Mutant E. coli kiz strains for production of pantothenic acid

Details Mutant E. coli kiz strains for production of pantothenic acid Mutant E. coli kiz strains for production of pantothenic acid

1999-10-12

2001-01-09

Mckelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

Reexamination Certificate

active

06171845

ABSTRACT:

FIELD OF THE INVENTION
Pantothenic acid is a vitamin of commercial importance which is used in cosmetics, medicine, human nutrition and animal nutrition.
BACKGROUND OF THE INVENTION
Pantothenic acid can be prepared by chemical synthesis, or biotechnologically by the fermentation of suitable microorganisms in suitable nutrient solutions. In the chemical synthesis, DL-pantolactone is an important compound. It is prepared in a multi-stage process from formaldehyde, isobutyraldehyde and cyanide. In further process steps, the racemic mixture is separated, D-pantolactone is subjected to a condensation reaction with &bgr;-alanine, and D-pantothenic acid is obtained.
An advantage of the fermentative preparation by microorganisms is the direct formation of the desired stereoisomeric D-form.
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 which comprises glucose, DL-pantoic acid and &bgr;-alanine, as shown in EPA 0 493 060. EPA 0 493 060 furthermore shows that in the case of
Escherichia coli
, the formation of D-pantothenic acid is improved by amplification of pantothenic acid biosynthesis genes contained on the plasmids pFV3 and pFV5, in a nutrient solution comprising glucose, DL-pantoic acid and &bgr;-alanine.
EPA 0 590 857 and U.S. Pat. No. 5,518,906 describe mutants derived from the
Escherichia constrain
IFO3547, such as FV5714, FV525, FV814, FV521, FV221, FV6051 and FV5069, which carry resistances to various antimetabolites, such as salicylic acid, &agr;-ketobutyric acid, &bgr;-hydroxyaspartic acid, O-methylthreonine and &agr;-ketoisovaleric acid and produce pantoic acid in a nutrient solution comprising glucose, and D-pantothenic acid in a nutrient solution comprising glucose and &bgr;-alanine. It is furthermore shown in EPA 0 590 857 and U.S. Pat. No. 5,518,906 that after amplification of the pantothenic acid biosynthesis genes contained on the plasmid pFV31 in the abovementioned strains, the production of D-pantoic acid in a nutrient solution comprising glucose and the production of D-pantothenic acid in a nutrient solution comprising glucose and &bgr;-alanine is improved.
In addition, WO 97/10340 shows that in strains of
Escherichia coli
which form pantothenic acid, pantothenic acid production can be increased further by increasing the activity of the enzyme acetohydroxy acid synthase II, an enzyme of valine biosynthesis.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an improved process for the preparation of pantothenic acid.
The vitamin pantothenic acid is a product of commercial importance which is used in cosmetics, medicine, human nutrition and animal nutrition. There is therefore a general interest in providing improved processes for the preparation of pantothenic acid. When D-pantothenic acid or pantothenic acid or pantothenate are mentioned in the present application, they are intended to include not only the free acid but also the salts of D-pantothenic acid, such as, for example, the calcium, sodium, ammonium or potassium salt.
The invention provides a process for the preparation and improvement of pantothenic acid-producing microorganisms by amplification, in particular over-expression, of nucleotide sequences which code for ketopantoate reductase, in particular sequences of the panE gene, individually or in combination with one another, and optionally, in addition, sequences of the ilvC gene.
The term “amplification” in this connection is intended to mean an increase in the intracellular activity of one or more enzymes which are coded by the corresponding DNA by increasing the number of copies of the gene(s), using a potent promoter or a gene which codes for a corresponding enzyme having a high specific activity, and optionally combining these measures.
In particular, it has been found that over-expression of the panE gene together with the genes panB, panC and panD, further improves the formation of pantothenic acid. To achieve the over-expression, the number of copies of the corresponding genes can be increased by means of plasmid vectors, such as, for example, pBR322 (Sutcliffe, COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 1979, 43: 77-90) or pUC19 (Viera, Gene 1982 19:259-268), or the promoter and regulation region upstream of the structural gene can be mutated. A known example of this is the lac-UV5 mutation of the lac promoter (Winnacker: Gene und Klone, Eine Einf{umlaut over (u)}hrung in die Gentechnologie [From Genes to Clones, Introduction to Gene Technology (Verlag Chemie, Weinheim, Germany, 1990). Expression cassettes which are incorporated upstream of the structural gene act in the same way. This method has been used, for example, by LaVallie et al. (BIO/TECHNOLOGY 11, 187-193 (1993) and in PCT/US97/13359. Alternatively, over-expression of the genes in question can be achieved by changing the composition of the media and the culture procedure. An example of this is the universally known regulation of the expression of the lac operon by glucose and lactose. The present inventors moreover have found that over-expression of the panE gene has an advantageous effect in strains which have resistance mutations to metabolites and antimetabolites, such as, for example, resistance to L-valine. It has furthermore been found that over-expression of the panE gene has an advantageous effect in strains which have defect mutations in genes of metabolic routes, such as, for example, the avtA or ilvE gene, which convert precursors of pantothenic acid or reduce the formation of pantothenic acid.
The microorganisms to which the present invention relates can synthesize pantothenic acid from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. These are fungi, yeasts or, in particular, Gram-positive bacteria, for example, of the genus Corynebacterium, or Gram-negative bacteria, such as, for example, those of the Enterobacteriaceae. Of the family of the Enterobacteriaceae, the genus Escherichia with the species
Escherichia coli
may be mentioned in particular. Within the species
Escherichia coli
there may be mentioned 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 IF03547 (Institute of Fermentation, Osaka, Japan) and mutants derived from these. Of the genus Corynebacterium, the species
Corynebacterium glutamicum
, which is known among specialists for its ability to form amino acids, is of particular interest. This species includes wild type strains, such as, for example,
Corynebacterium glutamicum
ATCC13032
, Brevibacterium flavum
ATCC14067
, Corynebacterium melassecola
ATCC17965 and others.
To isolate the ilvC gene and the panE gene, a mutant of, for example,
Escherichia coli
which carries a mutation in the ilvC gene and panE gene, is first prepared.
The nucleotide sequence of the ilvC gene of
Escherichia coli
is known (Wek and Hatfield, Journal of Biological Chemistry 261, 2441-2450 (1986)). Methods for isolation of chromosomal DNA are also known (Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). By choosing suitable primers, the ilvC gene can be amplified with the aid of the polymerase chain reaction (Innis et al., PCR protocols. A guide to methods and applications, 1990, Academic Press). It is then introduced into a plasmid vector. Possible plasmid vectors are those which can replicate in the corresponding microorganisms. For
Escherichia coli
, for example, the vectors pSC101 (Vocke and Bastia, Proceedings of the National Academy of Science U.S.A. 80 (21), 6557-6561 (1983)) or pKK223-3 (Brosius and Holy, Proceedings of the National Academy of Science USA 81, 6929 (1984)), for
Corynebacterium glutamicum
, fo

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