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
C435S252330
Reexamination Certificate
active
06184006
ABSTRACT:
BACKGROUND INFORMATION
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 D-form of pantothenic acid, 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, 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 are relative to 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 also shows that after the amplification of general 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 after increasing the activity of the enzyme acetohydroxy-acid synthase II by amplification of the ilvGM gene by means of plasmid pFV202, an enzyme of valine biosynthesis, 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.
It can not be gathered from the texts cited to what extent the cited strains produce pantothenic acid in a nutrient solution containing solely glucose or solely saccharose as substrate.
SUMMARY OF THE INVENTION
The present inventors had the objective of further improving strains of the family Enterobacteriaceae, especially of the genus Escherichia, which form pantothenic acid.
Pantothenic acid, or vitamin B, 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 included.
Subject matter of the invention is constituted by a method of producing D-pantothenic acid by fermentation of microorganisms of the family Enterobacteriaceae, especially of the genus Escherichia, which produce D-pantothenic acid, which method is characterized in that the microorganisms
a) Contain the plasmid pFV31 and/or pFV202, preferably pFV31, and in which
b) The panD gene and optionally other nucleotide sequences coding for aspartate-1-decarboxylase (E.C. 4.1.1.11) are enhanced, preferably overexpressed.
It is preferable to use microorganisms with DNA whose nucleotide sequence codes for panD with an origin from
E. coli,
and also from coryneform bacteria. A replicative DNA is preferable, in which
(i) The nucleotide sequence, shown in SEQ ID NO:1, codes for panD, or
(ii) The latter corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) The latter hybridizes with a sequence complementary to sequence (i) or (ii) and, optionally,
(iiii) The latter carries functionally neutral sense mutations in (i).
The fermentation preferably takes place in a nutrient solution which exclusively contains glucose or saccharose as substrate and is free of &bgr;-alanine and pantoic acid.
The production of pantothenic acid is improved in this manner in accordance with the invention. The addition of aspartate, &bgr;-alanine, ketoisovalerate, ketopantoic acid or pantoic acid and/or their salts is desired if necessary.
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. Glucose or saccharose is preferably used. This includes, in particular, Gram-negative bacteria such as, for example, those of the family Enterobacteriaceae. In the latter, the genus Escherichia 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 IFO3547 (Institute for Fermentation, Osaka, Japan) and mutants derived from them that have the capacity of producing pantothenic acid are suitable. Note in particular in this connection the strains 3547/pFV31, 5714/pFV31, 525/pFV31, 814/pFV31, 521/pFV31, FV221/pFV31, FV6051/pFV31, FV5069/pFV31, FV5069/pFV202 and those which are produced from the latter by conventional mutagenesis, selection, for example, for antimetabolic resistance such as azidothymidine resistance, thiaisoleucine resistance and screening. The strains FV5069/pFV31 and FV5069/pFV202 deposited as FERM BP 4395 and FERM BP 5227 in accordance with the Budapest Convention are especially suitable (see EP-A 0,590,857 and WO 97/10340).
In order to achieve an enhancement, especially an 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 also improved by measures for extending the life of m-RNA. Furthermore, the enzymatic activity is 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 be achieved by altering the composition of the media and conduction of the culture.
A skilled artisan will find instructions for this in, among others, Chang and Cohen (Journal of Bacteriology 134: 1141-1156 (1978)), in Hartley and Gregori (Gene 13: 347-353 (1981)), in Amann and Brosius (Gene 40: 183-190 (1985)), in de
Dusch Nicole
Kalinowski Jorn
Pfefferle Walter
Puhler Alfred
Rieping Mechthild
Achutamurthy Ponnathapu
Degussa-Huls Aktiengesellschaft
Kerr Kathleen
Pillsbury Madison & Sutro LLP
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