# Patent

## Title
Tetrahydropyrimidine dioxygenase gene, polypeptides encoded by said gene and method for producing the same

## Abstract

The present invention relates to enzymes having tetrahydropyrimidine dioxygenase activity, to the genes coding therefor, to the homologous and heterologous expression of these genes, to processes for preparing enzymes having tetrahydropyrimidine dioxygenase activity and the use of these enzymes for in vivo and in vitro production of hydroxylated tetrahydropyrimidine. In addition, the invention relates to the biotechnological use of the hydroxylated tetrahydropyrimidine prepared by one of the processes of the invention.

## Background
The present invention relates to enzymes having tetrahydropyrimidine dioxygenase activity, to the genes coding therefor, to the homologous and heterologous expression of these genes, to processes for preparing enzymes having tetrahydropyrimidine dioxygenase activity and the use of these enzymes for in vivo and in vitro production of hydroxylated tetrahydropyrimidine. In addition, the invention relates to the biotechnological use of the hydroxylated tetrahydropyrimidine prepared by one of the processes of the invention.
Following osmotic stress, microorganisms produce low molecular weight intracellular compounds which are referred to as osmolytes and which include the tetrahydropyrimidines. The tetrahydropyrimidines THP(B) (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and THP(A) (2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine) are produced by various microorganisms as intracellular components of the cytoplasm [Ventosa, A. et al. (1998). Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev. 62, 504-544] (FIG. 1). These microorganisms include the halophilic eubacteria, actinomycetes, bacillus species and brevibacteria. The THPs can be isolated from these organisms in pure form and be employed in various forms in biotechnology, such as, for example, in the stabilization of proteins both in solution and on freeze drying thereof [Lippert, K. and Galinski, E. A. (1992). Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl. Microbiol. Biotechnol. 37, 61-65.], in influencing the interactions of proteins and nucleic acids [Lapidot, A. et al. (1995). Tetrahydropyrimidine derivatives inhibit binding of a Tat-like, arginine-containing peptide, to HIV TAR RNA in vitro. FEBS Lett. 367, 33-38; Malin, G. and Lapidot, A (1996). Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress. J. Bacteriol. 178, 385-395; Malin, G. et al. (1999). Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type 11 restriction endonucleases as a model system. J. Biol. Chem. 274, 6920-6929.] and in the refolding of denaturated proteins. In addition, an increased salt and heat tolerance can be found on addition of THP(A) or THP(B) to culture media of various prokaryotes such as, for example, E. coli [Malin, G. and Lapidot, A. (1996). Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their effect on Escherichia coli in response to osmotic and heat stress. J. Bacteriol. 178, 385-395.].
The characterized THP(A) biosynthetic pathway, shown in FIG. 1 starts from L-aspartate-β-semialdehyde. In the subsequent transamination reaction, L-2,4-diamino-butyrate transaminase catalyzes the formation of L-2,4-diaminobutyrate (DABA). The subsequent N-acetylation of DABA is effected by L-2,4-diaminobutyrate acetyltransferase using acetyl-coenzyme A. The intramolecular condensation reaction catalyzed by N-acetyl-γ-L,2,4-diaminobutyrate cyclase results in THP(B) [Peters, P. et al. (1990). The biosynthesis of ectoine. FEMS Microbiol. Lett. 71, 157-162; Ono, H. et al. (1999). Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J. Bacteriol. 181, 91-99.]. By contrast, the steps to the biosynthesis of the 5-hydroxy derivative of THP(B), (THP(A)), are still unexplained.
Preparation of THPs by the processes known in the art is confined to the elaborate extraction of these compounds from the cells or culture filtrates from bacteria which produce THP(A) and/or THP(B). These processes are particularly cost-intensive because of the high salt concentrations in the culture medium.
Although a chemical synthesis of THPs is possible, this synthesis comprises a large number of synthetic steps and the use of an elaborate protective group technique, so that this complex process cannot be carried out economically, especially on a large scale. This particularly relates to the synthesis of THP(A) because the respective precursors cannot be obtained by chemical routes, and the consequence is additional difficulty for the chemical synthesis of THP(A).
The object of the invention is therefore to provide an improved process for preparing hydroxylated THPs, in particular THP(A).
The object of the invention is achieved by providing a process using a polypeptide having a tetrahydropyrimidine dioxygenase activity.
One aspect of the invention therefore relates to the nucleic acid sequences which code for polypeptides which have a tetrahydropyrimidine dioxygenase activity. These nucleic acid sequences comprise both DNA and RNA nucleic acid sequences and are selected from the following group of nucleic acid sequences:
(a) DNA nucleic acid sequence having the nucleotide sequence depicted in SEQ ID No.: 1;
(b) DNA nucleic acid sequence derived as a result of the degeneracy of the genetic code from the nucleotide sequence depicted in SEQ ID No.: 1;
(c) DNA nucleic acid sequence which hybridizes with at least one DNA nucleic acid sequence which has a DNA nucleic acid sequence according to (a) or (b);
(d) DNA nucleic acid sequence which is fragments, allelic or other variants of the DNA nucleic acid sequence having the nucleotide sequence depicted in SEQ ID No.: 1;
(e) RNA nucleic acid sequence derived from the DNA nucleic acid sequence according to (a) to (d); and
(f) nucleic acid sequence having a degree of homology with one of the sequences according to (a) to (e) of more than 60%.
DNA nucleic acid sequences coding for polypeptides having tetrahydropyrimidine dioxygenase activity mean both genomic DNA nucleic acid sequences and cDNA nucleic acid sequences. The degree of homology of the nucleic acid sequence according to (f) with one of the sequences according to (a) to (e) is at least 60%, preferably 80% and particularly preferably more than 90%.
A further aspect of the present invention relates to polypeptides which have tetrahydropyrimidine dioxygenase activity and which are preferably encoded by one of the nucleic acid sequences according to (a), (b), (c), (d), (e) or (f). Particular preference is given to a polypeptide (SEQ ID No.: 6) which is encoded by the DNA nucleic acid sequence shown in SEQ ID No.: 1.
In another aspect, the present invention relates to DNA or RNA nucleic acid sequences according to (a), (b), (c), (d), (e) or (f) which have been isolated from an archaebacterium, a prokaryote or a eukaryote. Particular preference is given to such nucleic acid sequences when they have been isolated from a halophilic eubacterium, an actinomycete, a bacillus or a brevibacterium. 
Preference is likewise given to nucleic acid constructs which comprise an operative unit which consists of a promoter sequence and a nucleic acid sequence according to (a), (b), (c), (d), (e) or (f).
Preference is likewise given to nucleic acid constructs which comprise an operative unit which consists of a promoter sequence and a nucleic acid sequence according to (a), (b), (c), (d), (e) or (f), and additionally comprise a nucleic acid sequence which codes for one or more secretion signals.
In a further aspect, the present invention relates to replicative recombinant vectors which comprise a nucleic acid sequence according to (a), (b), (c), (d), (e) or (f).
The invention further relates to cells which comprise a vector which undergoes autonomous replication in the cell and which comprises a nucleic acid sequence according to (a), (b), (c), (d), (e) or (f). Particular preference is given in this connection to bacteria cells, yeast cells or plant cells.
Preferred cells have a nucleic acid sequence according to (a), (b) (c), (d), (e) or (f) integrated into the genome by non-natural recombination. Particular preference is given in this connection to cells in which the non-natural recombination is into the genome of a bacterial cell, of a yeast cell or of a plant cell.
In an alternative aspect, the present invention relates to the use of an oligonucleotide as nucleic acid probes for identifying genes, where the nucleotide sequence of the oligonucleotide has been derived from that of the nucleic acid sequences according to (a), (b), (c), (d), (e) or (f), and the oligonucleotides are used for identifying chromosomal or extrachromosomal genes which are present in archaebacteria, prokaryotes or eukaryotes and which code for a polypeptide which has tetrahydropyrimidine dioxygenase activity.
The invention also relates to a plant which has been transformed with a recombinant DNA which comprises a DNA sequence as claimed in any of claims 1 to 4. Such a plant is distinguished by increased osmotic tolerance.
The invention further relates to a process for preparing a polypeptide having tetrahydropyrimidine dioxygenase activity, entailing cultivation of cells which comprise a nucleic acid section having a nucleic acid sequence according to (a), (b), (c), (d), (e) or (f) or a nucleic acid construct of the type explained above, in isolation of the polypeptide having tetrahydropyrimidine dioxygenase activity.
The method used for isolation of the polypeptide will depend, in a manner known to the skilled worker, on whether the cells used for the preparation comprise a nucleic acid section having a nucleic acid sequence according to (a), (b), (c), (d), (e) or (f) or a nucleic acid construct of the type explained above. With the first possibility, the prepared polypeptide can be isolated only after preceding disruption of the cells, and with the second possibility, in which the cells comprise a nucleic acid construct comprising a secretion signal, it would be possible to isolate the prepared polypeptide directly from the culture medium.
Another aspect of the invention relates to the use of a polypeptide having tetrahydropyrimidine dioxygenase activity for preparing hydroxylated tetrahydropyrimidine.
The preparation preferably takes place with the aid of a polypeptide having tetrahydropyrimidine dioxygenase activity in a living cell, the living cell being a bacterial cell, a yeast cell or a plant cell. The living cell containing the polypeptide having tetrahydropyrimidine dioxygenase activity may be present in a culture medium which contains tetrahydropyrimidine. In such a case, the living cell should be permeabilized, preferably before the preparation, in order to favor contact between the polypeptide having tetrahydropyrimidine dioxygenase activity and the tetrahydropyrimidine.
A preferred embodiment of the invention is described below, although this is not intended to be understood in such a way that a restriction of the protection desired by the inventor can be inferred therefrom.
In order to find THP biosynthesis genes in Streptomyces chrysomallus, L-2,4-diaminobutyrate acetyltransferase, an enzyme of THP biosynthesis from Streptomyces chrysomallus, was purified. Oligonucleotide sequences were derived from tryptic peptide sequences of the L-2,4-diaminobutyrate acetyltransferase and employed for screening a cosmid bank. The gene of L-2,4-diaminobutyrate acetyltransferase and flanking regions were sequenced. Four open reading frames were found inter alia on the BamHI/EcoRI fragment, 8.7 kb in size. These genes, which code for THP biosynthesis enzymes, are referred to hereinafter as thpA, thpB, thpC and thpD (FIG. 2). The open reading frame referred to as thpD codes for an enzyme which has a THP(B) dioxygenase activity and a molecular weight of 32.7 kDa. This enzyme is an α-ketoglutarate-dependent dioxygenase which catalyzes the irreversible hydroxylation of THP(B) to THP(A). The thpD gene was expressed in E. coli and Streptomyces, and the corresponding protein (THP(D)) was purified by methods known in the art.
Use of the homologously or heterologously expressed THP(D) protein makes in vitro production of THP(A) possible. Complete hydroxylation of THP(B) to THP(A) is observed by incubation of the THP(D) protein in the presence of THP(B), α-ketoglutarate, ascorbic acid, iron(II) sulfate and catalase.
The gene which codes for the tetrahydropyrimidine dioxygenase is located on a BamHI/EcoRI fragment 8.7 kb in size. Expression of the Streptomyces chrysomallus genes present on this DNA fragment in microorganisms makes it possible to synthesize hydroxylated tetrahydropyrimidine.
Expression of the thpD gene in microorganisms which produced THP(B) makes in vivo production of the 5-hydroxy derivative of tetrahydropyrimidine (THP(A)) possible.
This process can be applied to a large number of microorganisms such as, for example, actinomycetes, bacilli or halophilic bacteria, as long as they have nucleic acid sequences which code for polypeptides having THP dioxygenase activity. With this proviso it is possible for the expression additionally to be carried out in any other prokaryotic or eukaryotic expression system.
The invention is explained in more detail below by means of examples.


EXAMPLE 1
Detection of L-2,4-diaminobutyrate acetyltransferase from Streptomyces chrysomallus 
A nucleotide sequence was derived from an internal peptide sequence of the L-2,4-diaminobutyrate acetyltransferase previously purified from Streptomyces chrysomallus (FIG. 3). This nucleotide sequence was radiolabelled and employed as probe for screening a cosmid bank. Parts of the corresponding cosmid were subcloned and sequenced. Four open reading frames inter alia were found thereby, with thpA coding for L-diaminobutyrate acetyltransferase, thpB coding for L-aspartate-α-semialdehyde transaminase, thpC coding for N-acetyl-γ-L-2,4-diaminobutyrate cyclase and thpD coding for THP(B) dioxygenase (FIG. 2). Expression in other microorganisms is possible, in Streptomyces lividans serves as exemplary organism (see Example 2).
EXAMPLE 2
Expression of thpD in Streptomyces lividans 
For expression of thpD in Streptomyces lividans, the thpD gene was cloned as SphI/HindIII fragment into the expression vector pSPIJ002 (FIG. 5). The resulting expression plasmid was called pSPIJthpD. The SphI and HindIII restriction cleavage sites were introduced by PCR mutagenesis (35 cycles; 1 min 95° C.; 90 sec 55° C.; 65 sec 72° C.; see Table 1 for primers). The plasmid pQE30thpD served as template for the PCR. This plasmid is a pQE30 derivative which contains the thpD gene as BamHI/HindIII PCR fragment (35 cycles; 1 min 95° C.; 90 sec 55° C.; 65 sec 72° C.; see Tab. 1 for primers). The primers used as shown in Table 1 as SEQ ID No.: 2-5. Ligation and transformation took place by standard methods.
EXAMPLE 3
Purification of hexa-His-THP(D) from Streptomyces lividans 
YEME medium (34.0 g/l sucrose, 10 g/l glucose, 3 g/l yeast extract, 3 g/1 malt extract, 5 g/l Bacto peptone and 0.2% MgCl2) was inoculated to an OD600 of 0.05 from a preculture of the Streptomyces lividans strain (transformed with pSPIJthpD, see Example 2) and incubated at 28° C. in a shaker. The cells were harvested after 72 hours. The cells were resuspended in disruption buffer (100 mM phosphate buffer pH 8.0, 10% glycerol, 1 mM benzamidine, 1 mM PMSF, 10 mM imidazole and 300 mM NaCl) and disrupted by passing through a French press cell (16 000 psi; equivalent to 1.105×108 Pa) twice. The cell-free crude extract was centrifuged in an SS34 rotor at 12 000 rpm for 30 min. The cell-free supernatant was chromatographed on an Ni-NTA matrix. The hexa-His-THP(D) protein elutes in the region of 30-100 mM imidazole.
EXAMPLE 4
In vitro synthesis of 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A))
THP(B) dioxygenase activity is determined using the following reaction mixture:

















Potassium phosphate buffer, pH 8,
10
mM



THP (B)
5
mM



α-Ketoglutarate
5
mM



Ascorbic acid
5
mM



Iron (II) sulfate
1
mM



Hexa-His-THP (D) variable
100
μl.



in a total volume of










Incubation takes place at 30° C. for one hour. The product is analyzed by chromatography on an NH2-Nucleosil HPLC column. Elution took place with 70% acetonitrile/H2O. The elution profile is depicted in FIG. 4. Complete hydroxylation of THP(B) to THP(A) can be achieved with use of catalase (bovine liver, from Sigma).
FIG. 1: Biosynthesis of THP(B) starting from L-aspartate β-semialdehyde. A: L-2,4-diaminobutyrate transaminase, B: L-2,4-diaminobutyrate acetyltransferase, C: N-acetyl-diaminobutyric acid cyclase.
FIG. 2: Restriction map of the sequenced region and the location of the THP biosynthesis genes found. The corresponding base position is indicated under the restriction enzymes.
FIG. 3: Internal tryptic peptide sequence of the DABA acetyltransferase from Streptomyces chrysomallus and oligonucleotide sequence derived thereform (170498A and 170498B). The encoding DNA sequence and amino acid sequence shown for 170498A is SEQ ID No: 7 and SEQ ID No: 9, respectively. The encoding DNA sequence and amino acid sequence shown for 170498B is SEQ ID No: 8 and SEQ ID No: 10, respectively.
FIG. 4: HPLC of THP(A) and THP(B) on an NH2-Nucleosil column. In each case 1/10 of the reaction mixture which contained 5 mM THP(B), 5 mM a-ketoglutarate, 5 mM ascorbic acid, 1 mM FeSO4 and 2 mg/ml THP(D) fusion protein (A) or buffer D (B) was loaded. Elution took place with 70% acetonitrile at a flow rate of 1 ml/min.
FIG. 5: pSPIJ002: shuttle vector produced by ligation of pSP72 (BglII) and pIJ702 (BglII). This expression vector can be used both in E. coli and in Streptomyces. 
Tab. 1: PCR primers used for constructing plasmid pQE30thpD and pSPIJthpD.
PQE30thpD
FTHPD (30-mer):
5′-GCC TGA GGA TCC ATG ACC ACC GAA GTA CGC-3′
(SEQ ID NO.: 2)
RTHPD (27-mer):
5′-CCC GCC CGT GAA GCT TGC TAC TTC ACC-3′
(SEQ ID NO.: 3)
PSPIJthpD
FSPIJthp (30-mer):
5′-GAG GAG GAA TTC AGC ATG CGA GGA TCG CAT-3′
(SEQ ID No.: 4)
RSPIJthp (25-mer):
5′-AGT CCA AGC TCA GCT AAT TAA GCT T-3′
(SEQ ID No.: 5)

## Claims

1. An isolated nucleic acid that encodes a polypeptide having tetrahydropyrimidine dioxygenase activity, wherein said nucleic acid is a DNA nucleic acid having the nucleotide sequence depicted as SEQ ID NO: 1.

2. The nucleic acid of claim 1, wherein the nucleic acid has been isolated from an archaebacterium, a prokaryote cell or a eukaryote cell.

3. The nucleic acid of claim 2, wherein the nucleic acid has been isolated from a halophilic eubacterium, an actinomycete, a bacillus or a brevibacterium.

4. A nucleic acid construct, wherein the construct comprises in an operative unit a promoter sequence and the nucleic acid of claim 1.

5. The nucleic acid construct of claim 4, wherein the construct further comprises a nucleic acid sequence which codes for one or more secretion signals.

6. An isolated polypeptide encoded by the nucleic acid of claim 1 wherein said polypeptide has tetrahydropyrimidine dioxygenase activity.

7. A replicative recombinant vector comprising the nucleic acid of claim 1 or the nucleic acid construct of claim 4.

8. An isolated cell comprising the replicative recombinant vector of claim 7.

9. An isolated cell having a genome, wherein the nucleic acid of claim 1 is integrated into the genome by non-natural recombination.

10. The cell of any one of claims 8 or 9, wherein the cell is a bacterial cell, a yeast cell or a plant cell.

11. A plant transformed with a recombinant DNA comprising the nucleic acid of claim 1.

12. A method of preparing a polypeptide having tetrahydropyrimidine dioxygenase activity, comprising:
providing a cell according to claim 8, wherein the cell produces a polypeptide having tetrahydropyrimidine dioxygenase activity;
cultivating the cell in a medium; and
isolating the polypeptide from the cell.

13. The method of claim 12 wherein the cell has been permeabilized, and wherein tetrahydropyrimidine is present in the culture medium.