Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Reexamination Certificate
1999-05-26
2001-02-06
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
C435S252320, C435S252330, C435S252300, C435S254110, C435S325000, C435S419000, C435S320100, C536S023100, C536S023200
Reexamination Certificate
active
06184007
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. DL-pantolactone is an important intermediate stage in the chemical synthesis. It is produced in a multi-stage process from formaldehyde, isobutylaldehyde and cyanide. In further method steps the racemic mixture is separated and D-pantolactone condensed with &bgr;-alanine and D-pantothenic acid obtained. The advantage of the fermentative production with microorganisms resides in the direct formation of the desired stereoisomeric D-form, which is free of L-pantothenic acid.
Various types of bacteria such as, for example,
Escherichia coli, Arthrobacter ureafaciens, 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 from
E. coli
, which are contained on the plasmids pFV3 and pFV5, in a nutrient solution containing glucose, DL-pantoic acid and &bgr;-alanine.
EP-A 0,590,857 and U.S. Pat. No. 5,518,906 describe mutants derived from
Escherichia coli
strain IFO3547 such as FV5714, FV525, FV814, FV521, FV221, FV6051 and FV5069 which carry resistance genes against various antimetabolites such as salicylic acid, &agr;-ketobutyric acid, &bgr;-hydroxyaspartic acid, O-methylthreonine and &agr;-ketoisovaleric acid and produce D-pantothenic acid in a nutrient solution containing glucose, pantoic acid and in a nutrient solution containing glucose and &bgr;-alanine. E-A 0,590,857 and U.S. Pat. No. 5,518,906 also show that after the amplification of the pantothenic-acid biosynthesis genes contained on the plasmid pFV31, in the strains cited above, the production of D-pantoic acid is improved in a nutrient solution containing glucose and that the production of D-pantothenic acid is improved in a nutrient solution containing glucose and &bgr;-alanine.
Moreover, WO97/10340 shows that the production of pantothenic acid can be further increased in strains of
Escherichia coli
forming pantothenic acid by elevating the activity of the enzyme acetohydroxy-acid synthase II, an enzyme of valine biosynthesis.
SUMMARY OF THE INVENTION
It is an object of the invention to provide novel bases for improved methods for the fermentative production of pantothenic acid.
Pantothenic acid, or Vitamin B3, 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 novel 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, among other things, a method for the fermentative production of D-pantothenic acid using microorganisms which, in particular, already produce D-pantothenic acid and in which the panD gene coding for L-aspartate-1-decarboxylase (E.C. 4.1.1.11) is enhanced, especially overexpressed individually or in combination with the genes panB and/or panC. The invention relates to corresponding recombinant DNA sequences like those documented in the claims. The invention also includes methods for the fermentative production of D-pantothenic acid which are carried out using the improved microorganisms produced according to the invention which produce D-pantothenic acid.
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 and optionally combines 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. They can be fungi or yeasts or Gram-positive bacteria, for example, of the genus Corynebacterium, or Gram-negative bacteria such as, for example, those of the Enterobacteriaceae. In the family of Enterobacteriaceae the genus Escherichlia with the species
Escherichia coli
is to be cited in particular. Within the species
Escherichia coli
the so-called K-12 strains such as, for example, the strains MG1655 or W3110 (Neidhard et al.:
Escherichia coli
and Salmonella. Cellular and Molecular Biology (ASM Press, Washington, D.C.)) or the
Escherichia coli
wild-type strain IFO03547 (Institute for Fermentation, Osaka, Japan) and mutants derived from them are to be cited. In the genus Corynebacterium especially the species
Corynebacterium glutamicum
is to be cited, which is known to the skilled artisan for its ability to produce amino acids. This species includes wild-type strains such as, for example,
Corynebacterium glutamicum
ATCC13032,
Brevibacterium flavum
ATCC14067,
Corynebacterium melassecola
ATCC17965 and mutants derived from them.
The present inventors discovered that microorganisms produce pantothenic acid in an improved manner after overexpression of the novel panD gene, especially from
Corynebacterium glutamicum
, coding for L-aspartate-1-decarboxylase (E.C. 4.1.1.11).
The inventors discovered in addition that the overexpression of the panD gene has an advantageous effect in strains in which the genes panB and panC coding for ketopantoate hydroxymethyltransferase and pantothenate synthetase are additionally present in an overexpressed state either individually or together.
In order to achieve an overexpression, the copy number of the corresponding genes can be elevated or the promoter and regulatory region, which is located upstream from the structural gene, can be 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 can be present either in plasmids with different copy number or be integrated in the chromosome and amplified. Alternatively, an overexpression of the genes concerned can furthermore be achieved by altering the composition of the media and conduction of the culture.
An 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. 4,601,893, in Schwarzer and Pühler (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 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). In addition, an expert in the art will find instructions in Chang and Cohen (Journal of Bacteriology 134: 1141-1156 (1978)), in Hartley and Gregori (Gene 13: 347-353 (1981)), in Amann and Brosi
Dusch Nicole
Kalinowski Jorn
Puhler Alfred
Achutamurthy Ponnathapu
Degussa-Huls Aktiengesellschaft
Pillsbury Madison & Sutro LLP
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