Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Reexamination Certificate
1999-05-26
2001-01-23
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
C435S252300, C435S252320, C435S252330, C435S419000, C435S325000, C435S320100, C435S254110, C536S023100, C536S023200
Reexamination Certificate
active
06177264
ABSTRACT:
BACKGROUND OF THE INVENTION
Pantothenic acid is a commercially significant vitamin which is used in cosmetics, medicine, human nourishment and in animal nourishment.
Pantothenic acid can be produced by chemical synthesis or biotechnologically by the fermentation of suitable microorganisms in suitable nutrient solutions. The advantage of the biotechnological production with microorganisms resides in the formation of the desired stereoisomeric D-form of pantothenic acid.
Various types of bacteria such as, for example,
Escherichia coli, Corynebacterium erythrogenes, Brevibacterium ammoniagenes
and also yeasts such as, for example,
Debaromyces castellii
can produce D-pantothenic acid in a nutrient solution containing glucose, DL-pantoic acid and &bgr;-alanine, as is shown in EP-A 0,493,060. EP-A 0,493,060 also shows that the formation of D-pantothenic acid is improved in the case of
Escherichia coli
by amplification of pantothenic-acid biosynthetic genes by means of the plasmids pFV3 and pFV5.
EP-A 0,590,857 is relative to strains of
Escherichia coli
which carry resistances against various antimetabolites such as, salicylic acid, &agr;-ketobutyric acid, &bgr;-hydroxyaspartic acid, etc. and produce D-pantoic acid and D-pantothenic acid in a nutrient solution containing glucose and &bgr;-alanine. EP- 0,590,857 also describes that the production of D-pantoic acid and D-pantothenic acid in
E. coli
can be improved by amplification of pantothenic-acid biosynthetic genes (not defined in detail), from
E. coli
which are contained on the plasmid pFV31.
Moreover, WO 97/10340 shows that the production of pantothenic acid can be further increased in mutants of
Escherichia coli
forming pantothenic acid by elevating the activity of the enzyme acetohydroxy-acid synthesis II, an enzyme of valine biosynthesis.
SUMMARY OF THE INVENTION
The invention addresses the problem of making available novel and improved methods for the fermentative production of D-pantothenic acid with the aid of coryneform bacteria.
The vitamin pantothenic acid is a commercially significant product which is used in cosmetics, medicine, human nourishment and in animal nourishment. There is therefore general interest in making available improved methods of producing pantothenic acid.
When D-pantothenic acid or pantothenic acid or pantothenate are mentioned in the following text not only the free acid but also the salts of D-pantothenic acid such as, for example, the calcium salt, sodium salt, ammonium salt or potassium salt are meant.
Subject matter of the invention includes optionally recombinant DNA from Corynebacterium, which can be replicated in microorganisms of the genus Corynebacterium, containing at least one of the following nucleotide sequences:
a) Encoding the panB gene (ketopantoate hydroxymethyltransferase), in the SEQ ID NO:1,
b) Encoding the panC gene (pantothenate synthetase), set forth in SEQ ID NO:1, especially the panBC operon and, if necessary,
c) Encoding the ilvD gene (dihydroxy-acid dehydratase), prepared via the SEQ-ID No. 4.
Subject matter of the invention also includes replicative DNA according to cited claim
1
with:
(i) The nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:4,
(ii) Sequences which correspond to variants of the particular sequences (i) within the degeneracy of the nucleic acid code or
(iii) Sequences which hybridize with the sequences complementary to particular sequences (i) or (ii), and optionally
(iiii) functionally neutral sense mutations in (i).
Coryneform microorganisms, especially of the genus Corynebacterium, transformed by the introduction of one or several replicative DNA pieces are likewise included in the invention.
Subject matter of the invention also includes a method of producing D-pantothenic acid using especially coryneform bacteria which already produce this acid and in which the genes panB and panC are enhanced, in particular by overexpression individually or in combination with one another, optionally combined with a defect mutation in the ilvA gene or with an enhancement of the genes ilvBN, ilvC or ilvD.
The concept “enhancement” describes in this connection the elevation of the intracellular activity of one or several enzymes in a microorganism which are coded by the corresponding DNA in that, for example, the copy number of the gene(s) is increased, a strong promoter is used or a gene is used which codes for a corresponding enzyme with a high activity or optionally a combination of these measures.
The microorganisms constituting the subject matter of the present invention can produce pantothenic acid from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol, especially from glucose or saccharose. This involves coryneform bacteria, for example, of the genera Corynebacterium or Arthrobacter. In the genus Corynebacterium the species
Corynebacterium glutamicum
is mentioned in particular, which is known for its ability to form amino acids. This species includes wild-type strains such as, for example,
Corynebacterium glutamicum
ATCC13032,
Brevibacterium flavum
ATCC14067,
Corynebacterium melassecola
ATCC17965 and strains derived from them.
The present inventors discovered that D-pantothenate is produced in an improved manner after enhancement, especially overexpression, of the newly isolated D-pantothenate biosynthetic genes panB and panC individually or in common (panBC operon) from
Corynebacterium glutamicum,
which code for the enzymes ketopantoate hydroxymethyltransferase and pantothenate synthetase.
The inventors further determined that an enhanced expression of the novel valine biosynthetic gene ilvD from
Corynebacterium glutamicum,
which codes for the enzyme dihydroxy-acid dehydratase, contributes to an elevated formation of D-pantothenate. According to the invention, in addition to this gene the enhanced expression of the ilvBN genes, which code for the enzyme acetohydroxy-acid synthase, and of the ilvC gene, which codes for the enzyme isomeroreductase, also brings about an elevated formation of D-pantothenate in
Corynebacterium glutamicum.
In order to achieve an enhancement (overexpression), for example, the copy number of the corresponding genes is elevated or the promoter and regulation region, which is located upstream from the structural gene, is mutated. Expression cassettes which are inserted upstream from the structural gene operate in the same manner. It is additionally possible to increase the expression in the course of the fermentative formation of D-pantothenate by inducible promoters. The expression is likewise improved by measures for extending the life of m-RNA. Furthermore, the enzymatic activity is likewise enhanced by preventing the degradation of the enzymatic protein. The genes or gene constructs are present thereby either in plasmid vectors with different copy number or are integrated in the chromosome and amplified. Alternatively, an overexpression of the genes concerned can be achieved by altering the composition of the media and conduction of the culture. The expert in the art will find instructions for this in, among others, Martin et al., (Bio/Technology 5, 137-146 (1987)), in Guerrero et al., (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga, (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al., (Gene 102, 93-98 (1991)), in European patent EPS 0,472,869, in U.S. Pat. No. 4,601,898, in Schwarzer and P{umlaut over (u)}hler (Bio/Technology 9, 84-87 (1991)), in Reinscheid et al., (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al., (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) or in the manual “Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington, D.C., USA, 1981) and in known textbooks of genetics and molecular biology.
In order to isolate the genes panB and panC from
C. glutamicum,
at first a gene bank of this microorganism is established in
E. coli
. The establishme
Eggeling Lothar
Sahm Hermann
Thierbach Georg
Achutamurthy Ponnathapu
Degussa-Huls Aktiengesellschaft
Kerr Kathleen
Pillsbury Madison & Sutro LLP
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