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WO2009001315A2 - Utilisation d'une classe de gènes codant pour les lysophospholipide acyl transférases pour une application en agriculture, biotechnologie et médecine - Google Patents

Utilisation d'une classe de gènes codant pour les lysophospholipide acyl transférases pour une application en agriculture, biotechnologie et médecine Download PDF

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WO2009001315A2
WO2009001315A2 PCT/IB2008/052563 IB2008052563W WO2009001315A2 WO 2009001315 A2 WO2009001315 A2 WO 2009001315A2 IB 2008052563 W IB2008052563 W IB 2008052563W WO 2009001315 A2 WO2009001315 A2 WO 2009001315A2
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acid sequence
nucleic acid
polypeptide
polypeptides
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WO2009001315A3 (fr
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Ulf STÅHL
Kjell STÅLBERG
Hans Ronne
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Staahl Ulf
Staalberg Kjell
Hans Ronne
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides

Definitions

  • This invention relates to the use of a novel class of enzymes and their encoding genes in agricultural, biotechnological and biomedical applications. More specifically, the invention relates to the use genes encoding enzymes with acyl-CoA : lysophospholipid acyltransferase (LPLAT) activity.
  • acyl-CoA lysophospholipid acyltransferase
  • Phospholipids are phosphoglycerides in which two fatty acid side chains (acyl groups) are esterified to the hydroxyl groups on carbon- 1 and carbon-2 of a glycerol molecule, while a polar head group is attached to carbon-3 through a phosphoester bond.
  • the simplest phospholipid is phosphatidic acid (PA) which has a phosphate group as its polar head group.
  • Lipids with more complex head groups include phosphatidylcholine (PC), phosphatidyletanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI). They all share a phosphate group, through which the rest of the head group is attached to the glycerol backbone.
  • Biological membranes are made up of phospholipid bilayers, in which the polar head groups reside on the surface, and the acyl groups in the hydrophobic interior.
  • the most common phospholipid in biological membranes is PC, but the phospholipid composition of membranes may vary significantly between different species, tissues and organelles.
  • the acyl groups in a given phospholipid may also differ between different molecules.
  • Phospholipids are synthesized in vivo by two different pathways (Vance and Vance, 2004;
  • the first pathway is de novo synthesis from phosphatidic acid (PA) which can either be dephosphorylated to diacylglycerol (DAG) which is the precursor for the two major eukaryotic phospholipids PC and PE, or activated to CDP- DAG which is the precursor for PS, PG and PI.
  • PA phosphatidic acid
  • DAG diacylglycerol
  • CDP- DAG CDP- DAG
  • the DAG molecule is condensed with the activated head groups, CDP-choline and CDP- ethanolamine
  • PS, PG and PI biosynthesis the activated CDP-DAG molecule is condensed with serine, glycerol-3 -phosphate or inositol.
  • phosphatidylglycerolphosphate is formed which is subsequently dephosphorylated to produce PG.
  • phospholipids can also be synthesized by acylation of the corresponding acyl lysophospholipids, which lack an acyl chain at either the sn-1 or sn-2 position. This second pathway leaves the head group intact.
  • There are two lysophospholipids for each phospholipids (one that has a free hydroxy group at the sn-1 and the other at the sn-2 position): lysophosphatidylcholine (LPC) for PC, lysophosphatidylethanolamine (LPE) for PE, lysophosphatidylserine (LPS) for PS, lysophosphatidylglycerol (LPG) for PG, lysophosphatidylinositol (LPI) for PI and lysophosphatidicacid (LPA) for PA.
  • LPC lysophosphatidylcholine
  • LPE lysophosphatidylethanolamine
  • LPS ly
  • LPA is synthesized by acylation of glycerol-3-phosphate, which is the first step in the de novo synthesis of phospholipids.
  • the other lysophospholipids (and to some extent also LPA) are, however, generated by deacylation of the corresponding phospholipids, a reaction that is catalysed by the reverse action of the LPCAT enzyme or by phospholipase A.
  • the second pathway for phospholipid biosynthesis is therefore a cyclic process, known as the Lands cycle, in which phospholipids are deacylated and then resynthesized by acylation of the resulting lysophospholipids (Lands 2000).
  • acyl group composition of phospholipids which is known as phospholipid remodelling.
  • the acyl group composition of phospholipids can in turn affect membrane fluidity, which also changes with temperature. Phospholipid remodelling is therefore important for the adaptation of organisms to changes in the temperature, and also to other changes in the environment.
  • polyunsaturated fatty acids and a number of more unusual fatty acids including hydroxylated, acetylenic, epoxy, conjugated, cyclopropen, and methyl branched fatty acids, are synthesised on precursor fatty acids that are attached to a phospholipids, in particular to phosphatidylcholine.
  • the fatty acids produced on phospholipids are then channelled to other lipids via the acyl-CoA pool by deacylation of the phospholipid and reacylation to free CoA.
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the Lands cycle thus allows acyl groups to move back and forth between the acyl-CoA pool and the phospholipids.
  • the engineering of an efficient synthesis and accumulation of fatty acids that are modified on phospholipids in oil producing organisms such as oil crop plants therefore requires a rapid exchange of acyl groups between the acyl- CoA pool and the phospholipids.
  • Fatty acids that are synthesized in this way include nutritionally important omega-3 and omega-6 fatty acids, such as EPA, DHA and arachidonic acid (AA), and industrially important fatty acids, including hydroxylated, acetylenic, epoxy, conjugated, cyclopropen and methyl branched fatty acids.
  • LPAATs LPA acyltransferases
  • Plants have both cytosolic (microsomal) and plastidic LPAATs.
  • Arabidopsis thaliana has five LPAAT genes, one of which encodes a plastidic enzyme (Kim and Huang, 2004).
  • the SLCl gene was shown to encode a microsomal LPAAT (Nagiec et al., 1993).
  • Tafazzin a mitochondrial protein that is conserved in eukaryotes from yeast to man, was claimed to be an LPCAT (Testet et al., 2005), but recent experiments suggest that it is instead a CoA-independent phospholipid transacylase which catalyzes the transfer of acyl groups between PC and the mitochondrial phosphoplipid cardiolipin (Xu et al., 2006).
  • Yeast has an LPCAT activity similar to that found in plants and animals (Richard and McMaster, 1998). We have identified the gene responsible for this LPCAT activity as YOR175c.
  • the YOR175c encoded enzyme has a very broad substrate specificity, being able to acylate not only LPC, but also LPE, LPG, LPS, LPI and LPA. Accordingly, it is a general lysophospholipid acyltransferase, and we refer to this novel enzyme as LPLAT.
  • the yeast LPLAT protein (the YORl 75c gene product) is not closely related to any of the earlier reported LPCATs or LPAATs. Instead, it is related to several proteins from other eukaryotes, including plants, animals and fungi, whose functions were previously unknown. Yeast LPLAT and its relatives in other eukaryotes form a distinct subfamily within the MBOAT superfamily of putative membrane bound acyltransferases (Hoffman, 2000), but are much more similar to each other than to other MBOAT proteins. This makes it likely that the LPLAT-related proteins from other eukaryotes are true orthologs of yeast LPLAT and thus possess LPLAT activity. Consistent with this, we found that two LPLAT orthologs from Arabidopsis thaliana, the Atlgl2640 and Atlg63050 gene products, also possess LPLAT activity when expressed in yeast.
  • Figure 1 Evolutionary dendrogram showing the Saccharomyces cerevisiae YORl 75c encoded protein and some of its closest homologs in other eukaryotes. The dendrogram was made by aligning the most conserved part of the proteins, corresponding to amino acid residues number 286 to 392 of the YORl 75c encoded protein (SEQ ID 32), a region where all these proteins can be aligned without ambiguities. Pairwise distances were calculated from this alignment. The resulting branch lengths were used to construct the tree using the Neighbor-joining method (Saitou and Nei, 1987) as implemented in the ClustalX software (Thompson et al., 1997) with the Skip Gapped Positions and Correct for Multiple Substitution options enabled.
  • SEQ ID 32 amino acid residues number 286 to 392 of the YORl 75c encoded protein
  • the bar represents an evolutionary distance (PAM value) of 0.2.
  • the sequences used were: Human MBOATl, NMJ)01080480.1 (SEQ ID NO 8); Zebrafish MBOATl, XM_702282.2 (SEQ ID NO 16); Human MB0AT2, XMJ)01129292.1 (SEQ ID NO 10); Zebrafish MB0AT2, BX255963.
  • Proteins from different species without previously assigned names are labelled LPLAT in the figure, to indicate their predicted enzyme activities. This should not be interpreted as excluding other, already named proteins, such as MBOATl, from being members of the same protein family and also possessing LPLAT activities. It should further be noted that there are many more proteins that belong to the LPLAT family of proteins than those shown in the figure. For a longer list of proteins that are closely related to YORl 75c, and thus are expected to possess LPLAT activities, see Table 2. Figure 2. Lysophospholipid acyltransferase activity in crude extracts from wild type (WT) and yorl75c knockout yeast cells.
  • Figure 3 Metabolism of 14 C-labelled 16:0-CoA incubated with crude extracts from wild type (WT) and yorl75c knockout yeast cells in the presence of GPC.
  • Figure 4. Lysophosphatidylcholine acyltransferase activity in crude yeast extracts from the y or 175c knockout strain transformed with either the empty vector pYES2, pYES2- YORl 75c, pYES2/NT-Atlgl2640 or pYES2/NT-Atlg63050.
  • yeast Saccharomyces cerevisiae referred to as "yeast” below
  • yeast gene YOR175c
  • SEQ ID NO 2 its encoded enzyme
  • Saccharomyces cerevisiae extracts is dependent on YOR175c (Examples 2-6).
  • the YOR175c encoded protein is further able to acylate LPA, and our results show that it accounts for most of the LPA acylating activity in Saccharomyces cerevisiae extracts supplied with exogenous
  • This invention further describes a highly conserved family of proteins that are related to the YOR175c gene product (Example 1).
  • Members of this protein family (some of which are shown in Figure 1 or listed in Table 2) are present in many different eukaryotes from amoebas to man.
  • members of the LPLAT family of proteins show extensive similarity to each other also outside the MBOAT motif, which suggests that they may have similar substrate specificities and/or biological functions.
  • This invention further describes two other members of the LPLAT family of proteins, the products of the Arabidopsis thaliana genes Atlgl2640 (SEQ ID NO 3) and Atlg63050 (SEQ ID NO 5), which also possess acyl-CoA dependent acyltransferase activities with several different lysophospholipid substrates, similar to YOR175c.
  • both these plant proteins are able to acylate LPC, LPE, LPG, LPS and LPI when expressed in yeast (Examples 8 and 9).
  • the MBOAT superfamily of proteins includes several other protein families that possess other types of enzymatic activities (Hoffman, 2000). Our finding that several members of the LPLAT family of proteins, a distinct subfamily within the larger MBOAT superfamily, have
  • LPLAT activities is a novel finding, and the use of these proteins, their enzymatic activities, and their encoding genes for different applications is the basis of this invention.
  • members of the LPLAT protein family from organisms as diverse as plants, animals and fungi possess LPLAT activities suggests that other members of the LPLAT family, some of which are shown in Figure 1 or listed in Table 1, also will be found to possess such activities, which could be used for the applications that we describe below.
  • Derivatives of polypeptides are also considered.
  • One type of derivatives are mutated versions of a polypeptide.
  • Such mutated polypeptides may contain one or several point mutations, where amino acid residues have been changed, or deletions or insertions of one or several amino acid residues.
  • the methods for producing such mutated polypeptides are known to those skilled in the art, and may involve either site directed mutagenesis or random in vivo or in vitro mutagenesis of the nucleic acid sequence encoding the polypeptide.
  • Another type of derivatives are hybrid proteins, comprising fragments from two or more proteins that have been joined into one polypeptide.
  • hybrid proteins may be swap constructions, in which homologous parts of two or more related proteins have been exchanged for each other, but they can also be fusions between unrelated proteins or fragments thereof.
  • Swap constructions may possess novel properties, such as new substrate specificities in the case of enzymes, which differ from those of the parental molecules. Fusions between unrelated proteins may be used to physically link different activities, or to target an activity to a specific location in the cell.
  • the methods for producing hybrid proteins are also known to those skilled in the art.
  • a functional fragment is a fragment of a polypeptide which retains at least some of its biological activity, in the case of enzymes the enzymatic activity.
  • a functional fragment may be obtained by making deletions in the nucleic acid sequence encoding the polypeptide, using methods known to those skilled in the art.
  • a functional fragment of an enzyme always includes the active site, but other parts of the polypeptide may also be essential for the enzymatic activity.
  • the properties of a functional fragment of an enzyme may differ from those of the parental molecule. For example, it may be differently regulated or possess an altered substrate specificity if either a regulatory or a specificity-determining part of the the enzyme was removed in order to create the functional fragment.
  • One aspect of the invention relates to the transgenic expression of the products of the
  • a direct consequence of altering the total LPLAT activity or changing its acyl specificity is to facilitate phospholipid turnover, a process in which phospholipids are deacylated by the reverse action of the LPLAT activity or cleaved by phospholipase A to produce lysophosholipids and free fatty acids, after which the resulting lysophospholipids are acylated by LPLAT to produce new phospholipids.
  • phospholipid remodelling is important both in biological responses to stress conditions, and in biotechnological applications where the goal is to increase the yield of nutritionally or industrially important fatty acids whose biosynthesis in part occurs while the acyl group is incorporated in a phosholipid molecule.
  • Phospholipid remodelling will help to move acyl groups that have been modified on phospholipids into the acyl-CoA pool, where some biosynthetic steps such as fatty acid elongations and acylation to different acyl acceptors occurs e.g. acylation of intermediates in both phospholipid and neutral lipid synthesis.
  • transgenically expressing members of the LPLAT family of proteins in organisms used for oil production, including but not limited to oil seeds, in order to increase the overall yield of oil.
  • transgenic expression of members of the LPLAT family of proteins with unique specificities for certain nutritionally or industrially important acyl groups may help to improve the yield of lipids containing such acyl groups.
  • Different organisms vary in the acyl group composition of their lipids, and some organisms, such as castor bean (Ricinus communis), Lequerella fendleri and microalgae are enriched in oil with nutritionally or industrially important fatty acids.
  • Such organisms have lipid synthesizing enzymes that are adapted to their unique fatty acid content, and LPLAT from such an organism will show an enhanced specificity for the corresponding unique acyl groups.
  • Transgenic expression of such an LPLAT will improve the yield of nutritionally or industrially important fatty acids, particularly in cases where the production of such fatty acids is achieved by transgenic expression of acyl group modifying enzymes from other organisms in a crop plant whose endogenous LPLAT lacks specificity for the corresponding acyl groups.
  • This LPLAT gene is expressed in a desired transgenic organism and tissue, such as, but not limited to, oil seeds, together with gene(s) encoding enzyme(s) capable of synthesizing the desired fatty acid and/or genes encoding acyl-CoA utilising enzymes acylating the desired fatty acid into intermediates used in oil biosynthesis.
  • a desired transgenic organism and tissue such as, but not limited to, oil seeds
  • oils such as triacylglycerols and wax esters
  • transgenically expressing members of the LPLAT family of proteins in different organisms including but not limited to crop plants, in order to increase the total LPLAT activity in the tissues of these organisms and thus facilitate phospholipid remodelling.
  • the purpose of this is to enhance the ability of these organisms to adapt to different types of environmental stresses, such as changes in the temperature, a type of adaptation which is known to be facilitated by phospholipid remodelling.
  • a second aspect of the invention relates to the use of the products of the YOR175c, Atlgl2640, or Atlg63050 genes, or any other member of the LPLAT family of proteins, together with enzyme assays such as those described in Examples 2-10 for the purpose of identifying or developing small molecules that may enhance or inhibit the activities of these enzymes, or modify their substrate specificities.
  • Such a process may use chemical genomics, in which a large number of small molecules are screened for their effects on the enzymatic activity, or in silico drug design, in which the structure of members of the LPLAT family of proteins is used to predict candidate small molecules that may modulate the enzymatic activity.
  • RNAs, morpholinos and other molecules that selectively target LPLAT mRNAs or LPLAT genes through their nucleotide sequences, thus changing the expression of LPLAT proteins.
  • small molecules that can alter the rates at which AA, dihomo-gamma-linoleic acid (DGLA) and EPA are incorporated into phospholipids through their effects on the expression of members of the LPLAT family of proteins, or on their enzymatic activities.
  • DGLA dihomo-gamma-linoleic acid
  • EPA dihomo-gamma-linoleic acid
  • Phospholipids containing any of these three fatty acids function as precursors in the biosynthesis of eicosanoids, a group of biologically active molecules that includes prostaglandins, thromboxanes, prostacyclins and leukotrienes.
  • eicosanoids Biological processes that are regulated by eicosanoids include inflammation, the immune response and signal transduction in the central nervous system.
  • eicosanoids eicosanoid analogs or drugs that affect eicosanoid biosynthesis in medicine. Modulating the rates at which different eicosanoids are synthesized in vivo by modulating the LPLAT activity would provide an alternative to direct administration of eicosanoids for medical treatment, many of which are difficult to synthesize and highly unstable.
  • MB0AT4 plays a key role in the control of feeding, adiposity and insulin secretion through its ability to acylate (octanoylate) the appetite- stimulating peptide hormone ghrelin (Gutierrez et al., 2008; Yang et al., 2008). This modification is essential for ghrelin function (Bednarek et al., 2000).
  • Octanoylated ghrelin promotes food intake and adiposity, and also suppresses insulin secretion and impairs glucose tolerance (Kojima and Kangawa, 2005; Cummings and Overduin, 2007). It has therefore been proposed that MB0AT4 (GOAT) may provide a critical molecular target in developing novel therapeutics for obesity and type 2 diabetes (Gutierrez et al., 2008).
  • One way to find such novel drugs would be to transiently co-express MB0AT4 and ghrelin in a suitable system, such as embryonic kidney cells (Gutierrez et al., 2008), and then test the effects of different small molecules on the acylation of ghrelin that is secreted from these cells, using immunoprecipitation followed by mass spectrometric analysis (Gutierrez et al.,
  • Examples 2-10 could be modified to use ghrelin or a ghrelin- derived peptide that is still recognized by MB0AT4 as a substrate, which would make it possible to assay the ghrelin-acylating activity of MB0AT4 obtained by expression in yeast or another suitable expression system in vitro, for use in a drug screen.
  • Such an assay could use either radiolabeled ghrelin or radiolabeled acyl-CoA, after which radiolabeled acylated ghrelin would be detected using methods known to those skilled in the art, such as high pressure liquid chromatography or mass spectrometry. It is likely that the assay conditions that we describe in Examples 2-10 also would work with MB0AT4 given the fact that they work with several other members of the LPLAT protein family.
  • radiolabeled acylated ghrelin (or a radiolabeled acylated ghrelin-derived peptide) could be detected after co-expression of MB0AT4 and ghrelin (or a ghrelin-derived peptide) in yeast or another suitable cell system, followed by feeding of the transgenic cells with radiolabeled fatty acids.
  • Such an assay could also be used as a basis for a drug screen, and might avoid problems relating to effects of drugs on secretion.
  • the yeast YORl 75c protein can acylate a number of different lysophospholipids (Examples 2-10; Stahl et al., 2008), using acyl donor groups of different lengths (Tamaki et al., 2007), but it can also acetylate the for yeast unnatural substrate lysoPAF (Tamaki et al., 2007).
  • a simple method to find molecules that can inhibit or modulate the acyltransferase activity of MB0AT4 would therefore be to screen a library of small molecules for their effects on LPLAT activity in extracts from a yeast yorl75c knockout strain that lacks endogenous LPLAT activity, but expresses a mammalian MB0AT4 protein from a yeast promoter.
  • the assays used could include those described in Examples 2-10, but could also use other lysophospholipid substrates and/or other acyl group donors if found to be more suitable.
  • Examples 2-10 can still be used to simplify the development of an inhibitor, activator or modulator of MB0AT4 activity.
  • the procedure in this case would be to use one or several members of the LPLAT family that are known to possess LPLAT activity in the screen described above, to find general inhibitors, activators or modulators of this family of enzymes. Any small molecules with such activity could then be tested for its ability to also inhibit, activate or modulate the MBOAT4-dependent acylation of ghrelin.
  • a general inhibitor, activator or modulator of the LPLAT family of enzymes could also serve as a suitable starting point for development of a drug that specifically targets MB0AT4.
  • PI protein kinase C
  • a third aspect of the invention relates to the use of the nucleotide sequences of genes encoding members of the LPLAT family of proteins, together with enzyme assays such as those described in Examples 2-10 for the purpose of developing diagnostic methods aimed at identifying individuals (humans, animals or plants) with genetic diseases or other genetically determined traits that are linked to mutations in, or alleles of, these genes.
  • diseases or traits may include inherited metabolic disorders or increased risk for such disorders, inflammatory and auto-immune disorders that involve disturbances in eicosanoid biosynthesis and prostaglandin signaling, and neoplastic conditions, including tumor progression, that involve increased Pi-dependent signalling.
  • Such diagnostic methods could also be used to select individuals with desirable traits, such as but not limited to an increased ability to accumulate certain lipids, in animal and plant breeding.
  • a fourth aspect of the invention relates to the use of the products of the YOR175c, Atlgl2640, or Atlg63050 genes, or any other member of the LPLAT family of proteins, together with enzyme assays such as those described in Examples 2-10 for the purpose of developing mutant versions of the proteins with increased enzymatic activities or altered substrate specificities. Such mutants may be obtained by several methods that are known to those skilled in the art.
  • One example is in vitro mutagenesis of a yeast shuttle plasmid encoding a member of the LPLAT family of proteins followed by a screen in which the resulting mutant plasmid library is transformed into a yeast strain lacking the YORl 75c gene, after which enzyme assays are performed on extracts from the yeast transformants.
  • enzyme assays are performed on extracts from the yeast transformants.
  • Another example is structure-based protein engineering, where amino acid residues that are important for substrate specificity or other properties of the enzyme are identified based on sequence alignments and/or predicted or known secondary or tertiary structures of the protein(s), after which the effects of mutations in these amino acid residues on enzyme activities are tested using a suitable expression system such as the yeast yorl75c deletion mutant.
  • a third example is the creation of functional fragments of members of the LPLAT family of proteins, which retain enzyme activity even though parts of the protein have been removed.
  • Such functional fragments may possess a modified (usually broader) substrate specificity if a specificity-determining domain was removed, or an increased activity if a regulatory domain was removed.
  • the solubility of the enzyme, its intracellular location, and other properties may also be altered in this way. What parts of the proteins that can be removed while retaining enzymatic activity can be determined experimentally, by testing different N-terminal, C- terminal or internal deletion mutants in a suitable expression system such as the yeast yorl75c deletion mutant.
  • a fourth example is the creation of hybrid proteins by fusions between different members of the LPLAT family of proteins.
  • Such fusion proteins encoded by the corresponding gene fusions, may possess novel and unique properties if, for example, the catalytic domain is derived from one parent and a regulatory domain from another parent.
  • the properties of such fusion proteins can be tested in a suitable expression system such as the yeast yorl75c deletion mutant.
  • Transgenic expression of such mutant or engineered LPLAT proteins in organisms used for oil production will improve the yield of nutritionally and industrially important fatty acids, particularly in cases where the production of such fatty acids is achieved by transgenic expression of acyl group modifying enzymes from other organisms in an organism whose endogenous LPLAT lacks specificity for the corresponding acyl groups.
  • Example 1 The yeast and Arabidopsis LPLAT proteins belong to a distinct and well conserved subfamily among the MBOAT proteins, which is found in all eukaryotes. Genes and their encoded proteins can be grouped into families that share a common ancestor, i. e. a gene (or encoded protein) from which the members of such a family arose either by species divergence (orthologous genes and proteins) or gene duplications within a species (paralogous genes and proteins). In the case of enzymes, members of a given protein family usually possess similar enzymatic activities, and enzymes that are closely related to each other are more likely to share the same substrate specificity than more distantly related enzymes.
  • Sequences that are related to each other can be aligned, and the alignment can be used to deduce the order of divergence of the related proteins from a common ancestral molecule. This order of divergence can be displayed as a dendrogram or phylogenetic tree, which groups the proteins into subfamilies (branches) depending on how closely related they are to each other (Saitou and Nei, 1987).
  • the overall similarity between different member proteins may be too low to permit an unambiguous alignment of all sequences and the construction of an phylogenetic tree, but the fact that the proteins are related to each other can still be inferred from shorter regions of sequence similarity (conserved sequence motifs) in functionally important parts of the proteins.
  • conserved sequence motifs conserved sequence motif around the active site
  • Figure 1 shows an evolutionary tree computed from an alignment of selected protein sequences from different eukaryotes that are related to yeast LPLAT (the YOR175c gene product).
  • yeast LPLAT the YOR175c gene product
  • proteins that are related to LPLAT are found not only in plants, animals and fungi, but also in primitive eukaryotes such as Enteroamoeba histolytica.
  • LPLAT homologs which include the human MBOAT 1, 2 and 4 proteins, are clearly more similar to each other than to other members of the MBOAT superfamily. It is therefore to be expected that also other LPLAT homologs, including but not limited to the human MBOAT 1, 2 and 4 proteins, will possess LPLAT activity if tested under suitable conditions.
  • acyl-CoA cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT) (Hoffman, 2000), which are only distantly related to the LPLAT family of proteins, and which possess entirely different enzymatic activities.
  • ACAT cholesterol acyltransferase
  • DGAT diacylglycerol acyltransferase
  • Figure 1 The purpose of Figure 1 is to examplify members of the LPLAT family of enzymes, and not to provide an exhaustive list of these proteins. For a longer list of polypeptides with expected LPLAT activities, see Table 2 below.
  • Example 2 Lysophosphatidylcholine acyltransferase (LPCAT) activity in crude yeast extracts is dependent on the YOR 175c gene.
  • the cell slurry and about 1 ml of 0.5 mm zirconia/silica beads were mixed in 2.5 ml Eppendorf tubes and shaken in a Mini Beadbeater (Biospec Products, INC.) for 3 x 1 minute with 3 min cooling on an ice bath in between.
  • the extract was centrifuged at 1000 g for five minutes at 4 0 C and the supernatant, referred to as crude extract, was frozen in aliquots at -70 0 C. Protein concentrations were determined using the Bradford method with BSA as protein standard.
  • Lysophosphatidylcholine acyltransferase (LPCAT) assay was tested by incubating 7 ⁇ l of crude extract of yeast cells (50 ⁇ g of protein) in a total volume of 100 ⁇ l containing 40 mM Tris/HCL pH 7.6, 0.1 mM [ 14 C] 18: 1-CoA 5000 dpm/nmol (GE Heltcare, Amersham Biosciences) and 0.1 mM of sn-l-16:0-LPC, for 10 minutes at 30 0 C. The assays were stopped by Blight & Dyer extraction (Blight & Dyer 1959).
  • the lipid areas were located by brief exposure to I 2 vapours and identified by means of appropriate standards.
  • the distribution of radioactivity on the TLC plates was analyzed by phosphorimager. For quantification, spots were scraped of the silica gel plate, after that they had been sprayed gently with water (for a more easy removal of the silica gel).
  • the radioactivity in each spot was determined by scintillation counting using toluene/ethanol (2:1, v/v) with 0.4% 2(l-biphenol)-5-(4- biphenol)-l, 3, 4-oxadizole as scintillation fluid and an LKB Wallace 1290 Rackbeta Liquid scintillation counter.
  • Lysophosphatidylethanolamine acyltransferase (LPEAT) activity in crude yeast extracts is dependent on the YOR175c gene.
  • LPEAT activity in the crude extract from the y or 175c knockout strain was only 0.04 nmol/min/mg which is about 3% of the activity found in the wild type extract. This result strongly suggests that the YORl 75c gene encodes an enzyme with LPEAT activity, which is responsible for almost all detectable LPEAT activity in S. cerevisiae crude extracts.
  • Lysophosphatidylglycerol acyltransferase (LPGAT) activity in crude yeast extracts is dependent on the YORl 75c gene.
  • LPSAT Lysophosphatidylserine acyltransferase
  • Lysophosphatidylinositol acyltransferase (LPIAT) activity in crude yeast extracts is dependent on the YOR 175c gene.
  • LPIAT activity in the crude extract from the y or 175c knockout strain was only 0.05 nmol/min/mg which is about 8% of the activity found in the WT crude extract. This result strongly suggests that the YORl 75c gene encodes an enzyme with LPIAT activity, which is responsible for most of the LPIAT activity in S. cerevisiae crude extracts.
  • Lysophosphatidic acid acyltransferase (LPAAT) activity in crude yeast extracts is partially dependent on the YORl 75c gene.
  • Example 8 Acyl-CoA derived 14 C-label accumulates as LPC when glycerophospho-choline is added to a crude extract from a yeast strain that lacks the YOR 175c gene.
  • GPC acyltransf erase (GPCAT) assay Crude extracts of yeast cells (50 ⁇ g of protein) were incubated in a total volume of 100 ⁇ l containing 25 mM Tris/HCL pH 7.6, 8 mM NaF, 4 mM MgCl 2 , 2 mg/ml BSA, 1 mM DTT, 2 mM glycerophosphocholine (GPC) and 0.1 mM [ 14 C] 16:0-CoA 5000 dpm/nmol (GE Healthcare, Amersham Biosciences) for 10 minutes at 30 0 C. The assays were stopped and further treated as described in Example 2.
  • GPCAT GPC acyltransf erase
  • the GPCAT activity was calculated from the radioactivity detected in the LPC spot plus half of the radioactivity detected in the PC spot.
  • the PC radioactivity is added is because the radioactive LPC that is formed by the GPCAT reaction is further metabolized by the LPCAT activity present in yeast extracts to form PC.
  • the reason for dividing this number by 2 is that the PC molecules will be labelled in both the sn-1 and sn-2 positions.
  • Example 9 Expression of YOR175c, Atlgl2640 or Atlg63050 can restore LPCAT activity in the yorl75c knockout yeast strain.
  • YORl 75c open reading frame encodes a lysophospholipid acyltransferase
  • Atlgl2640 and Atlg63050 encode functional lysophospholipid acyltransferases
  • the thermal cycling conditions used for amplifying YOR175c were 26 cycles at 95 0 C for 1 min, 55 0 C for 1 min and 72 0 C for 2 min.
  • the thermal cycling conditions were 30 cycles at 94 0 C for 1 min, 57 0 C for 1 min, and 72 0 C for 2 min.
  • Atlgl2640 atg gat atg agt tea atg gct g tta ttc ttc ttt acg C gg ttt
  • the amplified DNA fragments were cut out from a 1% agarose gel and purified using a Gel Extraction Kit (Qiagen).
  • the YORl 75c fragment was cloned into the pYES2.1/V5-His-TOPO vector (Invitrogen) and the two A. thaliana genes were cloned into the pCR2.1-TOPO vector (Invitrogen).
  • the three cloned inserts were verified by DNA sequencing.
  • the YORl 75c coding fragment was amplified using its endogenous stop codon, thus removing the C- terminal tag derived from pYES2. l/V5-His-TOPO. The two A.
  • thaliana genes in the pCR2.1- TOPO vector were subcloned into pYES2/NTC (Invitrogen).
  • the genes were cut out from pCR2.1-TOPO vector by EcoRl digestion and then ligated to £c ⁇ RI digested and dephosphorylated pYES2/NTC vector.
  • the resulting inserts are expressed in frame with a N- terminal tag containing one 6 x HIS and one EXPRESS epitope.
  • the pYES2/NTC-Atlgl2640 and pYES2/NTC-Atlg63050 clones were verified by restriction digestion and sequencing of the cloning borders. Transformation and cell cultivation.
  • l-YOR175c, pYES2/NTC-Atlgl2640, pYES2/NTC-Atlg63050 and the empty vector (pYES2) as a control were transformed into the y or 175c knockout yeast strain using standard methods. Transformants were selected on synthetic yeast media lacking uracil. Liquid precultures in 5 ml of synthetic media lacking uracil and containing 2% glucose were grown over night and used to inoculate 50 ml of synthetic yeast media lacking uracil and containing 2% galactose. The cells were inoculated at an OD 6 Oo of 0.1 and were then grown for 16 hours to an OD 60 O of about 2.5. At this point, 5 ml of 20% galactose was added to each culture, and the cultures were then cultivated for an additional four hours before harvest. Cells were harvested and crude extract prepared as described above in Example 2.
  • LPCAT assay The LPCAT activity in crude extracts from transformed yeast strains were assayed as described above in Example 2 with the exceptions that 0.1 mM [ 14 C] 16:0-CoA 5000 dpm/nmol (GE Healthcare, Amersham Biosciences) was used instead of [ 14 C] 18: 1-CoA, that 0.2 mM instead of 0.1 mM of LPC was added, and that the incubation time was 5 min instead of 10 min.
  • Atlgl2640 and Atlg63050 can also catalyze the acyl-CoA dependent acylation of LPE.
  • thaliana enzymes Atlgl2640 and Atlg63050 crude extract from the yorl75c knockout yeast strain transformed with pYES2/NTC-Atlgl2640, pYES2/NTC-Atlg63050 or the empty vector (pYES2) as a control, were incubated with different lysophospholipids in the presence of 14 C-labeled 18:1- CoA ( Figure 5).
  • Figure 5 The results show that the two A. thaliana enzymes can catalyze the acylation of LPE, LPG, LPS and LPI in addition to LPC. They are thus lysophospholipid acyltransferases with broad substrate specificities, similar to the YORl 75c protein.
  • SEQ ID NO 7 >MBOAT 1 ( human )
  • SEQ ID NO 10 >MBOAT2 (human) encoded protein
  • transgenic expression of: (i) a polypeptide shown in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26,
  • SEQ ID NO 28 SEQ ID NO 30 or SEQ ID NO 32, or (ii) any of the polypeptides listed in Table 2, or (iii) a derivative or functional fragment of any of the polypeptides referred to under (i) or (ii), for its ability to provide lysophospholipid acyltranferase (LPLAT) activities as described in Examples 2-10, i.e.
  • LLAT lysophospholipid acyltranferase
  • fatty acid species are the nutritionally important omega-3 and omega-6 fatty acids, such as EPA, DHA and AA, and industrially important hydroxy and epoxy

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Abstract

Cette invention porte sur l'utilisation d'une nouvelle enzyme et de son gène codant dans des applications biotechnologiques et biomédicales. Plus spécifiquement, l'invention porte sur l'utilisation de gènes de levure et de plante codant pour une enzyme ayant une activité acyl-CoA : lysophospholipide acyltransférase (LPLAT), ainsi que sur des gènes apparentés dans d'autres eucaryotes qui codent pour des enzymes ayant des activités analogues. Par l'expression transgénique de ces enzymes, ou enzymes homologues avec une spécificité appropriée de groupe acyle provenant d'autres organismes, le taux de transfert de groupes acyle désirés entre l'ensemble acyl-CoA et les phospholipides peut être augmenté. L'invention porte également sur la manière dont les LPLAT ayant des spécificités acyle particulières peuvent être obtenus, qui peuvent être utilisées pour accumuler des quantités élevées d'acides gras désirés dans divers organismes. Ceci conduira à son tour à une biosynthèse accrue et à une accumulation d'acide gras importants des points de vue nutritionnel et industriel dans des organismes utilisés pour la production d'huile. Un taux accru de renouvellement des phospholipides améliorera également l'aptitude d'organismes transgéniques à s'adapter à des changements de l'environnement. Un autre mode de réalisation de l'invention peut être utilisé pour développer des médicaments qui ciblent la famille LPLAT d'enzymes pour l'utilisation dans le traitement de troubles métaboliques, ainsi que des troubles auto-immuns, inflammatoires et néoplasiques mettant en jeu des trajets de signalisation tels que les voies des prostaglandines, thromboxanes, prostacyclines, leucotriènes et phosphoinositides. Un mode supplémentaire de réalisation de l'invention décrit un procédé pour un diagnostic de traits génétiques affectant de tels troubles et autres traits d'importance génétiquement déterminés en médecine humaine ou vétérinaire, en agriculture ou dans la croissance de plantes ou d'animaux.
PCT/IB2008/052563 2007-06-26 2008-06-25 Utilisation d'une classe de gènes codant pour les lysophospholipide acyl transférases pour une application en agriculture, biotechnologie et médecine WO2009001315A2 (fr)

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WO2009140770A1 (fr) * 2008-05-21 2009-11-26 National Research Council Of Cananda Réduction de l'activité de la lyso-phosphatidylcholine acyltransférase
WO2010147900A1 (fr) 2009-06-16 2010-12-23 E. I. Du Pont De Nemours And Company Amélioration de la biosynthèse d'acide gras oméga-3 et oméga-6 polyinsaturé à chaîne longue par l'expression d'acyl-coa lysophospholipide acyltransférase
WO2010147907A1 (fr) 2009-06-16 2010-12-23 E. I. Du Pont De Nemours And Company Huiles à teneur élevée en acide éicosapentaénoïque à partir de souches optimisées améliorées de yarrowia lipolytica
WO2010147904A1 (fr) 2009-06-16 2010-12-23 E. I. Du Pont De Nemours And Company Souches optimisées améliorées de yarrowia lipolytica pour une production élevée d'acide éicosapentaénoïque
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WO2012027689A1 (fr) 2010-08-26 2012-03-01 E. I. Du Pont De Nemours And Company Cellules hôtes microbiennes recombinées pour la production élevée d'acide eicosapentaénoïque
US8524485B2 (en) 2009-06-16 2013-09-03 E I Du Pont De Nemours And Company Long chain omega-3 and omega-6 polyunsaturated fatty acid biosynthesis by expression of acyl-CoA lysophospholipid acyltransferases
WO2013192002A1 (fr) 2012-06-19 2013-12-27 E. I. Du Pont De Nemours And Company Production améliorée d'acides gras polyinsaturés par coexpression d'acyl-coa:lysophosphatidylcholine acyltransférases et de phospholipide:diacylglycérol acyltransférases
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AU2003259269A1 (en) * 2002-07-25 2004-02-16 Diomeda Life Sciences, Inc. Oncogenes useful for the identification of urinary bladder cancer
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US8461417B2 (en) 2008-05-21 2013-06-11 National Research Council Of Canada Reduction of lyso-phosphatidylcholine acyltransferase activity
WO2009140770A1 (fr) * 2008-05-21 2009-11-26 National Research Council Of Cananda Réduction de l'activité de la lyso-phosphatidylcholine acyltransférase
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EP2412804A4 (fr) * 2009-03-26 2012-09-12 Suntory Holdings Ltd Nouvelle lysophospholipide acyltransférase
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AU2014203166B2 (en) * 2009-03-26 2015-09-17 Suntory Holdings Limited Novel lysophospholipid acyltransferase
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CN102459627A (zh) * 2009-06-16 2012-05-16 纳幕尔杜邦公司 通过酰基-辅酶A溶血磷脂酰基转移酶的表达对长链ω-3和ω-6多不饱和脂肪酸的生物合成的改善
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WO2013192007A1 (fr) 2012-06-19 2013-12-27 E. I. Du Pont De Nemours And Company Acyl-coa:lysophosphatidylcholine acyltransférases mutantes
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JP2015519921A (ja) * 2012-06-19 2015-07-16 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニーＥ．Ｉ．Ｄｕ Ｐｏｎｔ Ｄｅ Ｎｅｍｏｕｒｓ Ａｎｄ Ｃｏｍｐａｎｙ 突然変異アシルＣｏＡ：リゾホスファチジルコリンアシルトランスフェラーゼ
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