AU5685400A - Dihydroorotate dehydrogenase sequence of corynebacterium glutamicum and the use thereof in microbial production of pyrimidine and/or compounds used with pyrimidine - Google Patents

Description

0091/00004 The sequence of the dihydroorotate dehydrogenase from Corynebacterium glutamicum and the use thereof in the microbial production of pyrimidines and/or pyrimidine related compounds 5 The present invention is concerned with the process for producing pyrimidines by fermentation with the aid of a genetically manipulated organism. This invention comprises the sequence of the dihydroorotate 10 dehydrogenase from Corynebacterium glutamicum and the use thereof for the microbial production of pyrimidines and/or pyrimidine-related compounds. The biosynthetic pathway for pyrimidines is essential 15 for all living organisms (a review article on this by Switzer, R.L. and Quinn, C.L. is to be found in Bacillus subtilis (editors: Sonenshein, A.L., Hoch, J.A. and Losick, R., American Society for Microbiology, Washington, D.C.), 1993, pp. 343-358. The 20 pyrimidine nucleotides are pyrimidine derivatives and, as such, activated precursors of DNA and RNA and for many biosynthetic pathways. In the pyrimidine nucleosides cytidine, uridine, deoxycytidine and deoxythymidine, a pyrimidine base is bonded to a 25 pentose, and the pyrimidine nucleotides are phosphate esters of the pyrimidine nucleosides. Pyrimidine nucleosides and pyrimidine nucleotides and derivatives thereof are also important starting compounds for synthesizing valuable drugs such as, for example,

CDP

30 choline, orotic acid or UMP (a review article on this by Kuninaka, A. is to be found in Biotechnology, vol. 6 (editors: Rehm, H.-J. and Reed, G.), VCH, Weinheim, Germany, 1996, pp. 561-612) 35 Many, but not all, microorganisms are able to synthesize their pyrimidine nucleotides both de novo and from pyrimidine bases and pyrimidine nucleosides supplied from outside. Pyrimidine bases and/or pyrimidine nucleosides normally do not occur inside 0091/00004 - 2 cells. However, under some conditions of growth they may be formed in excess and are then secreted into the culture medium. For this reason, microorganisms can be employed for the fermentative production of pyrimidine 5 nucleotides and/or related compounds. The biosynthetic efficiency of microorganisms for pyrimidine nucleotides can be optimized by genetically manipulating the pyrimidine biosynthetic pathway. 10 Genetic manipulation means in this connection that the number of gene copies and/or the rate of transcription of the genes for the pyrimidine synthetic pathway is increased. As a consequence of this, the proportion of gene product and the intracellular enzymatic activity 15 increases. An increased enzymatic activity leads to increased conversion of compounds supplied in the nutrient medium into pyrimidine nucleotides and/or related compounds and thus increases the synthetic efficiency. Thus, it has been possible to show that, 20 for example, an increase in the activity of dihydroorotate dehydrogenase, which catalyzes the oxidation of (S)-dihydroorotate to orotate - this is the fourth stage in de novo pyrimidine biosynthesis for pyrimidine nucleotides - increases the efficiency of 25 UMP synthesis in Corynebacterium ammoniagenes (Nudler, A.A., Garibyan, A.G. and Bourd, G.I. (1991) FEMS Microbiol. Lett. 82:263-266). The invention is concerned with the novel pyrD gene for 30 the dihydroorotate dehydrogenase of the pyrimidine biosynthetic pathway from Corynebacterium glutamicum and its use for preparing pyrimidine nucleotides and/or pyrimidine-related compounds. 35 One part of the invention comprises the pyrD gene product. SEQ ID NO. 2 describes a polypeptide sequence. The pyrD gene encodes a polypeptide of 322 amino acids with a molecular weight of 33953. The present invention- 0091/00004 - 3 is, however, also concerned with functional derivatives of this polypeptide which can be obtained by replacing one or more amino acids in SEQ ID NO. 2, preferably up to 25% of the amino acids, most suitably up to 15%, by 5 deletion, insertion or substitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymic activity of the derivative is still of the same order of magnitude as that of the polypeptide having the 10 sequence SEQ ID NO. 2. Another part of the invention comprises the polynucleotide sequences which encode the polypeptides described above. The polynucleotide sequences can be 15 generated starting from sequences isolated from Corynebacterium glutamicum (i.e. SEQ ID NO. 1), in which these sequences are modified by site-directed mutagenesis or, after back-translation of the corres ponding polypeptide using the genetic code, a total 20 chemical synthesis is carried out. These polynucleotide sequences can most suitably be employed in the form of gene constructs for the transformation of host organisms, preferably of 25 microorganisms. These gene constructs consist of at least one copy of one of the polynucleotides together with at least one regulatory sequence. Regulatory sequences comprise promoters, terminators, enhancers and ribosome binding sites. 30 Preferred host organisms for transformation with these gene constructs are Corynebacterium and Bacillus species, but any eukaryotic microorganisms can also be employed for this purpose, preferably strains of yeast 35 of the genus Ashbya, Candida, Pichia, Saccharomyces and Hansenula. Another part of the invention comprises the process for 0091/00004 - 4 preparing pyrimidines and pyrimidine derivatives by cultivation of a host organism which is transformed in the manner described above, as the subsequent isolation of the pyrimidines. A pyrimidine derivative means a 5 compound having a pyrimidine ring which can be prepared by transforming a host organism with one of the polynucleotides corresponding to the present invention. The processes and procedures for cultivating micro 10 organisms and for isolating pyrimidines from a microbial production are familiar to trained staff. The following examples describe how the invention arose and its application in the genetic manipulation of 15 microorganisms for increased efficiency of production of pyrimidine nucleotides and/or related compounds. Example 1 20 Construction of a genome library from Corynebacterium glutamicum ATCC 13032 DNA from the genome of Corynebacterium glutamicum ATCC 13032 can be obtained by standard methods which 25 have already been described, for example by J. Altenbuchner and J. Cullum (1984, Mol. Gen. Genet. 195:134-138). The genome library can be prepared by standard protocols (for example: Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual, Cold 30 Spring Harbor Laboratory Press) using any cloning vector, for example pBluescript II KS- (Stratagene) or ZAP ExpressTm (Stratagene). It is moreover possible to use any size of fragments, preferably Sau3AI fragments with a length of 2-9 kb, which can be incorporated in 35 cloning vectors with digested BamHI.

0091/00004 - 5 Example 2 Analysis of the nucleic acid sequence to the genome library 5 Individual E. coli clones can be selected from the genome library produced in example 1. E. coli cells are cultivated by standard methods in suitable media (e.g. LB supplemented with 100 mg/l ampicillin), and the 10 plasmid DNA can then be isolated. If genome fragments from the DNA of Corynebacterium glutamicum are cloned into pBluescript II KS- (see example 1), the DNA can be sequenced with the aid of the oligonucleotides 5'-AATTAACCCTCACTAAAGGG- 3 ' and 5'-GTAATACGACTCACTATA 15 GGGC-3'. Example 3 Computer analysis of the sequences of isolated nucleic 20 acids The nucleotide sequences can join together for example with the aid of the BLASTX algorithm (Altschul et al. (1990) J. Mol. Biol. 215:403-410) . It is possible in 25 this way to discover novel sequences and elucidate the function of these novel genes. Example 4 30 Identification of an E. coli clone which comprises the gene for dihydroorotate dehydrogenase (EC 1.3.3.1) Analysis of the E. coli clones as described in example 2, which was followed by analysis, as described 35 in example 3, of the sequences obtained thereby, revealed a sequence as described by SEQ ID NO. 1. Use of the BLASTX algorithm (see example 3) revealed that this sequence was similar to the dihydroorotate 0091/00004 - 6 dehydrogenase (PyrD; EC 1.3.3.1) from various organisms. The greatest similarity was with the dihydroorotate dehydrogenase from Mycobacterium leprae (SWISSPROT p 4 6 7 2 7 ; 67% agreement at the amino acid 5 level). Example 5 Use of the gene for the dihydroorotate dehydrogenase 10 (pyrD) from Corynebacterium glutamicum for producing pyrimidine and/or pyrimidine-related compounds The gene for the dihydroorotate dehydrogenase from Corynebacterium glutamicum can be introduced with the 15 aid of suitable cloning and/or expression systems into Corynebacterium glutamicum or into any other microorganism. It is possible to produce genetically manipulated microorganisms which differ from the wild type in the activity or the number of copies of the 20 genes. These novel, genetically manipulated strains can be employed for producing pyrimidine and/or pyrimidine related compounds. Sequence list 25 (I) General information (1) Applicant 30 (A) Name: BASF-LYNX Bioscience AG (B) Street: Im Neuenheimer Feld 515 (C) City: Heidelberg (D) Country: Germany (E) Postal code: 69120 35 (F) Telephone: 06221/4546 (G) Fax: 06221/454770 0091/00004 - 7 (2) Title: The sequence of the dihydroorotate dehydrogenase from Corynebacterium glutamicum and the use thereof in the microbial production of pyrimidines and/or pyrimidine-related compounds 5 (3) Number of sequences: 2 (4) Type of computer-readable form: 10 (A) Medium type: diskette (B) Computer: IBM PC compatible (C) Operating system: Windows NT (D) Software: Microsofteword 97 SR-1 15 (I) Information on SEQ ID NO. 1: (1) Sequence characteristics: (A) Length: 966 20 (B) Type: nucleic acid (C) Strand type: double strand (D) Topology: linear (2) Molecule type: DNA 25 (3) Hypothetical: no (4) Antisense: no (5) Source: (A) Organism: Corynebacterium glutamicum 30 (6) Description of the sequence: SEQ ID NO. 1: 0091/00004 - 8 ATGAAACTGATC~-T~ TCCCCC-GAT:TGCTACTC AACCAAGGGTTCCCGC TGAACACGCAGTAGAGCTACCGCCTCTGCAICT'ITTAGGT CACTCGT&CCGTTACTTCTCC~AATCGTTCCACGC GGCTGTGAACTTTGCGACCAATCCTCGCCGCAGTGCAG'cCACCACCGTCCCGTCTG AC~TAc~cTGAGAT~CCGCCCA<TAcGCAGCCGATCTTTGGA'r' ACGGTCTGCGCAACATCGCACGCTCTGGGCAGCGTTGGAGTGGACT)A (I) Information for SEQ ID NO. 2: 5 (1) Sequence characteristics: (A) Length: 322 (B) Type: amino acid (C) Strand type: one chain 10 (D) Topology: linear (2) Molecule type: amino acid (3) Hypothetical: no (4) Antisense: no 15 (5) Source: (B) Organism: Corynebacterium glutamicwn (6) Description of the sequence: SEQ ID NO. 2: 20 MEKZI-AVHDOSLCQEFGVTFPRPLGLAAGDKNASMADAWGAVGF'GYAELG'rV1A5PQPGNPTP RLRPDINNMTCAVKLNRSDIIIKXLA VDRSSL DLDLVVSNPLDAESRIAAVETVVVIFLDDIAALV' XLAC :VATNTTI SREGLNTPSGEVF) IGAGGI SGAPVMARLEVLKRLYARVGKEMVLISVGGIS TPEQAWERITSGATLLVGyvTPFIYGGPDWIRlIHLGIAKOLKAHGLRNX).fAVGSELEWN

Claims (5)

1. A polypeptide having dihydroorotate dehydrogenase activity and selected from the following group: 5 (a) a polypeptide having an amino acid sequence as described in SEQ ID NO. 2, (b) a polypeptide which has been modified by. 10 comparison with (a) by deletion, insertion or substitution of one or more amino acids.

2. A polynucleotide which encodes a polypeptide corresponding to claim 1. 15

3. A gene construct which comprises at least one copy of a polynucleotide corresponding to claim 2, together with at least one regulatory sequence. 20

4. A host organism which is transformed with a gene corresponding to claim 3.

5. A process for producing pyrimidines and pyrimidine derivatives, in which a host organism corres 25 ponding to claim 4 is cultivated and subsequently the pyrimidine or the pyrimidine derivative is isolated.