262-1267 (2018), which is hereby incorporated by reference in its entirety. Accordingly, to develop more efficient LPL production systems, CRISPR / Cas9 gene editing can be used to knock out the genes shown in Figure 14 in the strain S. cerevisiae CEN.PK2-1C (Mata ura3-52 trp1-289 Ieu2-3,112 his3D1 MAL2-8c SUC2), the best characterized and most widely used yeast strain for synthetic biology from Euroscarf. The Multiplex, Markerless Genome Editing Using CRISPR / Cas9 approach can be used (Lee et al., 2015). Moreover, to promote production of omega LPLs with fatty acid lengths over C18:1 (Oleic acid), the yeast strains can be further engineered by introducing into the genome heterologous genes that encode fatty acid elongases and desaturases from different omega-3 fatty acid producing organisms. Such genes include elongase 2 (ELO2), delta-9-desaturase (D9D), delta-12-desaturase D12D, w3-Desaturase (FAD), delta-6-desaturase (D6D), delta-6-elongase (D6E), delta-5-desaturase (1D5D), C20 / 22 elongase (ELO2), and delta-5-desaturase (FAD4), which are shown in Figure 15. Each enzyme has been successfully engineered into host cells and are known to promote omega-3 and / or omega-6 fatty acid production (see Yu et al., 2017, Tavares et al., 2011 , Meyer et al., 2024 and Qiu et al., 2001 , which are hereby incorporated by reference in their entirety). Integration of the above-mentioned genes into a host cell can be achieved by employing the established EasyClone2.0 system in which the heterologous genes are integrated at well-defined chromosomal locations that allowed gene overexpression without compromising yeast growth. To obtain high expression of the heterologous genes, all genes integrated into the genome can be synthesized according to the optimal codon usage of S. cerevisiae and expressed under a strong constitutive yeast promoter, TDH3 (from glyceraldehyde 3-phosphage dehydrogenase) for example. This will result in yeast strains that are better able to produce key phospholipids having a variety of useful fatty acids (i.e. PC-18:1, PC-18:2, PC-22:5, and PC-22:6) for LPL production, as quantified with lipidomics.

[0560] In order to convert these strains into “LPC secreting” strains, codon optimized human TMEM150 family proteins such as TMEM150A (HsTmem150A), TMEM150B, TMEM150C, DRAM1 or DRAM 2 or yeast SFK1 (ScSfkl) can be integrated at Ieu2 locus under promoters with varied strengths, PTDH3 (strong), PTEF1 (moderate), and Pcyd (weak), respectively. In addition, to circumvent possible negative effects on cell fitness, yeast strain that express HsTMEM150A orScSFKI under the control of an inducible promoter such as copper-inducible CUP1 promoter, which is inexpensive for use in an industrial setting can be constructed.

[0561] As a proof of concept, the inventors tested the strains “MaD9D MaD12D”, “MaD9d MaD12D TMEM150b”, “skFAD3 MaD12D” and “skFAD3 MaD12D TNEN150b” (as shown in Table 4) fortheir ability to produce the omega LPLs Linoleic acid (C18:2) and Linolenic acid (C18:3). As shown in Figure 15, expression of MaD9D and MaD12D is predicted to increase flux toward linoleic acid, and skFAD is predicted to increase flux towards linolenic acid.

[0562] 008933087Each strain was grown according to the workflow in Figure 11. Figure 16 demonstrates that yeast expressing TMEM150B together with the indicated fatty acid desaturases allows for secretion of LPC 18:2 and LPC 18:3 into media. In particular, expression of MaD9d and MaD12D alongside TMEM150b shifted the main recoverable 18-carbon LPC from 18:1 (oleic acid) to 18:2 (linoleic acid). Likewise, expression of MaD12D and skFAD3 alongside TMEM150b shifted the main recoverable 18-carbon LPC from 18:1 to 18:3 (linolenic acid). Both “MaD9d MaD12D TMEM150b” and “skFAD3 MaD12D TNEN150b” shifted the main 18-carbon LPE’s and LPS’s from 18:1 to 18:2.

[0563] Example 9. Alpha- and beta-cyclodextrin capture of secreted LPC / LPE

[0564] The S. cerevisiae strain sfk1A lem3A TMEM150B was cultured in liquid media containing respective cyclodextrin (5mM) for 24 h. Cells were removed by centrifugation at 6000g, and media were filtered with 0.22pm filter. Media containing lipid bound cyclodextrins were subjected to lipidomic analysis.

[0565] Figure 17 shows that cyclodextrins can effectively capture secreted LPC, LPE and LPS from the growth media, a-cyclodextrin captured ~6 pM LPC 18:1 and ~3.5 pM LPC 16:1, and p-cyclodextrin captured ~5.5 pM LPC 18:1 and ~3 pM LPC 16:1. Both a- and p-cyclodextrin outperformed BSA which captured -2 pM LPC 18:1 and ~0.3 pM LPC 16:1 (Figure 17A).

[0566] Similarly, a- and p-cyclodextrin outperformed BSA in capture of LPE, with a-cyclodextrin and p-cyclodextrin capturing -4 pM and -3.5 pM LPE 18:1, respectively. In contrast, the capture of LPE 18:1 by BSA was negligible (Figure 17B).

[0567] Finally, a- and p-cyclodextrin outperformed BSA in capture of LPS. a-cyclodextrin captured -0.4 pM LPS 18:1 and -0.1 pM LPS 16:0, and p-cyclodextrin captured -0.3 pM LPS 18:1 and -0.05 pM LPS 16:0. In contrast, the capture of LPS 18:1 by BSA was -0.2 pM, whereas capture of LPS 16:0 was negligible (Figure 17B).

[0568] These results are significant because it they demonstrate that cyclodextrins can be used to capture LPC / LPE secreted from culture media. This is important because the use of cyclodextrins provides an affordable and food-grade reagent for purification of LPC / LPE in lieu of albumin, a more expensive reagent.

[0569] 008933087References

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[0628] 008933087

Claims

Claims:

1. A method of producing a lysophospholipid (LPL), the method comprising:(i) providing an enzyme from the TMEM150 protein family, and(ii) contacting the enzyme with a phospholipid to allow the enzyme to hydrolyse a fatty acid from the phospholipid,thereby producing the LPL.

2. The method of claim 1, wherein the enzyme from the TM EM 150 protein family is selected from TMEM150B or a functional homologue thereof; TMEM150A or a functional homologue thereof; SFK1 or a functional homologue thereof; TMEM150C or a functional homologue thereof; DRAM1 or a functional homologue thereof; or DRAM2 or a functional homologue thereof.

3. The method according to any one of the preceding claims, wherein the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS), or any combination thereof; and / orwherein the phospholipid comprises a monounsaturated fatty acid (MU FA) or a polyunsaturated fatty acid (PUFA), optionally wherein the MUFA or PUFA is an omega-3 or omega-6 fatty acid; and / orwherein the phospholipid comprises a MUFA or a PUFA covalently bound at the sn2 position, optionally wherein the enzyme hydrolyses the fatty acid from the sn1 position of the phospholipid.

4. The method according to claim 3, wherein the MUFA is oleic acid, or the PUFA is selected from, linoleic acid, linolenic acid (ALA), eicosapentaenoic acid (EPA) docosahexaenoic acid (DHA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), docosapentaenoic acid (DPA), or any combination thereof.

5. The method according to any one of the preceding claims, wherein the enzyme is situated in a membrane, optionally wherein the membrane forms part of a:i) cell;ii) liposome or proteoliposome;iii) lipid nanoparticle; oriv) micelle, optionally a detergent micelle.

6. The method according to any one of the preceding claims, wherein the method further comprises a step of harvesting the LPL; optionally008933087wherein the harvesting step comprises contacting the LPL with a transporter moiety which can bind to amphipathic lipids, phospholipids and / or LPLs; further optionally, wherein the transporter moiety is selected from an albumin protein, alpha-fetoprotein protein, a vitamin D-binding protein, a cyclodextrin, an a- cyclodextrin, a p-cyclodextrin, an apolipoprotein, apolipoprotein A-1, apolipoprotein B, apolipoprotein D, apolipoprotein E, apolipoprotein F, and apolipoprotein M, or any combination thereof.

7. A cell that is engineered to express an enzyme from the TM EM 150 protein family; optionallywherein the enzyme from the TMEM150 protein family is selected from TMEM150B or a functional homologue thereof; TMEM150A or a functional homologue thereof; SFK1 or a functional homologue thereof; TMEM150C or a functional homologue thereof; DRAM1 or a functional homologue thereof; or DRAM2 or a functional homologue thereof.

8. The cell according to claim 7, wherein the enzyme from the TMEM150 protein family is TMEM150B, or a functional homologue thereof.

9. The cell according to claim 7 or 8, wherein the cell produces a greater amount of LPL compared to a non-engineered parent cell, optionally wherein the LPL is selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS) or any combination thereof; and / orwherein the cell is engineered to increase production of monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), or saturated fatty acids (SFA), or any combination thereof, compared to a non-engineered parent cell, optionally wherein the MUFA or the PUFA is an omega-3 or an omega-6 fatty acid.

10. The cell according to claim 9, wherein,(i) the SFA is selected from palmitic acid and / or stearic acid;(ii) the MUFA is oleic acid; and / or(iii) the PUFA is linoleic acid, linolenic acid (ALA), stearidonic acid (SDA) eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or any combination thereof.

11. The cell according to any one of claims 7 to 10, wherein the cell is engineered to have increased expression of any combination of the following, or functional homologues thereof, compared to a non-engineered parent cell;008933087elongase 2 (ELO2), delta-9-desaturase (D9D), delta-12-desaturase (D12D), w3-desaturase (FAD), delta-6-desaturase (D6D), delta-6-elongase (D6E), delta-5-desaturase (D5D), C20 / 22 elongase (ELO2), and delta-5-desaturase (FAD4),S. cerevisiae elongase 2 (ScELO2), Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), S. kluyveri w3- Desaturase (skFAD), Ostreococcus tauri delta-6-desaturase (OtD6D), Mortierella alpina delta-6-elongase (MaD6E), Paramecium tetraurelia delta- 5-desaturase (Ptet1D5D), Ostreococcus tauri C20 / 22 elongase (OtELO2), and Thraustochytrium sp delta-5-desaturase (TaFAD4).

12. The cell according to any one of claims 7 to 11 , wherein the cell is engineered to have increased expression of one or more of delta-9-desaturase (D9D), delta- 12-desaturase (D12D), and w3-desaturase (FAD), or functional homologues thereof.

13. The cell according to any one of claims 7 to 12, wherein the cell is engineered to have increased expression of one or more of Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), and S. kluyveri w3-Desaturase (skFAD).

14. The cell according to any one of claims 7 to 13, wherein the enzyme from the TMEM150 protein family is TMEM150B, or a functional homologue thereof, and wherein the cell is engineered to have increased expression of Mortierella alpina delta-9-desaturase (MaD9D), Mortierella alpina delta-12-desaturase (MaD12D), and S. kluyveri w3-Desaturase (skFAD).

15. The cell according to any one of claims 7 to 14, wherein the cell is engineered to increase production of a phospholipid compared to a non-engineered parent cell; optionally wherein the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), or any combination thereof; further optionally wherein the phospholipid comprises a MLIFA or PLIFA covalently bound at the sn1 and / or sn2 position of the phospholipid; optionally wherein:(i) the phospholipid comprises a MLIFA which is an omega-3 or an omega-6 fatty acid, optionally oleic acid; and / or(ii) the phospholipid comprises a PLIFA which is an omega-3 fatty acid or an omega-6 fatty acid, optionally selected from linoleic acid, linolenic acid (ALA), stearidonic acid (SDA), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), or any combination thereof.

16. The method according to claim 5 or 6, or the cell according to claim 15, wherein the cell is engineered to reduce beta oxidation of fatty acids, triglyceride (TAG) formation, diacylglycerol (DAG) formation, and / or expression of a phospholipase gene.00893308717. The method or the cell according to claim 16, wherein the cell is engineered to reduce expression of any combination of the following, or functional homologues thereof, compared to a non-engineered parent cell;sfk1, Iem3, pox1, dga1, Iro1, pah1, Ipp1, dpp1, plb1, plb2 are1, are2, faa1, faa2.

18. The method according to claim 5 or 6, or the cell according to any one of claims 15 to 17, wherein the cell is engineered to increase expression of one or more enzymes of the Kennedy pathway, and / or lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT).

19. The method according to claim 5, 6 or 16 to 18, or the cell according to any one of claims 7 to 18, wherein the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, an insect cell, a fungal cell, a bacterial cell, an algal cell, a plant cell, a protist cell, or a prokaryotic cell.

20. The method or the cell according to claim 19, wherein;(i) the human cell is from an immortalised cell line; optionally a HeLa cell;(ii) the insect cell is from Spodoptera frugiperda; optionally a Sf-9 cell; and / or(iii) the fungal cell is a yeast cell, optionally a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.

21. A composition comprising at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, 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 99% lysophospholipid (LPL) components.

22. The composition according to claim 21, wherein the LPL components are produced by the method or the cell according to anyone of claims 1 to 21.

23. The composition according to claim 21 or claim 22, wherein the LPL components comprise LPC, LPE and / or LPS.

24. The composition according to any one of claims 21 to 23, wherein the LPL components comprise one or more MUFA and / or a PUFA.

25. The composition according to claim 24, wherein the MUFA and / or the PUFA comprise omega-3 and / or omega-6 fatty acids.00893308726. The composition according to claim 25, wherein the fatty acids comprise eicosatetraenoic acid (ETA), oleic acid, linoleic acid, linolenic acid (ALA), stearidonic acid (SDA), eicosapentaenoic acid (EPA) and, docosapentaenoic acid (DPA) or docosahexaenoic acid (DHA), or any combination thereof.

27. The composition according to any one of claims 21 to 26, wherein the LPL component comprises LPE 20:4 (ETA), LPC 22:6 (DHA), LPE 22:6 (DHA), LPS 22:6 (DHA), LPC 18:3 (ALA), LPE 18:3 (ALA), LPS 18:3 (ALA), LPC 20:5 (EPA), LPE 20:5 (EPA), LPS 20:5 (EPA), LPC 20:4 (ETA) and LPS 20:4 (ETA), or any combination thereof.

28. The composition according to any one of claims 21 to 27, wherein the composition comprises a transport moiety which can bind to amphipathic lipids, phospholipids and / or LPLs, optionally wherein the transport moiety is selected from an albumin protein, alpha-fetoprotein protein, a vitamin D-binding protein, a cyclodextrin, an a- cyclodextrin, a p-cyclodextrin, an apolipoprotein, apolipoprotein A-1, apolipoprotein B, apolipoprotein D, apolipoprotein E, apolipoprotein F, and apolipoprotein M, or any combination thereof.

29. The composition according to claim 28, wherein the transport moiety comprises a cyclodextrin selected from a-cyclodextrin and a p-cyclodextrin, ora combination thereof.

30. The method according to claims 1 to 6 or 16 to 20, the cell according to claims 9 to 20, the composition according to claims 21 to 29, wherein the LPL is selected from Table 1, Table 2, and / or Table 3 or any combination thereof.008933087

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