Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
  • C12N9/1007—Methyltransferases (general) (2.1.1.)

Definitions

• Methyltransferases nucleic acid molecules encoding methyltransferases, their recombinant expression and uses thereof
• the present invention relates to proteins which are capable of functioning as methyltransferases. More, specifically, the present invention relates to methyltransferases which are capable of carrying out at least one of the following reactions : the conversion of glycine to sarcosine (N- methylglycine) , sarcosine to N,N-dimethyl glycine and N,N- dimethyl glycine to betaine (N,N,N-trimethylglycine) in the presence of a methyl group donor. Furthermore, the present invention relates to nucleic acid molecules encoding such methyltransferase proteins, recombinant organisms which are capable of expressing said nucleic acids as well as the use of said recombinant organisms .
• Betaine N,N,N-trimethylglycine
• Betaine is synthesized or accumulated in living cells in response to abiotic stress (salinity, desiccation or low temperatures) (Mc Gue and Hanson, 1990; Csonka, 1989; Yancey et al . , 1982; Wyn Jones et al . , 1977; Gorham, 1995; Bohnert and Jensen, 1996) . Due to its physical properties, betaine is an osmolyte and thus it is able to restore and maintain osmotic balance of living cells. In addition, it has been demonstrated that betaine stabilizes and protects cell membranes (Coughlan and Heber, 1982) and other macromolecules (i.e. enzymes) in the cell (Papagiorgiou and Murata, 1995) .
• Betaine is synthesized by a number of microbes.
• these microbes are usually capable of accumulating betaine or a precursor, choline, from the culture medium (Boch et al., 1994; Perroud and Rudulier, 1985; Kempf and Bremer, 1995; Glaasker et al., 1996; Peter et al . , 1996; and Kappes et al., 1996) .
• Practically all halophilic bacteria are able to use betaine as at least one of their osmolytes in order to survive in the high ionic strength environment .
• betaine In animal cells, betaine also acts as a methyl group donor. The most important function is to the ability to methylate homocysteine back to methionine, which can then further be used as a methyl group donor when metabolized to S-adenosyl methionine (SAM) . It has been demonstrated that orally administered betaine relieves diarrhea and dehydration in many animals and inhibits invasion of gut epithelium by coccidia parasite (Ferket, 1994; Augustine and McNaughton, 1996) . As a methyl group donor betaine has been shown to be lipotropic, thus decreasing the amount of fat in chicken meat (Saunderson and MacKinlay, 1990; Barak et al . , 1993) .
• the most extensively studied betaine biosynthesis pathway is the two-step oxidation reaction of choline to betaine via betaine aldehyde. This metabolic pathway has been demonstrated to exist in number of microbes, plants and animal cells.
• the choline-betaine pathway of E. coli (La ark et al., 1991) and Pseudomonas aeruginosa (Nagasawa et al . , 1976) comprises an oxygen-dependent choline dehydrogenase, which catalyzes the oxidation of both choline to betaine aldehyde and betaine aldehyde to betaine.
• the E. coli choline dehydrogenase gene has been cloned and sequenced.
• oxidation of choline to betaine can be catalyzed by a choline oxidase found for example in some Corynebacteria, Brevibacterium and Alcaligenes species
• the second oxidation step from betaine aldehyde to betaine may also be catalyzed by a betaine aldehyde dehydrogenase.
• a betaine aldehyde dehydrogenase has also been found in a number of organisms (Pseudomonas aeruginosa (30) ) . Also some plants have this enzyme (Hanson et al., 1985) . In plants, the enzyme has been demonstrated to have wider substrate specificity and thus it also catalyzes other reactions (Trossat et al . , 1997) . The gene has been cloned from E.
• E. coli (Lamark et al., 1991), spinach (Weretilnyk and Hanson, 1990) and also from barley (Ishitani et al . , 1995) .
• the E. coli gene has also successfully been expressed in transgenic tobacco (Holmstr ⁇ m et al . , 1994).
• Betaine is used as feed additive in feed industry.
• transgenic plants producing high amounts of betaine in vivo would have better nutritional value.
• Feed crops e.g. maize or soybean
• betaine could therefore directly be used in feed without the need of betaine supplementation.
• betaine is synthesized by many plants, there are several commercially important crops such as potato, rice, tomato and tobacco which do not accumulate betaine.
• Bulow and co-workers (1995) were the first to demonstrate that expression of the E. coli choline dehydrogenase in tobacco improves the salt tolerance and freezing tolerance (Holmberg, 1996) of transgenic potato and tobacco due to endogenously synthesized betaine.
• Arabidopsis Hidet al., 1997)
• rice Neakamura et at., 1997; Guo, 1997) . Therefore, expression of the methyltransferases in plants can facilitate stress tolerance and improve the productivity of the plants when grown under conditions of water stress or freezing and cold temperatures.
• betaine has shown to induce pathogenesis-related protein expression in plants (Xin et al . , 1996) as well as increasing the resistance of plants to attack by pathogenic fungi or nematodes (Blunden et al . , 1996; Wu et al . ,1997) and may decrease the incidence of nematode (e.g. Meloidogne javanica and M. incognita) attack in plants. Therefore, transgenic plants producing endogenous betaine, can be more resistant to fungal pathogens. Moreover, the betaine synthesis in the plants may be coupled to the systemic resistance genes which are induced when the plant is attacked by pathogens .
• Endogenously synthesized betaine may also affect the viability of microbes and therefore it would improve their performance in various biotechnical processes. For instance, in high cell density fermentation or immobilized cell systems, the production microbes are subjected to considerable environmental stress. Betaine has successfully been used in fermentation media to increase the product yield in amino acid production. For instance, betaine has shown to relieve stress and improve yield of lysine producing Brevibacterium lactofermentum (Kawahara et al . , 1990). Betaine is also commercially sold for the purpose (Nutristim®, Cultor Corp) . Thus, endogenously synthesized betaine can improve productivity in biotechnical processes where the cells are subjected to abiotic stress.
• Microbes also suffer from stress when subjected to high temperatures or when cells are freeze-dried or frozen.
• the viability of yeast or bacterial cells may be dramatically reduced in these processes used in e.g. frozen dough manufacturing or preservation of lactic acid bacterium starters. Therefore, it would be highly advantageous if one could improve the viability of microbes subjected to freeze-thaw or freeze-drying processes by accumulating betaine inside the cells.
• exogenously applied betaine improves the viability of microbes in extreme pH (Smirnova and Oktyabrsky, 1995; Chambers and Kunin, 1985) .
• Improved performance of beneficial, probiotic microbes organisms in animal digestive tract can be utilized in animal nutrition.
• introduction of a betaine synthesis pathway can improve the stress tolerance of "probiotic" lactic acid microbes which efficiently bind to gut epithelium in cells, providing a way to balance the microbial population in the GI tract and to improve pathogen resistance.
• Betaine has been shown to stabilize proteins in the cells .
• cytoplasmic accumulation of betaine will reduce the formation of inclusion bodies (Blackwell and Horgan, 1991; Bhandari and Gowrishankar, 1997) which is a problem often encountered when heterologous proteins are expressed in E. coli.
• co-expression of the genes of betaine biosynthesis with the protein of interest should result in better solubility of the heterologous protein and reduce the amount of inclusion bodies .
• the use of the glycine methylation pathway may have a number of advantages over the oxidative synthesis from choline.
• Glycine is synthesized by practically all organisms and as an amino acid, this metabolite is present in high concentrations in the cells.
• the availability of intracellular choline may limit betaine biosynthesis.
• the metabolism and formation of glycine in cells is known and the genes of this metabolic pathway have been cloned, thus allowing the engineering of the glycine pathway.
• an object of the present invention is to provide proteins which are capable of acting in a biosynthetic pathway from glycine to betaine as well as methods for the purification and production of said proteins.
• a further object of the present invention is to provide nucleic acid molecules which, when transformed into a host organism, encode proteins which are capable of acting in a biosynthetic pathway from glycine to betaine.
• a further object is to provide recombinant microorganisms which are capable of expressing one or more proteins which are capable of acting in a biosynthetic pathway from glycine to betaine for the production of betaine and precursors thereof .
• an object of the present invention is to provide recombinant plants which express one or more proteins which are capable of acting in a biosynthetic pathway from glycine to betaine for the production of betaine and precursors thereof .
• an object of the present invention is to provide a method for the production of betaine and precursors thereof, for example sarcosine and dimethyl glycine, in recombinant organisms .
• a further object is to provide recombinant organisms which have an increased concentration of intracellular betaine and are useful in the fields of recombinant heterologous protein production, agriculture, etc.
• a further object of the present invention is to provide nucleic acid probes and method for identifying and cloning genes which encode proteins which are capable of participating in a biosynthetic pathway from glycine to betaine.
• a further object of the present invention is to provide methods for improving the general growth and/or productivity of an organism including enhancing stress tolerance, for example, salt tolerance, freezing tolerance and cold tolerance, enhancing resistance to drought, water stress and attack by pathogens in organisms.
• stress tolerance for example, salt tolerance, freezing tolerance and cold tolerance
• an object of the present invention is to provide recombinant microorganisms which have improved viability in culture, enhanced pH tolerance in culture, result in decreased inclusion body formation when expressing a heterologous protein, result in increased solubility, stability and/or yield of a heterologous protein expressed in said organism.
• the inventors have identified, isolated and purified proteins which are capable of carrying out at least one of the following reactions in a metabolic pathway from glycine to betaine: the conversion of glycine to sarcosine (N- methylglycine) , sarcosine to N,N-dimethyl glycine and N,N- dimethyl glycine to betaine (N,N,N-trimethylglycine) in the presence of a methyl group donor.
• These proteins are designated herein as methyltransferases based on their ability to transfer a methyl group from a methyl group donor to a methyl group acceptor.
• the above mentioned reactions are individual steps in a three-step methylation reaction pathway of glycine to betaine in certain microorganisms, for example, Ectothiorhodospira halochloris and Actinopolyspora halophila.
• a methyltransferase of the present invention (designated hereinafter as glycine-sarcosine methyltransferase or GSMT) has been isolated from Ectothiorhodospira halochloris and Actinopolyspora halophila which is capable of catalyzing methylation reactions that convert glycine to dimethyl glycine, i.e. convert glycine to sarcosine (N-methyl glycine) and sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
• a methyltransferase according to the present invention (designated hereinafter as sarcosine- dimethylglycine methyltransferase or SDMT) has been isolated from Ectothiorhodospira halochloris and Actinopolyspora halophila which is capable of catalyzing methylation reactions that convert sarcosine to betaine, i.e. convert sarcosine (N-methyl glycine) to dimethyl glycine (N,N- dimethyl glycine) and dimethyl glycine to betaine.
• GSMT glycine-sarcosine me- methyltransferase lycine
• the methyltransferases of the present invention are capable of utilizing S-adenosyl methionine (hereinafter also referred to as SAM) as a methyl group donor in the above reactions.
• SAM S-adenosyl methionine
• Figure 1 Formation of methylation products from glycine, sarcosine and dimethyl glycine substrates using the A . halophila cell extract. The retention times of the standards are shown by arrows .
• FIG. 1 Analysis of purified methyl transferases on SDS- PAGE.
• Figure 3 The determination of the isoelectric point of A . halophila SDMT by isoelectric focusing.
• FIG. 5 The temperature dependence of A. halophila SDMT activity.
• Figure 6 In vi tro synthesis of betaine by using the purified E. halochloris GSMT and A. halophila SDMT enzymes. The retention times of the standards are shown by arrows.
• Figure 7 The schematic structure of the betaine operons of A . halophila and E. halochloris . GSMT; glycine sarcosine methyltransferase. SDMT; sarcosine dimethyl glycine methyltransferase. SAMS; S-adenosyl methionine synthase.
• Figure 8 The nucleotide and amino acid sequence of the E. halochloris betaine operon.
• the arrows indicate the amino acids encoding GSMT, SDMT and SAMS.
• the underlined regions indicate regions which are hybridized with the primers used to construct the expression vectors in heterologous organisms.
• the * indicates a stop codon.
• Figure 9 The nucleotide and amino acid sequence of the A . halophila betaine operon.
• the underlined regions indicate regions which are hybridized with the primers used to construct the expression vectors in heterologous organisms.
• the * indicates a stop codon.
• Figure 10 Schematic presentation of the expression plasmid used in expression of the methyl transferases.
• the insert was ligated to vector digested with Ncol/Bgrlll .
• FIG. 11 The growth curves of E. coli transformants carrying the E. halochloris GSMT gene (EGSM) .
• Transformant carrying only the cloning vector (PQE-60) was used as the control .
• FIG. 12 The growth curves of E. coli transformants carrying the E. halochloris GSMT and SDMT genes (EhFU) . Transformant carrying only the cloning vector (PQE-60) was used as the control.
• a methyltransferase for example GSMT, capable of catalyzing the conversion of glycine to sarcosine ( ⁇ -methyl glycine) and/or the conversion of sarcosine to dimethyl glycine ( ⁇ , ⁇ - dimethyl glycine) .
• said methyltransferase comprises an amino acid sequence selected from the group consisting of:
• a fragment of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO : 6 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO: 6 which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
• a derivative of an amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO: 6 is designated as any mutation, deletion, addition, substitution, insertion or inversion of one or more amino acids in the of amino acid sequence as depicted in SEQ ID NO: 2 or SEQ ID NO : 6 which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
• a derivative of a methyltransferase which is capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO : 2 or SEQ ID NO: 6.
• the amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e. Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys , Arg and His.
• the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids , Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids .
• a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 2.
• a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 6.
• the methyltransferase of the present invention capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) can exist in the form of an active enzyme or as a zymogen.
• the term 'zymogen' designates a protein molecule or fragment or derivative thereof (as defined above) which is synthesized in an inactive form and is capable of being activated in vitro or in vivo by the chemical or enzymatic cleavage of one or more peptide bonds .
• a preferred zymogen of the methyltransferase of the present invention capable of catalyzing the conversion of glycine to sarcosine (N-methyl glycine) and/or the conversion of sarcosine to dimethyl glycine (N,N-dimethyl glycine) comprises the amino acid sequence as depicted in SEQ ID NO: 2 and SEQ ID NO: 3, wherein the N-terminus of SEQ ID NO: 3 is joined to the C- erminus of SEQ ID NO: 2.
• a further embodiment of the present invention provides a methyltransferase, for example SDMT, capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine (N,N-dimethyl glycine) and/or dimethyl glycine to betaine.
• a methyltransferase for example SDMT, capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine (N,N-dimethyl glycine) and/or dimethyl glycine to betaine.
• said methyltransferase comprises an amino acid sequence selected from the group consisting of :
• a fragment of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 which is capable of catalyzing the conversion of sarcosine (N- methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine to betaine.
• a derivative of an amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 is designated as any mutation, deletion,- addition, substitution, insertion or inversion of one or more amino acids, or combination thereof, in the of amino acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 7 which is capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine.
• a derivative of a methyltransferase which is capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO: 3 or SEQ ID NO: 7.
• amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e. Ala, Val, Leu, lie, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu, Lys, Arg and His.
• the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids, Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids .
• a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 3.
• a methyltransferase according to the invention has the amino acid sequence depicted in SEQ ID NO: 7.
• the methyltransferase of the present invention capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine can exist in the form of an active enzyme or as a zymogen.
• the term 'zymogen' designates a protein molecule, fragment or derivative thereof (as defined above) which is synthesized in an inactive form and is capable of being activated in vitro or in vivo by the chemical or enzymatic cleavage of one or more peptide bonds .
• a preferred zymogen of the methyltransferase of the present invention capable of catalyzing the conversion of sarcosine (N-methyl glycine) to dimethyl glycine and/or the conversion of dimethyl glycine (N,N-dimethyl glycine) to betaine comprises the amino acid sequence as depicted in SEQ ID NO: 2 and SEQ ID NO: 3, wherein the N-terminus of SEQ ID NO: 3 is joined to the C-terminus of SEQ ID N0:2.
• the methyltransferases of the present invention for example naturally occurring GSMT and SDMT or recombinantly produced GSMT and SDMT and fragments or derivatives thereof as well as zymogens of these naturally occurring or recombinant proteins, are preferably isolated to a state free from other proteins originating from the organisms from which they are isolated; more preferably, to a pure state, most preferably to a homogeneous state .
• SAM S-adenosyl methionine
• SAMS S-adenosyl methionine synthase
• said SAMS comprises an amino acid sequence selected from the group consisting of:
• a fragment of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO : 8 is designated as any fragment of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 8 which is capable of catalyzing the conversion of S-adenosyl methionine from methionine and ATP (adenosine triphosphate) .
• a derivative of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO : 8 is designated as any mutation, deletion, addition, substitution, insertion or inversion of one or more amino acids, or combination thereof, in the of amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 8 which is capable of catalyzing the conversion of S-adenosyl methionine from methionine and ATP.
• a derivative which is capable of catalyzing the conversion of methionine to S- adenosyl methionine has about 60 % homology, preferably about 70 % homology, more preferably about 80 % homology, and most preferably about 90 % homology to the corresponding amino acid sequence depicted in SEQ ID NO: 4 or SEQ ID NO: 8.
• the amino acids constituting these changes are selected from the 20 standard naturally-occurring amino acids found in proteins, i.e.
• the mutation (s) and/or substitution (s) are conservative, for example, Ala, Val, Leu, lie, Pro, Phe, Trp, or Met residue (s) are replaced with one of these amino acids, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, residue (s) are replaced with one of these amino acids, Asp or Glu are replaced with one of these amino acids and Lys, Arg or His are replaced with one of these amino acids .
• the SAMS of the invention has the amino acid sequence depicted in SEQ ID NO: 4.
• the SAMS of the invention has the amino acid sequence depicted in SEQ ID NO: 8.
• the SAMS of the present invention for example naturally occurring SAMS or recombinantly produced SAMS and fragments or derivatives thereof, are preferably isolated to a state free from other proteins originating from the organisms from which they are isolated; more preferably, to a pure state, most preferably to a homogeneous state.
• the present invention also relates to a nucleic acid molecule which is capable of encoding a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
• a nucleic acid molecule which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) comprises a nucleotide sequence selected from the group consisting of:
• a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO: 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
• the term 'standard conditions' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989) .
• the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background.
• plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment of the above gene of interest .
• the probe can be prepared for example by PCR as described in Example 6.
• Hybridization can be carried out at 42°C in 50 mM Tris-HCl, pH 7.5 , 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989) , 100 ⁇ g herring sperm DNA and 125 ⁇ g/ml polyA.
• the filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989) . The filters can then be exposed to x-ray film to monitor the number of positive clones .
• the washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained.
• the positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example.
• the cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
• a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO: 1 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence comprising nucleotide 208 to 1047 of SEQ ID NO:l or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5.
• a fragment of a nucleotide sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 or a nucleotide sequence from nucleotide 221 to 1024 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N-dimethyl glycine) .
• a preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l.
• a further preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO : 5
• nucleic acid molecules according to the invention comprise the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 or the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l, the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 being more preferred.
• the present invention also relates to a nucleic acid molecule which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine.
• a nucleic acid molecule which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine comprises a nucleotide sequence selected from the group consisting of:
• a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO: 1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 and encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine.
• any nucleotide sequence for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 1048 to 1902 of S
• the term 'standard conditions' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989). Basically, the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background. For example, plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment, of the above gene of interest .
• the probe can be prepared for example by PCR as described in Example 6.
• Hybridization can be carried out at 42°C in 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989) , 100 ⁇ g herring sperm DNA and 125 ⁇ g/ml polyA.
• the filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989). The filters can then be exposed to x-ray film to monitor the number of positive clones .
• the washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained.
• the positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example.
• the cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
• a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 1048 to 1902 of SEQ ID N0:1 or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l or a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
• a fragment of a nucleotide sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or a nucleotide sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine to glycine betaine.
• a preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l.
• a further preferred nucleic acid molecule according to the invention comprises the DNA sequence from 1031 to 1867 of SEQ ID NO:5.
• nucleic acid molecules according to the invention comprise the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO : 5 or the DNA sequence from nucleotide 208 to 1902 of SEQ ID NO:l, the DNA sequence from nucleotide 221 to 1867 of SEQ ID NO: 5 being more preferred.
• a further aspect of the present invention provides a nucleic acid molecule which encodes an enzyme capable of converting S-adenosyl methionine from methionine and ATP.
• a nucleic acid molecule which encodes an enzyme capable of converting S-adenosyl methionine from methionine and ATP comprises a nucleotide sequence selected from the group consisting of:
• a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 is designated as any nucleotide sequence, for example DNA or RNA, preferably DNA, which hybridizes under standard conditions to a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 and encodes enzyme capable of converting S-adenosyl methionine from methionine and ATP.
• the term ' standard conditions ' designates a standard procedure used in heterologous hybridization to screen for genes with enough sequence homology (Maniatis, 1989) .
• the hybridization filters are washed first in low stringency conditions and the stringency is gradually increased (usually by increasing the washing temperature) in order to select the positive signals from the background.
• plaques or colonies of a gene bank to be screened can be transferred onto nitrocellulose membranes and hybridized with a PCR fragment, oligonucleotide or any other cloned DNA containing a fragment of the above gene of interest .
• the probe can be prepared for example by PCR as described in Example 6.
• Hybridization can be carried out at 42 °C in 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 M NaCI, 0.5% SDS, 0.1% sodium pyrophosphate, lOx Denhardt ' s solution (Maniatis, 1989), 100 ⁇ g herring sperm DNA and 125 ⁇ g/ml polyA.
• the filters can be first washed with low stringency conditions, for example, at 37°C using 3x SSC, 0.5% SDS and 10% sodium pyrophosphate (Maniatis, 1989) . The filters can then be exposed to x-ray film to monitor the number of positive clones.
• the washing temperature will be raised in 2-5°C intervals up to a temperature at which the background becomes invisible and only a few positive plaques are obtained.
• the positive plaques or colonies can then be purified and the DNA can be isolated for Southern blot hybridization to check the size of the cloned insert for example .
• the cloned DNA obtained can be sequenced. On the basis of the sequence homology, it can be concluded that the DNA contains the gene of interest .
• a nucleotide sequence which hybridizes to a nucleic acid molecule comprising a DNA sequence from nucleotide 2027 to 2722 of SEQ ID N0:1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5 has about 60 %, preferably 70 %, more preferably 80 % and especially 90 % homology to a nucleotide sequence corresponding to a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO: 5.
• a fragment of a nucleotide sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 is designated as any nucleic acid fragment, for example DNA or RNA, preferably DNA, which encodes enzyme capable of converting S-adenosyl methionine from methionine and ATP.
• a preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO:l.
• a further preferred nucleic acid molecule according to the invention comprises the DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5.
• DNA probe for use in identifying and cloning a nucleic acid molecule encoding a methyltransferase comprising at least 15 nucleotide bases, preferably 20 or more nucleotide bases, of a nucleotide sequence selected from the group consisting of : (a) a DNA sequence from nucleotide 208 to 1047 of SEQ ID NO:l, a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5, a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l, a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5.
• Said DNA probes can be utilized in a method according to the invention for identifying and cloning a nucleic acid molecule encoding a methyltransferase comprising the steps of hybridizing said probe with a sample containing nucleic acid of an organism, detecting a nucleic acid molecule in said sample which hybridizes to said probe and isolating said detected nucleic acid molecule.
• Preferred methods include the use of the polymerase chain reaction (PCR) and Southern blotting techniques which are described herein and are familiar to the skilled person in the art.
• vectors for expression of the proteins according to the invention in prokaryotic and eukaryotic hosts.
• expression vectors for example phages, plasmids and DNA or RNA viruses, are capable of transforming and/or replicating and expressing the proteins of the present invention in prokaryotes and/or eukaryotes, for example bacteria, yeast, fungi and/or plants.
• Such expression vectors and methods for their construction are known to the skilled person and can be provided with nucleic acid elements for transcription, for example start codons, 'TATA' boxes, promoters, enhancers, stop codons, etc., and nucleic acid elements important for translation and processing of the nucleic acids transcribed from said vectors in a given host, for example ribosome binding sites, leader sequences for secretion of the proteins of the present invention, etc.
• One embodiment of the invention is an expression vector comprising a nucleic acid sequence which encodes a methyltransferase capable of converting glycine to sarcosine (N-methyl glycine) and/or sarcosine to dimethyl glycine (N,N- dimethyl glycine) and/or a nucleic acid sequence which encodes a methyltransferase capable of converting sarcosine (N-methyl glycine) to dimethyl glycine and/or dimethyl glycine (N,N-dimethyl glycine) to betaine.
• said expression vector comprises at least one nucleotide sequence selected from the group consisting of:
• an expression vector comprising a nucleotide sequence coding for an enzyme capable of catalyzing the synthesis of S-adenosyl methionine and at least one nucleotide sequence selected from the group consisting of:
• preferred expression vectors comprise a nucleotide sequence selected from the group consisting of:
• preferred expression vectors comprise the DNA sequence from nucleotide 208 to 1047 of SEQ ID NO : 1 and the DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO : 1 or the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5, an expression vector comprising the DNA sequence from nucleotide 221 to 1024 of SEQ ID NO : 5 and the DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO : 5 being more preferred.
• expression vectors comprising fragments and/or derivatives of the above mentioned sequences as well as other combinations of the above mentioned DNA sequences, for example a DNA sequence from nucleotide 208 to 1047 of SEQ ID N0:1 and a DNA sequence from nucleotide 1031 to 1867 of SEQ ID NO: 5 or a DNA sequence from nucleotide 221 to 1024 of SEQ ID NO: 5 and a DNA sequence from nucleotide 1048 to 1902 of SEQ ID NO:l or fragments and/or derivatives thereof are also subject matter of the present invention.
• expression vectors of the present invention can additionally comprise a nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of S-adenosyl methionine.
• E. halochloris and A. halophila GSMT and SDMT enzymes are located in a "betaine operon".
• E. halochloris the enzymes are encoded by two separate genes, whereas in A. halophila the two enzymes are coded by a single gene.
• the "betaine operon” contains a S-adenosyl methionine synthase (SAMS) gene.
• SAMS enzyme catalyzes the synthesis of S-adenosyl methionine (SAM) from methionine and ATP, and thus, it is useful in the methylation reactions of the methyltransferases of the invention because it increases the concentration of the enzyme substrate SAM. Therefore co-expression of the SAMS gene with one or more of the methyltransferase genes of the invention can be used to increase betaine synthesis in these organisms.
• the above mentioned expression vectors can additionally comprise a DNA sequence from nucleotide 2027 to 2722 of SEQ ID NO : 1 or a DNA sequence from nucleotide 2006 to 3004 of SEQ ID NO : 5 or fragments and/or derivatives thereof when the organism to be transformed is E halochloris or A. halophila.
• Preferred expression vectors of this type which also encode the methyltransferases of the present invention, comprise a DNA sequence from nucleotide 208 to 2722 of SEQ ID NO:l or a DNA sequence from nucleotide 221 to 3004 of SEQ ID NO: 5, the latter being more preferred.
• said nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of S-adenosyl methionine originates or is derived from the organism which is to be transformed with said expression vector.
• the organism to be transformed with a nucleic acid, for example an expression vector, according to the invention is E. coli
• nucleic acid for example an expression vector
• the organism to be transformed with a nucleic acid for example an expression vector
• a nucleic acid for example an expression vector
• Other nucleotide sequences which can be used for this purpose are the SAH hydrolase form Mesembryanthemum crystallinum (genebank accession number U79766; Arabidopsis thaliana, accession number AF059581; S. pombe, accession number AL022072) .
• the expression vectors can additionally comprise a nucleic acid molecule coding for an enzyme capable of increasing the intracellular amount of intracellular glycine.
• said nucleic acid molecule coding for an enzyme capable of directly or indirectly increasing the intracellular amount of glycine originates or is derived from the organism which is to be transformed with said expression vector.
• the expression vector can include a nucleotide sequence encoding the enzyme phosphoglycerate dehydrogenase (Bacillus subtilis, accession number L47648; S. pombe, accession number AL022243; Arabidopsis thaliana, accession number AB010407) , phosphoserine aminotransferase (E. coli, accession number AE000193, U00096; Bacillus subtilis, accession number Z99109, AL009126; S. pombe, accession number Z69944; Arabidopsis thaliana, accession number AL031135) , phosphoserine phosphatase (E. coli, accession number AE000509; S.
• phosphoglycerate dehydrogenase Bacillus subtilis, accession number L47648; S. pombe, accession number AL022243; Arabidopsis thaliana, accession number AB010407
• Further subject matter of the present invention is a recombinant prokaryotic or eukaryotic organism, for example, bacteria, yeast, fungus or plant, transformed with at least one nucleic acid molecule of the invention as defined above, for example, an expression vector according to the invention as defined above.
• a recombinant organism according to the invention is a bacterium
• said bacterium is preferably selected from the group consisting of E. coli, Bacillus, Corynebacteria, Pseudomonas and lactic acid bacteria and Streptomyces .
• yeast is preferably selected from the group consisting of Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula.
• the recombinant organism according to the invention is a fungus
• said fungus is preferably selected from the group consisting of Aspergillus, Trichoderma and Penicillium.
• the recombinant organism according to the invention is a plant including but not limited to cereals, legumes, oilseeds, vegetables, fruits, ornamentals and perennials
• said plant is preferably selected from the group consisting of lettuces, Capsicums, grasses, clovers, alfalfa, beans, sweet potatoes, cassava, yams, taro, groundnut, brassica, sugar beet, grapes, potato, tomato, rice, tobacco, rapeseed, maize, sorghum, cotton, soybean, barley, wheat, rye, canola, sunflower, linseed, pea, cucumber, carrot, ornamentals, perennial trees including citrus pear and almond and fruits including strawberry.
• plant' according to the invention is understood to include individual cells of a plant, plant seeds and callus material.
• Further subject matter of the present invention is a method for the production of a recombinant organism according to the invention comprising the steps of transforming a host prokaryotic or eukaryotic organism, preferably a bacteria, yeast or fungus, with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
• a host prokaryotic or eukaryotic organism preferably a bacteria, yeast or fungus
• this nucleic acid for example an expression vector
• this nucleic acid for example an expression vector
• transformation can be performed using these two nucleic acid molecules, e.g. expression vectors.
• the present invention relates to a methyltransferase obtainable by culturing wild- ype Ectothiorhodospira or Actinopolyspora or a recombinant prokaryotic or eukaryotic organism according to the invention and isolating said methyltransferase from the organism and/or the medium used to culture or process said organism as well as a method for the production of said methyltransferase comprising the above mentioned steps .
• a method for the purification of a methyltransferase capable of catalyzing the conversion of glycine to dimethyl glycine comprising the steps of subjecting a sample comprising the methyltransferase to a matrix containing adenosine, binding said methyltransferase to said matrix and eluting said methyltransferase from said matrix is also subject matter of the present invention.
• the above purification step can be combined with other methods of protein purification including ammonium sulfate precipitation, size exclusion chromatography, cation or anion exchange chromatography, hydrophobic interaction chromatography, etc.
• Suitable host organisms are practically all bacteria which can be transformed with foreign DNA (for instance E. coli, Bacillus, Corynebacteria, Pseudomonas, lactic acid bacteria and Streptomyces) yeast (for instance Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula) fungi (for instance Trichoderma, Aspergillus, Penicillium) or plants.
• foreign DNA for instance E. coli, Bacillus, Corynebacteria, Pseudomonas, lactic acid bacteria and Streptomyces
• yeast for instance Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida and Hansenula
• fungi for instance Trichoderma, Aspergillus, Penicillium
• subject matter of the present invention is a method for the production of betaine comprising the steps of culturing a recombinant organism according to the invention and isolating betaine from the organism and/or the medium used to culture or process said organism.
• sarcosine and/or dimethyl glycine comprising the steps of culturing an organism transformed with a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
• nucleotide sequence which is degenerate as a result of the genetic code to any of the nucleotide sequences defined in (a) , (b) and (c) , and isolating sarcosine and/or dimethyl glycine from said organism or the medium used to culture or process said organism.
• Further subject matter of the invention is a method for increasing the intracellular concentration of sarcosine, dimethyl glycine and/or betaine in an organism, enhancing the general productivity of an organism, enhancing the salt tolerance of an organism, enhancing the freezing or cold tolerance of an organism, and/or enhancing the resistance of an organism to drought and/or low water stress comprising the steps of transforming an organism with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
• said organism is a bacteria, yeast, fungus or plant as recited above .
• Further subject matter of the invention is a method for inducing pathogenesis-related proteins in a plant, increasing the resistance of a plant to attack by pathogens and/or increasing the nutritional value of a plant comprising the steps of transforming a plant with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
• said plant is a plant as recited above.
• Pathogens include but are not limited to Fusarium sp. which cases root, shoot and leaf diseases in several plant types, Rhizoctonia ⁇ p. and Pythium sp. which cause soil borne diseases in crops, Erysiphe sp. which cause mildew in several species, Phytophthora infestans which causes late blight in potato and tomato, Alternaria solani which causes early blight in potato, fungal diseases of soya caused by Cephalosporium sp., Diaporthe sp., Cerospora sp. Septoria ⁇ p. and Peronospora sp., nematodes for example Meloidogne javanica and M. incognita and insects.
• subject matter of the invention is a method for enhancing the pH tolerance and/or viability of a cultured microorganism comprising the steps of transforming a microorganism with at least one nucleic acid molecule of the invention as defined above, for example an expression vector according to the invention as defined above.
• said microorganism is a bacteria, yeast or fungus as recited above .
• microorganisms of the invention can also be used as hosts in the field of recombinant DNA technology for the expression of a heterologous protein of interest. Therefore, subject matter of the invention is a method for decreasing inclusion body formation, increasing the stability of a heterologous protein and/or increasing the production of a heterologous protein expressed in a microorganism comprising the step ⁇ of transforming a microorganism with at least one nucleic acid molecule of the invention as defined above, for example an expres ⁇ ion vector according to the invention a ⁇ defined above, and tran ⁇ forming a microorganism with a nucleic acid molecule capable of expres ⁇ ing ⁇ aid heterologou ⁇ protein. Said microorganism can be transformed with the nucleic acid molecule, for example an expression vector, according to the invention before, during or after the microorganism is transformed with a nucleic acid molecule capable of expressing said heterologous protein.
• the growth medium of Actinopolyspora halophila ATCC 27976 used in all cultivations was the "complex medium" described by Sehgal and Gibbons (1960) . Inoculum was grown at 37°C in a shake flask with agitation at 180 rpm until the late exponential growth phase. Then, 8 1 of the above medium with 10 g/l glucose was inoculated with 800 ml culture.
• the pH in the fermentor (Biostat M (Braun) laboratory fermentor) wa ⁇ maintained at pH 6.5-7.5 with 0.5 M H 2 S0 4 and 1 M NaOH. Agitation and aeration rates were 400 rpm and 10 1/min., respectively.
• the cultivation temperature was 37°C.
• Cells were grown to late exponential phase and harve ⁇ ted by centrifugation at 15,000 g for 15 min. Cell ⁇ were stored at -75°C. Before disruption, the cells were thawed and suspended in Buffer I (22 % (w/v) sucrose, 27 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) , 50 mM Tris-HCl, pH 7.5) in a ratio of 1.5 ml buffer 1:1 g cells (wet weight) .
• Buffer I 22 % (w/v) sucrose, 27 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) , 50 mM Tris-HCl, pH 7.5
• the growth medium used in the cultivation of Ectothiorhodo ⁇ pira halochlori ⁇ ATCC 35916 is described by Tschichholz and Tr ⁇ per (1990) . Cultivation was carried out anaerobically at 42°C in 1 1 glas ⁇ bottle ⁇ with continuou ⁇ ⁇ tirring with magnetic stirrer. The cell ⁇ were illuminated during growth (5,000 - 10,000 lux). 100 ml of pre-inoculum wa ⁇ inoculated into 1 1 medium. Cell ⁇ were grown until late exponential pha ⁇ e and harve ⁇ ted by centrifugation at 28,000 g for 20 min.
• Buffer II (560 mM Tris-HCl, pH 7.5, 4 mM 2-mercaptoethanol, 50 ⁇ M MgCl2, 160 ⁇ M EDTA) and disrupted with 1 mM PMSF and 1 mM dithiothreitol (DTT) .
• Buffer II was added in a ratio of 1.5 ml buffer II: 1 g cells (wet weight) .
• the cells were disrupted with a MSE Soniprep 150 sonicator.
• the suspension of A. halophila cells wa ⁇ ⁇ onicated in 20 ml batches (sonication pulses 30 s, cooling intervals 2 min) for 1 min / 2 ml cell suspension.
• the suspension of E. halochloris cells was sonicated in 5 ml batches (sonication pulse ⁇ 15 s; cooling intervals 2 min) for 1 min/1.5 ml cell su ⁇ pension.
• the cell debris was removed by centrifugation at 28,000 g at 1°C for 30 min.
• the cell free extracts were stored at -75°C.
• reaction mixture contained 25 ⁇ l of 0.1 M substrate (glycine, sarcosine or dimethylglycine) , 25 ⁇ l of Buffer II (see above) , 25 ⁇ l 4 mM S-adenosyl-L-methionine containing 45 nCi S-adenosyl-L- [methyl- 14 C] methionine (Amersham) in 1/10 Mcllvaine buffer (pH 3.0), and 25 ⁇ l enzyme sample (e.g. cell free extract) . The reaction was initiated by adding the enzyme. The reaction mixture was incubated for 30 min.
• substrate glycine, sarcosine or dimethylglycine
• Buffer II see above
• 25 ⁇ l 4 mM S-adenosyl-L-methionine containing 45 nCi S-adenosyl-L- [methyl- 14 C] methionine Amersham
• 25 ⁇ l enzyme sample e.g. cell free extract
• the cell extracts typically contain the following activities. Table 1. Methyltran ⁇ fera ⁇ e activitie ⁇ of A. halophila and E. halochloris cell extracts on different substrates.
• the reaction products were characterized by HPLC.
• the reaction mixture supernatants a ⁇ de ⁇ cribed above were filtered after centrifugation through a Mini ⁇ art NML 0.2 ⁇ m filter (Sartorius AG) and a 25 - 100 ⁇ l sample was analyzed on AminexHPX-87C cation exchange column (300 x 7.8 mm) (BioRad Laboratories) .
• the HPLC system used was a Varian 500 equipped with a HP 1047 (B) efractive index detector and a Water ⁇ VISP717 injector. A ⁇ bondapack C 18 -precolumn was u ⁇ ed in the ⁇ y ⁇ tem.
• Step 1 Ammonium sulphate fractionation. 25 ml of cell free extract (as described in example 1) was diluted to 90 ml and saturated ammonium sulphate in 50 mM Tris-HCl, pH 7.5, was added to achieve 20 % saturation. The solution was incubated for 30 min at 0°C and centrifuged at 15,000 g. The precipitate was discarded and the supernatant purified further.
• Step 2 Hydrophobic interaction chromatography.
• the supernatant from step 1 (105 ml) was applied to a Butyl Sepharose 4 FF (Pharmacia) (10 x 50 mm) column pre-equilibrated with 20 % (w/v) ammonium sulphate in 20 mM Tris-HCl, pH 7.5.
• the column was washed with 45 ml of 20% (w/v) ammonium sulphate in 20 mM Tris-HCl and eluted with a linear gradient of 20-0% ammonium sulphate.
• the volume of the gradient wa ⁇ 80 ml and the flow rate was 2 ml/ in. Fractions of 3 ml were collected.
• the active fractions (40 ml) were pooled.
• the ammonium sulphate was removed by gel filtration (Sephadex G-2S, Pharmacia) .
• Step 3 Ion exchange chromatography.
• the sample from step 2 (73 ml) was applied to a DEAE-Memsep 1000 HP (Millipore) (1.4 ml) column pre-equilibrated with 20 mM Tris-HCl, pH 7.5.
• the column was washed with 15 ml of buffer and eluted with a linear NaCI gradient (0 - 1 M) .
• the volume of the gradient was 60 ml and the flow rate was 3 ml/min. 2 ml fractions were collected
• the active fraction ⁇ (8 ml) were pooled and concentrated by ultrafiltration (A icon Centriplus 30; Ultrafree MC 10,000 NMWL filter unit Millipore) to 100 ⁇ l.
• Step 4 Gel filtration.
• the concentrated sample from step 3 (100 ⁇ l) was applied to a Superose 12 HR 30 (Pharmacia) column 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCI was u ⁇ ed a ⁇ the elution buffer with flow rate of 0.4 ml/min. 0.5 ml fraction ⁇ were collected.
• the fraction ⁇ containing glycine-sarcosine methyltransferase (GSMT) activity (1.5 ml) were collected and concentrated by ultrafiltration (Ultrafree MC 10,000 NWML filter unit, Millipore) to 100 ⁇ l .
• GSMT glycine-sarcosine methyltransferase
• the purity and the molecular weight were determined by gradient SDS-polyacrylamide gel electrophoresis according to the following procedure. Electrophoresis under denaturing conditions was carried out using pre-made polyacrylamide gel ⁇ lab ⁇ (12 % Tri ⁇ -glycine gel with 4 % stacking gel, Ready Gel ⁇ , Biorad) according to the in ⁇ tructions of the manufacturer. Mid range molecular weight standard from Promega was used. Staining of the gel was performed with 0.25% (w/v) Coomassie Blue R-250 (Promega) in 50 % (v/v) methanol and 10% (v/v) acetic acid. Stained gels were de ⁇ tained with 10% (v/v) methanol and 5% acetic acid (Laemmli, 1970) .
• the affinity column was prepared as follows. 5 'AMP-Sepharose 4B (Pharmacia) was treated with alkaline phosphata ⁇ e to remove the pho ⁇ phate group of the ligand. The gel wa ⁇ fir ⁇ t ⁇ wollen in water (5 ml of distilled water was u ⁇ ed per 1 g of dry 5 'AMP-Sepharo ⁇ e 4B) . The ⁇ wollen gel wa ⁇ then wa ⁇ hed with 200 ml of di ⁇ tilled water.
• CIP-buffer 10 mM MgCl 2 , 1 mM dithiothreitol, 50 mM NaCI, 10 mM Tri ⁇ -HCl, pH 7.9
• the molecular weight of A. halophila SDMT is approximately 32 kDa.
• the molecular weight was also determined by gel filtration with Superose 12 HR 30 (Pharmacia) column. The flow rate wa ⁇ 0.4 ml/ml.
• the elution buffer was 20 mM Tris-HCl pH 7.5 containing 150 mM NaCI.
• the molecular weight was calculated from a calibration curve made with a mixture of standard proteins .
• the mixture contained 0.5 mg/ml Blue Dextran (0.5 mg/ml), Ferritin (440 kDa), 7.0 mg/ml aldolase (158 kDa), 2.0 mg/ml ovalbumin (43 kDa) and 1.0 mg/ml chymotrypsinogen (25 kDa).
• the calculated molecular weight wa ⁇ 31.6 kDa, which indicates that the protein is a monomer.
• the isoelectric focusing wa ⁇ performed with Pharmacia Phast system using gels with pH-gradient from pH 3 to 9 (IEF 3-9) .
• a mixture of Pharmacia IEF standard proteins with pis from 3.5 to 9.3 were u ⁇ ed a ⁇ standards.
• the gels were stained by silver staining as described in example 1.
• the result ⁇ shown in Figure 3 show that the pi of the protein is approximately 4.1-4.2.
• the activity of the purified protein was determined as described in example 1 with glycine, sarcosine and dimethyl glycine.
• the data presented in table 2 demonstrates that the isolated protein catalyzes step methylation reaction from sarcosine to dimethyl glycine and from dimethyl glycine to betaine.
• the pH-optimum of the two methylation reaction ⁇ were determined by u ⁇ ing following buffers: 0.1 M piperazine buffer, pH 5.0; 0.1 M Bis-Tri ⁇ buffer, pH 6.0; 0.1 M Bis-Tris buffer, pH 7.0; 0.1 M Tris-HCl, pH 8.0; 0.1 M Tris-HCl, pH 9.0; 0.1 methanolamine, pH 10.
• the exact pH value ⁇ of the reaction mixture ⁇ were measured. It can be concluded from Figure 4 that the pH optimum of the both enzyme reactions is at pH 7.5.
• the temperature dependence of the enzymatic reaction ⁇ were determined with sarcosine and dimethyl glycine. As seen in Figure 5. the temperature optimum is approximately 45 - 50°C. When the temperature is elevated above 50°C, the enzyme is rapidly inactivated.
• Example 5 In vitro synthesis of betaine by using purified E. halochloris GSMT and A. halophila SDMT
• the purified GSMT from E. halochlori ⁇ and SDMT from A. halophila were concentrated by ultrafiltration (Ultrafree MC 10,000 NWML filter unit, Millipore) to protein concentrations 4.2 mg/ml and 5.6 mg/ml, respectively.
• the reaction mixture contained 50 ⁇ l 5.0 mM glycine, 50 ⁇ l 32 mM S-adenosyl methionine in water containing 640 nCi S-adenosyl-L- [methyl- 1 C] -methionine (Amersham) , 50 ⁇ l Buffer II (see example 1), 25 ⁇ l GSMT of E. halochloris and 25 ⁇ l SDMT of A. halophila.
• the reaction was initiated by adding the enzymes .
• the reaction mixture wa ⁇ incubated for 2 h at 37°C and the reaction was stopped by adding 150 ⁇ l of charcoal suspension.
• reaction mixtures were then incubated for 10 min at 0°C and centrifuged for 10 min in a Heraeu ⁇ table top centrifuge.
• the ⁇ upernatant ⁇ were filtered through Mini ⁇ art NML 0.2 ⁇ m filter (Sartorius AG) .
• the identification of the reaction products was performed by HPLC as described in example 2.
• the chromatogram is presented in Figure 6 and it show ⁇ peak ⁇ corresponding to the retention times of sarcosine, dimethylglycine and betaine.
• N-terminal and tryptic peptide ⁇ peptide ⁇ equences of the purified proteins were determined by using Perkin Elmer/Applied Biosy ⁇ tems Procise 494A protein ⁇ equencing system as described by Kerovuo et al . , 1998.
• the peptide ⁇ equences obtained are shown in table 3.
• the genomic DNA from both microbes was isolated essentially as described in Ausubel et al . (1991).
• the chromo ⁇ omal DNAs were partially digested with Sad and ligated to Sad digested dephosphorylated lambda ZapII arms (Stratagene, La Jolla, California, USA) and packaged to lambda particle ⁇ u ⁇ ing Gigapack III Gold packing extract (Stratagene, La Jolla California, USA) according to protocol provided by manufacturer.
• the chromo ⁇ omal DNA isolated from the organisms was used as the template DNA in the PCR reactions .
• the probes were made by PCR using following degenerate primers.
• the primers were designed according to Sambrook et al. (1989) .
• the amplification wa ⁇ performed under the following conditions.
• PCR-fragments obtained were labeled with rediprime DNA labelling sy ⁇ tem (Amersham Life Science) according to the instruction ⁇ given by the manufacturer.
• the E. halochloris GSMT is encoded by the first ORF of the fragment.
• the second ORF of E. halochloris clone contains a SDMT gene. This has also been demonstrated by expres ⁇ ing the gene in E. coli (Example 8) .
• A. halophila SDMT protein is coded by the 3 '-end of the first ORF of the A. halophila clone. The 5 '-end of the same ORF is very homologous to the E. halochloris GSMT.
• the "betaine operon” codes for a third gene which is homologous to number of S-adenosyl-methionine synthases.
• the operon ⁇ tructure is schematically shown in Figure 7.
• the nucleotide and amino acids sequences of the cloned genes have been shown in Figures 8 and 9.
• the gene coding for the E. halochloris GSMT was amplified by PCR.
• the purified plasmid used for DNA sequencing in example 6 was used as the template for the PCR reaction.
• the following primers were used in the PCR reaction:
• the 3 '-end of the primers are homologous to the 5'- and 3 ' -end of the GSMT gene.
• the 5 '-end oligonucleotide hybridizes to position 221-241 and the 3 '-end to the position 1001-1024. (See Figure 8) .
• the primer hybridizing to the 5'- end contains an extra Ncol restriction site such that the nucleotide A at position 224 in Figure 8 i ⁇ replaced by the nucleotide G in the primer and the 3 '-end primer contain ⁇ a Bglll site which were used for cloning.
• the amplification was performed in the following conditions : 34 cycles of 1 min at 94°C for denaturation. 1 min at 50 °C annealing and 2 min at 72°C for synthe ⁇ i ⁇ .
• GSMT transformants were grown overnight in 2.5 ml of LB broth containing 100 ⁇ g/ml ampicillin.
• E. coli XLI E. coli XLI
• the cell pellet was su ⁇ pended in 100 ⁇ l Buffer II containing 1 mM PMSF (See example 1) and the cell ⁇ were di ⁇ rupted with a MSE Soniprep 150 ⁇ onicator.
• the cell suspension was ⁇ onicated with ⁇ onication pulses of 5 ⁇ for 10 s .
• the sample ⁇ were cooled on ice between the pulses .
• the cell debris was removed by centrifugation at 13,000 rpm for 30 min at 4°C in a Heraeus table top centrifuge.
• the activitie ⁇ of the supernatants were determined a ⁇ in example 1.
• the activities using glycine and sarco ⁇ ine a ⁇ substrates were typically 3,000-5,000 dpm/30 min and 1,000-2,000 dpm/30 min., respectively.
• the strain ⁇ u ⁇ ed in the ⁇ e te ⁇ t ⁇ were the po ⁇ itive clone de ⁇ ignated EGSM and E. coli XLI Blue MRF' tran ⁇ formed with the cloning vector PQE-60.
• the growth medium u ⁇ ed in this test was the synthetic medium MM63 de ⁇ cribed by Lar ⁇ en et al . (1987) supplemented with 1.5 mil/1 of vitamin solution VA (Imhoff and Tr ⁇ per, 1977) and 100 ⁇ l/ml ampicillin.
• the bacterial strain ⁇ were grown to mid-exponential growth phases with shaking at 180 rpm at 37°C and centrifuged (1,000 g, 15 min) .
• the cells were resu ⁇ pended in the growth medium to ab ⁇ orbance of 0.9 at 600 nm.
• the gene encoding the E. halochloris SDMT was amplified by PCR.
• the purified plasmid used for DNA sequencing in example 6 was used a ⁇ the template for the PCR reaction.
• the following primer ⁇ were used in the PCR reaction:
• the primers are homologous to the 5'- and 3 ' -end of the E. halochloris SDMT gene.
• the 5' -end oligonucleotide hybridizes to position 1031-1054 and the 3 '-end to the position 1844- 1867 ( Figure 8) .
• the primer hybridizing to the 5' -end contains an extra Ncol restriction site and the 3 '-end primer a Bglll site which were used for cloning of the fragment.
• the cultivations and preparation of the cell-free extracts were performed essentially as described in example 7.
• the ⁇ onication of the cell extract pulses was shortened to 3 x 2 second interval ⁇ (total sonication time was 6 s).
• the activities using sarcosine and dimethyl glycine as substrate ⁇ were typically 20,000 dpm/30 min. with both ⁇ ub ⁇ trate ⁇ .
• the DNA construct made for this experiment contain ⁇ both GSMT and SDMT genes separated by a short (3 nucleotides long) linker.
• the DNA fragment was obtained by amplification of the purified pla ⁇ mid used for DNA sequencing in example 6.
• the primers are homologous to the 5 '-end of the E. halochloris GSMT and the 3 ' -end of the E. halochloris SDMT gene.
• the 5 '-end oligonucleotide hybridizes to position 221- 241 and the 3 ' -end to the position 1844-1867 (See Figure 8) .
• the primer hybridizing to the 5 '-end contains an extra Ncol restriction site and the 3' -end primer a Bglll site.
• the enzymatic activities were as ⁇ ayed as described in example I .
• the cell extracts of the transformant ⁇ clearly ⁇ howed activity with glycine, ⁇ arcosine and dimethylglycine.
• the activities of the cell extracts with the three substrate ⁇ were all over 20,000 dpm/30 min.
• the test was performed es ⁇ entially a ⁇ described in example 7.
• the po ⁇ itive clone de ⁇ ignated EhFU was used in this te ⁇ t.
• the growth medium used in this test was the synthetic medium MM63 described by Larsen et al. (1987) ⁇ upplemented with 1.5 ml/1 of vitamin solution VA (Imboff and Tr ⁇ per, 1977) and 100 ⁇ l/ml ampicillin.
• the medium contained 1% (wlv) glucose.
• the clone EhFU ( Figure 10) and the control strain (E coli XLl-Blue MRF' transformed with the cloning vector PQE-60) were grown to mid-exponential pha ⁇ e with ⁇ haking at 180 rpm at 37°C.
• the cell ⁇ were centrifuged at 1,000 g for 10 min and resuspended in the growth medium ⁇ o that the turbidity goot was 0.640. 5 ml of this cell suspension wa ⁇ inoculated to 50 ml of media containing 0.22 or 0.33 M NaCI and 25 mM L-methionine.
• the bacterial ⁇ train ⁇ were grown for 2 h with ⁇ haking at 180 rpm at 37°C and 1 mM IPTG was added.
• the cell pellets were suspended in 2 ml of water and kept in a boiling water bath for 10 min.
• the su ⁇ pen ⁇ ion was centrifuged for 15 min at 23,000 g and the supernatant collected and the pellet resuspended in water. This extraction was repeated twice.
• the three supernatants were combined.
• the volumes of supernatant ⁇ were measured and the supernatant ⁇ were filtered and analyzed by HPLC as described in example 1.
• the betaine produced inside the cells is presented in table 4.
• Example 10 Expression of the DNA fragment encoding the protein isolated as A. halophila SDMT in E. coli
• the gene sequencing results revealed that a single gene codes for the A halophila GSMT and SDMT.
• the fusion protein was not, however, successive ⁇ fully purified from the A. halophila cell extract ⁇ . Instead a protein with SDMT activity was isolated.
• the corresponding part of the GSMT-SDMT gene i ⁇ expres ⁇ ed in E. coli.
• the gene fragment encoding the SDMT enzyme activity was amplified by PCR.
• the genomic DNA from A. halophila isolated in example 6 was used as the template for the PCR reaction.
• the following primers were used in the amplification:
• the primers are homologous to the 5'- and 3 '-end of the ASDMT gene.
• the 5' -end oligonucleotide hybridizes to position 1048- 1068 and the 3' -end to the position 1879-1902 (See Figure 9) .
• the primer hybridizing to the 5 '-end contains an extra Ncol restriction site and the 3 '-end primer a Bglll site.
• Example 11 Expression of A. halophila GSMT-SDMT fusion protein in E. coli
• the primer ⁇ are homologous to the 5'- and 3 ' -end of the AGSMT-ASDMT gene.
• the 5 '-end oligonucleotide hybridizes to position 208-231 and the 3 '-end to the position 1879-1902 (See Figure 9).
• the primer hybridizing to the 5 '-end contains an extra Ncol restriction site ⁇ uch that the nucleotide A at po ⁇ ition 211 in Figure 9 i ⁇ replaced by the nucleotide G in the primer and the 3 '-end primer contain ⁇ a Bglll ⁇ ite.
• the purified pla ⁇ mid used for DNA-sequencing in example 6 was used as a template for the PCR reaction.
• the amplification was performed in following conditions: 34 cycles of 30 s at 94°C for denaturation, 1 min at 50°C annealing and 2 min at 72°C for ⁇ ynthesis.
• Ligation of the amplification product into Ncol/Bglll cut PQE-60 and the transformation of XL-1 Blue MRF' cells was performed as in example 7.
• the induction and preparation of the cell-free extracts was performed essentially as in example 9 except that the sonication pulses were shortened to 2 ⁇ and the total ⁇ onication time to 6 s .
• the cell-free extract was analyzed by SDS-polyacrylamide gel electrophoresis as in example 2.
• the pellet from the centrifuged suspension wa ⁇ suspended to 10 mM Tri ⁇ -HCl-buffer, pH 8.0 containing 8 M urea and 0.1 M Na3P0 4 to ⁇ olubilize the proteins of the pellet and centrifuged for 15 min in a Heraeus table top centrifuge at 13,000 rpm.
• the ⁇ upernatants were analyzed by SDS-polyacrylamide gel electrophoresis as in example 2.
• the enzymatic activities were assayed as described in example 1.
• the cell extracts of the transformant ⁇ clearly showed SDMT activity.
• the activities on sarcosine and dimethyl glycine were typically 10,000 dpm/30 min and 20,000 dpm/30 min., respectively. There was no activity on glycine.
• the SDS-polyacrylamide gel of the cell-free extract showed no major protein band of correct ⁇ ize.
• the insoluble pellet solubilized with 8 M urea showed a major band corresponding to the molecular weight of the GSMT-SDMT fusion protein.
• the results indicate that when A. halophila GSMT- SDMT is over-expres ⁇ ed in E. coli it forms inclusion bodies.
• a fraction of the protein - which corresponds the SDMT - is proteolytically cleaved and remains soluble in the cells .
• Example 12 Expression of E. halochloris GSMT and SDMT in tobacco and potato
• Tobacco and potato plants can be transformed by AgrroJacteriu-n mediated tran ⁇ formation system.
• Identical DNA construct can be used for both plants.
• the GSMT gene is first tran ⁇ formed into the plant u ⁇ ing a pla ⁇ mid containing a kanamycin re ⁇ istance marker. Positive transgenic plants obtained by screening for the enzyme activity are then used as host plants for second transformation of the SDMT gene. Another ⁇ election marker, hygromycin selection is used in the second transformation. Experiments are performed using stable transformants of the F x generation.
• the genes of E. halochloris GSMT and SDMT are amplified by PCR by using plasmid pEFU (see example 10) as the template.
• the primers used hybridize to the same regions of the DNA as shown in Fig. 8 (GSMT: primer 1 and primer 2; SDMT; primer 3 and primer 4) .
• the final DNA constructs are made using suitable restriction sites to transfer the genes to plant transformation vectors.
• PBin 19 based pGPTV vectors (Becker et al, 1992) are used which have a strong 35S promoter and the CaMv polyadenylation signal.
• Strain EHA 105 Hood, E.E. et al . , (1993) is used as a vector to transform tobacco basically as described by Rogers et al . (1986).
• Strain ClC58p-GV3850 Zambryski et al . , (1983); Van Larabece et al . , (1974) is used a ⁇ an alternative host to transform potato Solanum tuherosum (Desire) according to Dietze et al . (1995) .
• the transformants are analyzed by Southern blot analysis to check for the presence of the genes. PCR-amplified, DIG- labelled (Boehringer) 200 bp gene fragments are used as a probe. The enzymatic activities of the cell extracts of transgenic plant ⁇ and the levels of ⁇ arcosine, dimethyl glycine and betaine are analyzed as described in Example 1.
• Stress tolerance for example, tolerance to drought, salinity, cold or freezing, resistance to pathogens, etc., is determined according to methods known in the art, for example, methods described in the technological background section of the present application.
• Example 13 Expression of E. halochloris GSMT and SDMT in rice
• the plasmid construction ⁇ described in Example 13 are also used to transfer the GSMT and SDMT genes to rice by particle bombardment.
• the GSMT are transferred to rice first and positive regenerated tran ⁇ formants are used as host plants for the SDMT transformation.
• Immature Oryza sativa embryos of the Japonica variety Taipei 309 are aseptically isolated 10-14 days after pollination from greenhouse plants and plated scutulum site up on solid MS medium (Murashige and Skoog, 1962) containing 3% sucrose, 2 mg/1 2,4- dichlorophenoxyacetic acid and 50 mg/l cefotaxime (MSI) . After 4-6 days (28°C, darkness) embryos are transferred to ⁇ olid MS medium containing 10% ⁇ ucro ⁇ e, 2 mg/1 2,4- dichlorophenoxyacetic acid and 50 mg/1 cefotaxime (MS2) and ⁇ ubjected within 1 hour to microprojectile bombardment with a particle inflow gun.
• solid MS medium Morashige and Skoog, 1962
• MSI cefotaxime
• the DNA fragment containing the mehtyltransferase gene and the selective marker (5 ⁇ g) is precipitated on 1-3 mm gold particles (Aldrich) as described by Vain et al . , (1993) .
• Gold particles 400 mg per bombardment
• Embryo ⁇ are placed 16 cm below the ⁇ yringe filter. Twenty four hour ⁇ po ⁇ t-bombardment embryos are subjected to selection on solid media (containing hygromycin or kanamycin) and incubated at 28°C in the dark.
• Developing calli are isolated 3 to 6 weeks later, and transferred to a callus increa ⁇ ing media (R2 medium supplemented with: 6% ⁇ ucrose, MS vitamins, 100 mg/l inositol, 2 mg/l 2, 4-dichlorophenoxyacetic acid, 50 mg/l cefotaxime and 20 mg/l hygromycin B kanamycin) .
• the calli are incubated in thi ⁇ media at 28°C in the dark and ⁇ ubcultured weekly.
• Re ⁇ i ⁇ tant calli are tran ⁇ ferred to solid R2 regeneration media supplemented with 2% sucrose, 3% sorbitol, 20 mg/l hygromycin B, 1 mg/l zeatin, 0.5 mg/l indole-3-acetic acid, MS vitamins and 0.65% agarose.
• the callus tissue is maintained at 28°C with 12 h of light in order to enhance shoot formation.
• the calli are then subcultured every 3 week ⁇ until ⁇ hoot ⁇ had reached a length of 2-3 cm. They are transferred to half-strength MS rooting medium without hormones, supplemented with 1.5% sucro ⁇ e and 0.3% gelriteR (Sigma) .
• plantlets are tran ⁇ ferred directly to the green-house and planted in soil. Plantlets are grown in 7 liter aquaculture pot ⁇ with fertilizer enriched earth, 3 plants per pot (day: 12 h, 28°C, 80% humidity; night: 12h, 21°X, 60% humidity) until they flower and set seeds.
• Southern blot analy ⁇ i ⁇ is performed as described previously (Burkhardt et al., 1997).
• a PCR amplified, DIG-labelled (Boehringer) 200-bp fragment of the coding region of the GSMT or SDMT genes is used as a probe .
• Stress tolerance for example, tolerance to drought, salinity, cold or freezing, resistance to pathogens, etc., is determined according to methods known in the art, for example, methods described in the technological background section of the present application.
• Example 14 Expression of the E. halochloris GSMT and SDMT in yeast
• pYX242 plasmid (R&D systems, USA) was used for expressing the GSMT and SDMT genes in Saccharomyces cerevisiae .
• the plasmid used (pYX242) is a E. coli -Saccharomyces cerevisiae ⁇ huttle vector containing a bacterial origin of replication and ampicillin re ⁇ i ⁇ tance gene, a yeast ⁇ S. cerevisiae) origin of replication from 2 ⁇ m DNA, and the yeast LEU2 gene for selection in yeast.
• the two genes are expressed under the yeast triose phosphate isomerase (TPI) promoter.
• TPI yeast triose phosphate isomerase
• the DNA of the pla ⁇ mid pEFU described in Example 10 wa ⁇ used as the template of PCR reactions.
• the primers u ⁇ ed hybridize to DNA ⁇ equence ⁇ shown in Fig. 8 (primer 1 and primer 4) and thus amplify both the GSMT and SDMT genes.
• the PCR fragment was ligated to the promoter of the expression plasmid with standard methods. A fragment containing a TPI transcription terminator and a fragment containing the TPI promoter was ligated between the two genes. Thus, both gene ⁇ are expressed under the TPI promoter.
• the primers used in the amplification of the fragments ligated in the expression plasmid contained suitable restriction sites that were used in the cloning. S.
• the transformants were grown in YNB-medium supplemented with amino acid mixture without leucine (R &D systems product manual) .
• the cultivation was done overnight at 30°C by ⁇ haking at 180 rpm.
• 5 ml of culture supernatant was centrifuged (1,700 g, 10 min) and the cell pellet wa ⁇ suspended in 200 ⁇ l of the assay buffer (see example 1) supplemented with 1 mM PMSF.
• the cells were broken by vortexing with glass beads (10 x l min intervals) .
• the cells were kept on ice between the pulses.
• the cell debris was centrifuged down (30 min, 4°C, 10,000g).
• the methylase activities were assayed from the supernatant as described in example 1.
• the relative enzyme activities on different substrates were the following: glycine - 17,000 dpm/30 min; sarco ⁇ ine - 71,000 dpm/30 min and dimethyl glycine - 530,000 dpm/30 min.
• JP patent 87,190,078 Kojima, Y., Aisui, S., Ando, M. (1987) Kapper, R., Kempf, B., Bremer, E., (1996) J. Bacteriol. 178, 5071-5079

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