Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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GENES FROM CORYNEHACTERIUM GLUTAMICUM FOR THE BIOSYNTHESIS
OF FOLIC ACID AND THEIR USE FOR THE MICROBIAL PRODUCTION
OF FOLIC ACID
The present invention is concerned with the process for
producing folic acid by fermentation using a geneti-
cally manipulated organism. This invention consists of
the nucleotide sequences of four genes (folE', folP,
folB and folK) from Corynebacterium glutamicum for
folic acid 3~iosynthesis and the use thereof for the
microbial production of. folic acid. These four genes
form an operon and are transcribed in the following
sequence : folE, folP, folB, folK.
Folic acid is essential for animal organisms. Its
derivative tetrahydrofolate is a very versatile carrier
of activated one-carbon units in cells of the animal
organism. Folic acid consists of three groups: a sub
stituted pteridine ring, p-aminobenzoate and glutamate.
Mammals are unable to synthesize a pteridine ring. They
absorb folic acid from the diet and from microorganisms
in their intestinal tract. Folic acid deficiency leads
mainly to lesions in the mucous membranes.
The commercial importance of folic acid is in the
animal feed and human food markets. Folic acid is
employed mainly as a dietary supplement.
Microorganisms can be employed for the fermentative
production of folic acid. They can be optimized in
their efficiency of folic acid biosynthesis by genetic
manipulation of the folic acid biosynthetic pathway.
Genetic manipulation means in this connection that the
number of copies and/or the rate of transcription of
the genes of the folic acid biosynthetic pathway i-s
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increased. As a consequence thereof, the proportion of
gene product increases,, and thus the intracellular
enzymic activity doss too. Increased enzymic activity
leads to ~n increased rate of dietary (e. g. glucose)
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conversion into folic acid and thus also to an
increased product concentration. For genetic manipula-
tion, the nucleotide sequences of the genes of the
folic acid biosynthetic pathway must be identified.
This invention is concerned with four novel gene
sequences for folic acid biosynthesis from
Corynebacterium glutamicum and with their use for the
microbial production of folic acid.
One part of the invention comprises the fo1~ gene
product. SEQ ID N0. 2 describes a polypeptide sequence.
The folE gene product encodes a polypepti.de of 202
amino acids with a molecular weight of 22 029 Da. The
present invention is also concerned with functional
derivatives of this polypeptide obtainable by replacing
one or more amino acids, preferably up to 25~ of the
amino acids, most suitably up to 15~ of the amino
acids, in SEQ ID NO. 2 by deletion, insertion or sub-
stitution or by a combination of deletion, insertion
and substitution. The term functional derivative means
that the enzymatic activity of the derivative is still
of the same order of magnitude as that of the poly-
peptide having the sequence SEQ ID N0. 2.
Another part of the invention comprises the folP gene
product. SEQ ID N0. 4 describes a polypeptide sequence.
The folP gene product encodes a polypeptide of 285
amino acids with a molecular weight of 29 52~ Da. The
present invention is also concerned with functional
derivatives of this polypeptide obtainable by replacing
one or more amino acids, preferably up to 40~ of the
amino acids, most suitably up to 25~ of the amino
acids, in SEQ ID N0. 4 by deletion, insertion or sub-
stitution or by a combination of deletion, insertion
and substitution. The term functional derivative means
that the enzymatic activity of the derivative is still
of the same order of magnitude as that of the poly-
peptide having the sequence SEQ ID NO. 4.
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Another part of the invention comprises the folB gene
product. SEQ ID NO. 6 describes a polypeptide sequence.
The folB gene product encodes a polypeptide of 131
amino acids with a molecular weight of 14 020 Da. The
present invention is also concerned with functional
derivatives of this polypeptide obtainable by replacing
one or more amino acids, preferably up to 30~ of the
amino acids, most suitably up to 20~ of the amino
acids, in SEQ ID N0. 6 by deletion, insertion or sub-
stitution or by a combination of deletion, insertion
and substitution. The term functional derivative means
that the enzymatic activity of the derivative is still
of the same order of magnitude as that of the poly-
peptide having the sequence SEQ ID N0. 6.
Another part of the invention comprises the folK gene
product. SEQ ID N0. 8 describes a polypeptide sequence.
The folFC gene product encodes a polypeptide of 160
amino acids with a molecular weight of 18 043 Da. The
present invention is also concerned with functional
derivatives of this polypeptide obtainable by replacing
one or more amino acids, preferably up to 40~ of the
amino acids, most suitably up to 30~ of the amino
acids, in SEQ ID N0. 8 by deletion, insertion or sub-
stitution or by a combination of deletion, insertion
and substitution. The term functional derivative means
that the enzymatic activity of the derivative is still
of the same order of magnitude as that of the poly-
peptide having the sequence SEQ ID N0. 8.
Another part of the invention comprises the polynucleo-
tide sequences which encode the polypeptides described
above. The polynucleotide sequences can be generated
starting from sequences isolated from Corynebacterium
glutamicum (i.e. SEQ ID NaS. 1, 3, 5 and 7) in which
these sequences are modified by site-directed
mutagenesis or a total chemical synthesis is carried
out after back-translation of the corresponding
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polypeptide using the genetic code.
These polynucleotide sequences can preferably be
employed for the transformation of host organisms, and
in this connection preferably of microorganisms,
specifically in the form of gene constructs which
comprise at least one copy of one of these polynucleo
tides together with at least one regulatory sequence.
Regulatory sequences comprise promoters, terminators,
enhancers and ribosome binding sites.
Preferred host organisms for transformation with these
gene constructs are Corynebacterium and Bacillus
species. It is also possible to employ any eukaryotic
microorganism, preferably yeast strains of the genus
Ashbya, Candida, Pichia, Saccharomyces and Hansenula.
Another part of the invention comprises the process for
producing folic acid by cultivating a host organism
which is transformed in the manner described above, and
subsequently isolating the folic acid.
The processes and the procedures for cultivating micro
organisms and for isolating folic acid from a microbial
production are familiar to trained staff.
The invention is described. in more detail in the
following examples, as is its use for the genetic
manipulation of microorganisms, to increase the
efficiency of folic acid synthesis.
Example 1
Construction of a genome library from Corynebacterium
glutamicum ATCC 13032
DNA from the genome of Corynebacterium glutamicum
ATCC 13D32 can be obtained by standard methods which
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have already been described, for example by
J. Altenbuchner and J. Cullum (1984, Mol. Gen. Genet.
195: 234-13B). The genome library can be produced in
accordance with standard protocols (e.g. Sambrook, J.
et al. (1989) Molecular cloning: a laboratory manual,
Cold Spring Harbor Laboratory Press) with any cloning
vector, e.g. pBluescript II KS- (Stratagene) or ZAP
Expresses (Stratagene). It is moreover possible to use
any fragment size, preferably Sau3AI fragments with a
length of 2-9 kb, which can be incorporated into
cloning vectors with digested BamXI.
Examp 1 a 2
Analysis of the nucleic acid sequence of the genome
library
Individual E. coli clones can be selected from the
genome library constructed in example 1. ~'. coli cells
are cultivated by standard methods in suitable 'media
(e.g. LB supplemented with 100 mg/1 ampicillin), and
the plasmid DNA can then be isolated. Cloning of genome
fragments from the DNA of Corynebacterium glutamicum
into pBluescript II KS- (see example 1) allows the DNA
to be sequenced with the aid of the oligonucleotides
5'-AATTAACCCTCACTAAAGGG-3' and
5'-GTAATACGACTCACTATAGGGC-3'..
Example 3
Computer analysis of the sequences of the isolated
nucleic acids
The nucleotide sequences can be connected together for
example With the aid of the BLASTX algorithm (Altschul
et al. (1990) J. Mol. Biol. 215: 403-410). It is
possible in this way to discover novel sequences and
elucidate the function of these novel genes.
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Example 4
Identification of an E. coli clone which comprises a
nucleotide sequence of the gene for GTP cyclo
hydrolase I (EC 3.5.4.16)
Analysis of the E, coli clones as described in
example 2, which was followed by analysis, as described
in example 3, of the sequences obtained thereby
revealed a sequence which is described by SEQ ID N0. 1.
On use of the BLASTX algorithm (see example 3?, this
sequence.revealed similarity with GTP cyclohydrolases I
(FolE; EC 3.5.4.16) from various organisms. The
greatest similarity was with the GTP cyclohydrolase I
(FolE) from Mycobacterium tuberculosis (NRDB 006273;
72~ agreement at the amino acid level).
Example 5
Identification of an E. cola clone which comprises a
nucleotide sequence of the gene for dihydropteroate
synthase (EC 2.5.1.15)
Analysis of the E..coli clones as described in
example 2, which was followed by analysis, as described
in example 3, of the sequences obtained thereby
revealed a sequence which is described by SEQ ID N0. 3.
On use of the BLASTX algorithm (see example 3), this
sequence revealed similarity with dihydropteroate
synthases (FolP; EC 2.5.1.15) from various organisms.
The greatest similarity was with the dihydropteroate
synthase (FolP) from Mycobacterium tuberculosis
(NRDB 006274; 53~k agreement at the amino acid level).
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Example 6
Identification of an E. coli clone which cor~rises a
nucleotide sequence of the gene for dihydroneopt-erin
aldolase (EC 4.1.2.25)
Analysis of the E. coli clones as described in
example 2, which was followed by analysis, as described
in example 3, of the sequences obtained thereby
revealed a sequence which is described by SEQ ID NO. 5.
On use of the BLASTX algorithm (see example 3), this
sequence revealed similarity with dihydroneopterin
aldolases (FolB; EC 4.1.2.25), from various organisms.
The greatest similarity was with the dihydroneopterin
aldolase (FolB) from Mycobacterium tuberculosis
(NRDB 006275; 61~ agreement at the amino acid level).
Example 7
Identification of an E. coli clone which comprises a
nucleotide sequence of the gene for 2-amino-4-hydroxy-
6-hydroxymethyldihydropteridine pyrophosphokinase
(EC 2.7.6.3)
Analysis of the E. coli clones as described in
example 2, which Was followed by analysis, as described
in example 3, of the sequences obtained thereby
revealed a sequence which is described by SEQ ID N0. 7.
On use of the BLASTX algorithm (see example 3), this
sequence revealed similarity with 2-amino-4-hydroxy-
6-hydroxymethyldihydropteridine pyrophosphokinases
(FolK; EC 2.7.6.3) from various organisms. The greatest
similarity was with the 2-amino-4-hydroxy-6-hydroxy-
methyldihydropteridine pyrophosphokinase (FolK) from
Mycobacterium leprae (ENBL AL023093; 43~ agreement at
the amino acid level).
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Example 8
Use of the genes for GTP cyclohydrolase I, for
dihydropteroate synthase, for dihydroneopterin aldolase
and for 2-amino-4-hydroxy-6-hydroxymethyldihydro-
pteridine pyrophosphokinase from Corynebacterium
g~utamicum for producing folic acid
The genes for GTP cyclohydrolase I, for dihydropteroate
synthase, for dihydroneopterin aldolase and for
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine
pyrophosphokinase from Corynebacteriurri glutamicum can
be introduced with the aid of suitable cloning and
expression systems into Corynebacterium glutamicum or
into any other microorganism. Genetically manipulated
microorganisms which differ from the wild-type organism
in relation to the activity or the number of gene
copies can be produced. These novel genetically
manipulated strains can be employed for producing folic
acid.
Sequence list
(I) General information
(1) Applicant:
(A) Name: BASF-LYNX Bioscience AG
(B) Street: Im Neuenheimer Feld 515
(C) City: Heidelberg
(D) Country: Germany
(E) Postal code: 69120
(F) Telephone: 06221/4546
(G) Fax: 06221/454770
(2) Title: Genes from Corynebacterium glutamicum
for folic acid biosynthesis and their
us.e for the microbial production of
folic acid
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(3)~ Number of sequences: 8
SEQ ID N0. l: DNA (folE)
ATGAAGGAGACAACCGTGGATAACCACvCTGCAGTTCGCGAGTTCGATGAGGAGCGCGCAACAGC
TGCGATTCGTGAGTTGCTCATCGCTGTC~GTGAGGATCCAGATCGCGAAGGCCTGTTGGAAACCC
CAGCTCGAGTGGCTAGGGCGTACAAGGA.AACT'fTCGCGGGTCTGCATGAGGATCCCAGCACTGTG
CTGGAGAAGAGGTTCTCTGAGGGCCATGAAGAGTTGGTTCTGGTTCGTGAGATCCCGATTTACTC
CATGTGTGAGCACCACTTGGTGCCGTTCTTTGGCGTGGCGCAGATTGGTTACATTCCGGGTAAGT
CCGGCA,r~.GGTGACTGGCCTGTCCAAGCTGGCGCGTTTAGCGGATATGTTTGCTrIP.GCGACCTCRG
G'I"?'CAGGAGCGCTTGACCTCCCAAATTGCGGATGCTCTCGTCGAAAAGCTTGATGCCCAGGCCGT
GGCCGTGGTGATTGAAGCTGAGCACCi'GTGCATGGCCATGCCCGGAATCCGTAAGCCTGGTGCTG
TGACCACGACGTCTGCGGTGCGCGGCGGTTTTAAGR.ACp.ACCCTGCCTCCCGCGCTGa~GGTGTrC
TCCCTGATTCGGGGGCACTAA
SEQ ID N0. 2: amino acid (FolE)
P'~CETTVDNf~AAVREFLE~RATAAIn~LL IAVt;EJ FDtt~GLL ~T
PARVARAYK~TFAGLFiEDi~'!'Til
LEKTP S EGHEELVLVREI P I YSMCEHFi.L VFI'FGVAHIGYI PGKSGKVTGLSXLARLADI~'A3CRPQ
VQ~RLTSQ LADALVEKLDAQAVRWIEA~HLCMANRGIRKPGAVTTTSAVRGGFKNNAASRAEVF
SLiRGH
SEQ ID NO. 3: DNA (folP)
ATGAACGTATCCTCTTTGACCATCCCGGGACGCTGTTTGGTCATGGGAAT'I'GTCAATGT~CACTGA
GGATTCCTTTTCGGACGGTGGCAAGT'ACATTGACGTTGATCAGGCGATCGCGCATGCCAAGGAAT
TGGTGGCTGCTGGCGCCGACATGRTTGATGTCGGCGGCGAGTCCACCCGGCCTGGGGCAGTGCGC
GTCGACGCCTCCGTGGAACGGGACCGGGTTG?GCCGGTCATTAAGGCGCTTCACGACGCCGGCAT
CC.aCACTTCCGTAGACACCATGCG~vGCCTCCGTGGCGCAGGCTGCCGCGGGCGCTGGCGTCTCCA
TGATCAACGACGTCTCTGGCGGTTTGGCTGATCCTGAGATGTTTTCTGTCATGflCGGAAGCGCAA
ATTCCCGTGTGTTTGATGCAC:'GGCGCACCCTCC.AATTCGGTGATGCCGCAGGTCAGGCAGA?'CA
CGGTGGAGACGTTGTAGCCGATGTGCACGCAGTGCTTGATGATCTTGTGGCCCC..CGCCACCGCTG
CTGGTGTGGCCGAAAACCAGATCGTGCT'TGATCCAGGTTTGGGTTTTGCCAAATCACGTGAAGAC
AACTGGCGTTtGCTGCAAGCACTGCCCGAGTT'TATTTCTGGACCTTTL'CCCATCCTGGTGGGAGC
ATCCCGGAAGCGAiTCCTGGCTGGCGT.'GCGCAAAGACCGTGGCCTAGATGTCACCCCCATTGATG
CCGACCCAGCAACCGCAGCGGTGACCGCAGTGTCTGCACATATGGGAGCATGGGGTwTGCGCGTG
CACGATGTCCCAGTATCAAGGGAGGCTGTTGATGTTGCCGCATTGTGGCGAAGTGGAGGAACTCA
CCATGGCTGA
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SEQ ID NO. 4: amino acid (FolP)
t~IVSSLTIPGRCLVMGIVNVTEDSFSDG: tcYIDvDQA:.~AKEL'~rAAGADMIDVGGESTRPGAVR
VDASVER,DRWPVIKAJ~F3DAGIh3' SvDT?~tASVAQAAAGAGVSid'INDVSGGLADPEI~SVMAEAQ
I PVCLI~iWk T LQFGDAAGQADtiGGDWADVHAVLDDLVAP,ATAAGVA~~QIV'LDPGLGFAICSR~D
NWRLLQALpEFISGPFPILVGASRK.kFLAGVRXDRGLDVTPIDADPATAAtITAVSAf~IGAWGVRV
HI~VPVSRL~AVDVAAL4PRSGGTFyiG
SEQ ID N0. 5: DNA (FolB)
ATGGCTGATCGTATTG.AACTTP.AAGGCCTTGAATGCTTCGGACACCACGGTGTGTTCGACTTTGA
AAAAGAGCAAGGCCAGCCCTTCATTGTGGATGTCACCTGCTGGATGGATTTCGATGCCGCAGGTG
CCAC-CGATGACCTTTCCCACACCG'CACATTACGGCC-CGTTGC~CATTGTTGGTTGCTGAA.ATCGTG
GAAGGCCCATCCAGGGATTTGATCGAGACGGTGGCCACGGAATCTGCGGATGCTGTGATGGCTAA
ATTTGATGCGCTTCATGCGGTGGAAGTAACCATCCATAAGC~CCF.AAGCACGGATCCCACGTACTT
TTGCTGACGTCGCGGTGGTTOCCCGACGTTCCAGGRAATCCt~.TGGC'I'~CTGGAAGGAGCAACGCC
TAA
i0
SEQ ID N0. 6: amino acid (FolB)
MADkI ELICGL ~C FG~:r:GVFDFEICEQGQ c~F I VDV3'CWI~ FDAAGASDDLSDTVDYGALALLVAEIV
EGPSR.DL IETVATE SADAVMaXFDAL-t'.RV~VT I f:KPKA.P I PRTFADVAWARRSRKSi~A,AGRSNA
SEQ ID NO. 7: DNA (fold)
ATGCATGCAGTTTTGTCCATCGGTTCCAACATGGATGATCGCTACGCGCTGCTCAACACAGTGAT
CGAGGAATTCAAAGATGAGATCGTGGCGCAGTCTGCGATCTe~.CTCAACCCCACCGTGGGGCATT'G
AGGATCAGGATGAATTCC'iCAACGCAGTGCTCGTTGTTGAGGTTGAAGAAACCCCCATCGAGTTG
CTGCGCCGTgGCCAAAAACTCGAAGAAGCCGCCGAGCGGGTCCGCGTCCGCAAATGGGGGCCACG
CACCCTCGATGTGGATATCGTGCAGATCATTAAAGATGGGGAAGAGATCCTTTCTGAGGATCCCG
AACTGACCTTGCCACACCCTTGGGCTTGGCAGCGTGCCTTCGTGTT'GATCCCTTC,~GTTGGAA~GCA
GAACCTGATGCCGTCCTGCACCGCACGACCATTGCAGAACATGTGGATAATCTTGATCCGACAGA
CATTGAAGGTGTCACCA4GATTTAA
SEQ ID N0. 8: amino acid (folK)
M~IAVLSTGSNt~DRYALLNTVIEEFKD~IVAQSAIYSTPPWGIEDQDEFLNAVLWEVE~TPIEL
LRRGQKLEEAAERVRVRKWGPRTLDVDIVQZIKDGLEZLSEDPELTLPFi~PWAWQRAFVLIPW'.,EA
E FDAVLHGT :'IAEHVDN'.,,DPTD i'FGVTKI
