Process for the Preparation of L-Amino Acids by using
Coryneform Bacteria
Field of the Invention
The invention provides a process for the preparation of L- amino acids, in particular L-lysine, by using coryneform bacteria in which the fda gene, which codes for fructose bisphosphate aldolase, is attenuated.
State of the Art
L-amino acids, in particular L-lysine, find application in human medicine and in the pharmaceutical industry, in the food industry and, quite especially, in animal nutrition.
It is known that amino acids are prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. On account of its great importance, work on improving the production process is constantly in progress . Improvements to the process may concern measures pertaining to fermentation technology, such as, for example, stirring and provision with oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during fermentation, or the reprocessing into product-form by, for example, ion- exchange chromatography, or the intrinsic output properties of the micro-organism itself.
With a view to improving the output properties of these micro-organisms, methods of mutagenesis, selection and mutant selection are adopted. In this way, strains are obtained that are resistant to antimetabolites such as, for example, the lysine analogue S- (2-aminoethyl) cysteine or that are auxotrophic in respect of metabolites of regulatory significance and that produce L-amino acids.
Methods pertaining to recombinant DNA technology have also been employed for a number of years for the improvement of
strains of Corynebacterium glutamicuiti producing L-amino acid, by individual amino-acid-biosynthesis genes being amplified and by the effect on the production of L-amino acid being investigated.
Object of the Invention
The inventors have set themselves the task of making available new foundations for improved processes for the fermentative preparation of L-amino acids, in particular L- lysine, by using coryneform bacteria.
Summary of the Invention
When mention is made in the following of L-amino acids or amino acids, these expressions are intended to mean one or more amino acids, including their salts, selected from the group comprising L-asparagine, L-threonine, L-serine, L- glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L- methionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan and L- arginine. L-lysine is particularly preferred.
When mention is made in the following of L-lysine or lysine, these expressions are intended to mean not only the bases but also the salts such as, for example, lysine monohydrochloride or lysine sulfate.
The invention provides a process for the preparation of L- amino acids by using coryneform bacteria in which at least the nucleotide sequence (fda gene) coding for fructose bisphosphate aldolase is attenuated, in particular switched off or expressed at a low level.
This invention further provides a process for the preparation of L-amino acids in which the following steps are implemented:
a) fermentation of the coryneform bacteria producing the L-amino acid, in which at least the nucleotide sequence coding for fructose bisphosphate aldolase is attenuated, in particular switched off or expressed at
5 a low level;
b) enrichment of the L-amino acids in the medium or in the cells of the bacteria; and
c) isolation of the desired L-amino acids, whereby constituents of the fermentation broth and/or of the
10. biomass optionally remain in the end product in proportions or in their total quantities .
Detailed Description of the Invention
The strains that are employed preferably already produce L- amino acids, in particular L-lysine, before the attenuation 15 of the fda gene.
Preferred embodiments are to be found in the Claims .
The term 'attenuation1 in this context describes the diminution or switching-off of the intracellular activity of one or more enzymes (proteins) in a micro-organism that
20 are coded by the corresponding DNA, by use being made, for example, of a weak promoter or by use being made of a gene or allele that codes for a corresponding enzyme with a low activity or that inactivates the corresponding gene or enzyme (protein) and by these measures optionally being
25 combined.
By virtue of the measures of attenuation, the activity or concentration of the corresponding protein is lowered in general to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type 30 protein or of the activity or concentration of the protein in the initial micro-organism.
The micro-organisms that are the subject-matter of the present invention are able to produce amino acids from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerin and ethanol. It may be a question of representatives of coryneform bacteria, in particular of the genus Corynebacterium. In the case of the genus Corynebacterium, in particular the species Corynebacterium glutamicum should be mentioned, which is known amongst experts for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are especially the known wild-type strains
Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and mutants and strains prepared therefrom that produce L- a ino acids,
such as, for example, the L-lysine-producing strains
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 and Corynebacterium glutamicum DSM 5715.
It has been found that coryneform bacteria produce L-amino acids in improved manner after attenuation of the fda gene .
The nucleotide sequence of the fda gene of Corynebacterium glutamicum was published by Sinskey (Mol. Microbiol. 3 (11), 1625-1637 (1989)) and can also be drawn from the gene bank under Accession Number X17313.
The sequences, described in the stated passage, coding for fructose bisphosphate aldolase can be used in accordance with the invention. Moreover, use can be made of alleles of fructose bisphosphate aldolase that arise from the degenerate nature of the genetic code or as a result of functionally neutral sense mutations.
With a view to achieving an attenuation, either the expression of the fda gene or the catalytic properties of the gene products can be lowered or switched off. Both measures are optionally combined.
The gene expression can be reduced by suitable culturing or by genetic modification (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. Data relating to this can be found by a person skilled in the art in, for example, patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Noskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek et al . (Microbiology 142: 1297 (1996)) and in known textbooks on genetics and molecular biology such as, for example, the textbook by Knippers. (Molekulare Genetik, 6th Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or in that by Winnacker {Gene und Klone, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) .
Mutations that lead to a change in or a lowering of the catalytic properties of enzyme proteins are known from the state of the art; by way of examples, mention may be made
of the papers by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugi oto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Mδckel {Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms, Berichte des Forschungszentrums Jϋlichs, Jϋl-2906, ISSN09442952, Julich, Germany, 1994) . Synoptic accounts can be gathered from known textbooks on genetics and molecular biology such as, for example, that by Hagemann {Allgemeine Genetik, Gustav Fischer Nerlag, Stuttgart, 1986) .
Transitions, transversions, insertions and deletions enter into consideration by way of mutations. Depending on the effect of the amino-acid exchange on the enzyme activity, one speaks of missense mutations or nonsense mutations.
Insertions or deletions of at least one base-pair in a gene lead to frame-shift mutations, as a consequence of which false amino acids are incorporated or the translation terminates prematurely. Deletions of several codons typically lead to a complete loss of enzyme activity.
Instructions for the generation of mutations of such a type pertain to the state of the art and can be gathered from known textbooks on genetics and molecular biology such as, for example, the textbook by Knippers {Molekulare Genetik, 6th Edition, Georg Thieme Nerlag, Stuttgart, Germany, 1995) , that by Winnacker { Gene und Klone, NCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann {Allgemeine Genetik, Gustav Fischer Nerlag, Stuttgart, 1986) .
Customary methods for mutating genes of C. glutamicum are the methods of gene disruption and of gene replacement described by Schwarzer and Pύhler (Bio/Technology 9, 84-87 (1991)) .
In the case of the method of gene disruption, a central part of the coding region of the gene of interest is cloned
into a plasmid vector that- is able to replicate in a host (typically E. coli) but not in C. glutamicum. By way of vectors, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKlδmob or p 19mob (Schafer et al., Gene 145, 69- 73 (1994)), p lδmobsacB or pKl9mobsacB (Jager et al . , Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-T0P0 (Shu an (1994), Journal of Biological Chemistry 269:32678- 84; US Patent 5,487,993), pCR®Blunt (Invitrogen, Groningen, Netherlands; Bernard et al . , Journal of
Molecular Biology, 234: 534-541 (1993)) or pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516), for example, enter into consideration.
The plasmid vector, which contains the central part of the coding region of the gene, is subsequently converted by conjugation or transformation into the desired strain of C. glutamicum. The method of conjugation is described, for example, in Schafer et al . (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994) ) . After homologous recombination by means of a cross-over event, the coding region of the gene in question is interrupted by the vector sequence, and two incomplete alleles are obtained, from each of which the 3 ' - end or the 5 '-end is missing. This method was used, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) for the purpose of switching off the recA gene of C. glutamicum.
In the case of the method of gene replacement, a mutation such as, for example, a deletion, an insertion or a base- exchange in the gene of interest is produced in vitro. The allele that is produced is, in turn, cloned into a vector
that is non-replicative in respect of C. glutamicum and said vector is subsequently converted by transformation or conjugation into the desired host of C. glutamicum. After homologous recombination by means of a first cross-over event bringing about integration and by means • of a suitable second cross-over event in the target gene or in the target sequence bringing about an excision, the incorporation of the mutation or of the allele is obtained. This method was used, for example, by Peters-Wendisch et al . (Microbiology 144, 915 - 927 (1998)), in order to switch off the pyc gene of C. glutamicum by a deletion.
In this way a deletion, an insertion or a base-exchange can be incorporated into the fda gene.
In addition, for the production of L-amino acids it can be advantageous, in addition to the attenuation of the fda gene, to enhance, in particular to overexpress, one or more enzymes of the respective biosynthetic pathway, of glycolysis, of anaplerosis, of the citric-acid cycle, of the pentose-phosphate cycle, of the export of amino acid and optionally regulatory proteins.
The term ' enhancement ' in this context describes the increase in the intracellular activity of one or more enzymes or proteins in a micro-organism which are coded by the corresponding DNA by, for example, the copy-number of the gene or genes being increased, by use being made of a strong promoter or a gene that codes for a corresponding enzyme or protein with a high activity and by optionally combining these measures .
By virtue of the measures of enhancement, in particular overexpression, the activity or concentration of the corresponding protein is increased in general by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% .or 500%, maximally up to 1000% or 2000%, relative to that of the
wild-type protein or of the activity or concentration of the protein in the initial micro-organism.
The use of endogenous genes is generally preferred.- The term "endogenous genes" or "endogenous nucleotide sequences" is to be understood to mean the genes or nucleotide sequences, respectively, existing in the . population of a species .
Thus, for the preparation of L-lysine, in addition to the attenuation of the fda gene one or more of the genes selected from the group comprising
• the gene lysC coding for a feedback-resistant aspartate kinase (Accession No. P26512, EP-B-0387527; EP-A-0699759; WO 00/63388) ,
• the gene dapA coding for dihydrodipicolinate synthase (EP-B 0 197 335) ,
• the gene gap coding for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) ,
• simultaneously the gene pyc coding for pyruvate carboxylase (DE-A-198 31 609),
• the gene mqo coding for malate:quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395 - 403 (1998)) ,
• the gene zwf coding for glucose-6-phosphate dehydrogenase (JP-A-09224661) ,
• simultaneously the gene lysE coding for the lysine-export protein (DE-A-195 48 222),
• the gene zwal coding for the zwal protein (DE: 19959328.0, DSM 13115),
• the gene tpi coding for triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), and
• the gene pgk coding for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
is/are enhanced, in particular overexpressed.
Moreover, for the production of amino acids, in particular L-lysine, it can be advantageous, in addition to the attenuation of the fda gene, simultaneously to attenuate, in particular to reduce the expression of, one or more of the genes selected from the group comprising
• the gene pck coding for phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047),
• the gene pgi coding for glucose-6-phosphate isomerase (US 09/396,478, DSM 12969),
• the gene poxB coding for pyruvate oxidase (DE:1995 1975.7, DSM 13114),
• the gene zwa2 coding for the zwa2 protein (DE: 19959327.2, DSM 13113).
Finally, for the production of amino acids it can be advantageous, in addition to the attenuation of the fda gene, to exclude undesirable side reactions (Nakayama: Breeding of Amino Acid Producing Micro-organisms, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Nanek (eds.), Academic Press, London, UK, 1982).
The micro-organisms that are produced in accordance with the invention are likewise a subject of the invention and can be cultivated continuously or discontinuously in the batch process (batch cultivation) or in the fed-batch or repeated-fed-batch process for the purpose of producing L- a ino acids . A summary of known cultivation methods is
described in the textbook by Chmiel {Bioprozesstechnik 1 . Ξinfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas {Bioreaktoren und periphere Einrichtungen (Nieweg Nerlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used has to satisfy the demands of the respective strains in suitable manner. Descriptions of culture media of various micro-organisms are contained in the manual entitled Manual of Methods for General Bacteriology published by the American Society for Bacteriology (Washington D.C., USA, 1981).
Sugar and carbohydrates such as, for example, glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as, for example, soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as , for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerin and ethanol, and organic acids such as, for example, acetic acid can be used by way of carbon source. These substances can be used individually or as a mixture.
Organic nitrogenous compounds such as peptones, yeast • • extract, meat extract, malt extract, maize steep liquor, soybean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used by way of nitrogen source. The nitrogen sources can be used individually or. as a mixture.
Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium- containing salts can be used by way of phosphorus source. The culture medium must, moreover, contain salts of metals such as, for example, magnesium sulfate or iron sulfate, that are necessary for growth. Finally, essential growth substances such as amino acids and vitamins can be employed
in addition to the aforementioned substances. Besides, suitable precursors can be added to the culture medium. The stated feed materials can be added to the culture in the form of a single charge or can be fed in during the cultivation in suitable manner.
With a view to controlling the pH of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammoniacal liquor or acidic compounds such as phosphoric acid or sulfuric acid are employed in suitable manner. With a view to controlling the formation of foam, anti-foaming agents such as, for example, fatty-acid polyglycol esters can be employed. With a view to maintaining the stability of plasmids, suitable substances acting selectively, such as antibiotics for example, can be added to the medium. In order to maintain aerobic conditions, oxygen or oxygenous gas mixtures, such as air for example, are introduced into the culture. The temperature of the culture is normally around 20°C to 45°C and preferably 25°C to 40°C. The culture is continued for such time until a maximum of the desired product has formed. This objective is normally attained within a period from 10 hours to 160 hours.
Methods for the determination of L-amino acids are known from the state of the art. The analysis can be undertaken as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by anion-exchange chromatography with subsequent ninhydrin derivation, or it can be undertaken by reversed-phase HPLC, as described in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
The present invention is elucidated in more detail in the following on the basis of exemplary embodiments.
Example 1
Preparation of the expression vector pXK99Efda for IPTG- induced expression of the fda gene in C. glutamicum
1.1 Cloning of the f a gene
Chromosomal DNA is isolated from the strain ATCC 13032 in accordance with the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis of the known sequence of the fda gene for C. glutamicum the following oligonucleotides for the polymerase chain reaction are selected (see SEQ ID No. 1 and SEQ ID No. 2) :
fda for:
5 -GA TCT AGA-TTT TGG AGG AGA CAC CTT AT-3 %
fda int:
5 - CT AAG CTT-GAC AAC ACC GAT CTC AAC TT-3 N
In this connection the primers are selected in such a way that the amplified fragment contains the incomplete gene, beginning with the nativen ribosome binding site without promoter region, as well as the anterior region of the fda gene. In addition, the primer fda for contains the sequence for the cleavage site of the restriction endonuclease Xbal, and the primer fda int contains the sequence for the cleavage site of the restriction endonuclease Hindlll, which are marked by underlining in the nucleotide sequences represented above.
The primers that are represented are synthesized by MWG- Biotech AG (Ebersberg, Germany) , and the PCR reaction is carried out in accordance with the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with Pwo polymerase produced by Roche Diagnostics GmbH (Mannheim, Germany) . With the aid of the polymerase chain reaction the primers enable the amplification of a 529-bp DNA fragment that
bears the incomplete fda gene including the native ribosome binding site.
The 529-bp fda fragment is cleaved with the restriction endonucleases Xbal and HindiII and is subsequently isolated from the agarose gel with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) .
1.2 Construction of the expression vector pXK99E
The IPTG-inducible expression vector pXK99E is constructed in accordance with the state of the art. The vector is based on the Escherichia coli expression vector pTRC99A (Amann et al., Gene 69: 301-315 (1988)) and contains the trc promoter which is inducible by addition of the lactose derivative IPTG (isopropyl-β-D-thiogalactopyranoside) , the termination regions Tl and T2, the replication origin ColEl from E. Coli, the laclQ gene (repressor of the lac operon of E. coli), a multiple cloning site (mcs) (Norrander, J.M. et al. Gene 26, 101-106 (1983)) and the kana ycin- resistance gene aph(3')-Ila from E. coli (Beck et al . (1982), Gene 19: 327-336).
It has been found that the vector pXK99E is quite especially suitable for regulating the expression of a gene, in particular for bringing about the attenuated expression in coryneform bacteria. The vector pXK99E is an E. coli expression vector and can be employed in E. coli for the enhanced expression of a gene.
Since the vector cannot replicate independently in coryneform bacteria, it is preserved in the cell only when it integrates into the chromosome. The peculiarity of this vector in this connection is the use for the regulated expression of a gene after cloning of a gene segment from the anterior region of the corresponding gene into the vector, containing the start codon and the native ribosome binding site, and after subsequent integration of the
vector in coryneform bacteria, in particular C. glutamicum. By addition of metered amounts of IPTG to the nutrient medium the gene expression is regulated. In this connection, quantities from 0.5 uM up to 10 μM IPTG bring about a very weak expression of the corresponding gene, and quantities from 10 μM up to 100 μM bring about a slightly attenuated to normal expression of the corresponding gene.
The constructed E. coli expression vector pXK99E is transferred by means of electroporation (Tauch et al. 1994, FEMS Microbiol Letters, 123: 343-347) into E. coli DH5αmcr (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649). Selection of the transformants is undertaken on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) that has. been supplemented with 50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant in accordance with the customary methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915 - 927) , is cut with the restriction endonuclease Ncol, and the plasmid is examined by subsequent agarose-gel electrophoresis .
The plasmid construct that is obtained in this way is designated as pXK99E (Figure 1) . The strain that is obtained by electroporation of the plasmid pXK99E into the E. coli strain DH5αmcr is called E. coli
DH5alphamcr/pXK99E (= DH5αmcr/pXK99E) and was deposited on 31 July 2001 as DSM 14440 in the Deutsche Sammlung fiir Mikroorganismen und Zellkulturen (German Collection of Micro-Organisms and Cell Cultures, DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
1.3 Cloning of the fda fragment into the E. coli expression vector pXK99E
By way of vector, use is made of the E. coli expression vector pXK99E described in Example 1.2. DNA of this plasmid is cleaved completely with the restriction enzymes Xbal and HindiII and is subsequently dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, Product No. 1758250).
The approximately 520-bp fda fragment described in Example 1.1, which is obtained by means of PCR and cleaved with the restriction endonucleases Xbal* and Hindlll, is mixed with the prepared vector pXK99E, und the charge is treated with T4-DNA-Ligase (Amersham Pharmacia, Freiburg, Germany, product description T4-DNA-Ligase, Code No. 27-0870-04) . The ligation charge is transformed into the E. coli strain DH5αmcr (Hanahan, In: DNA Cloning. A Practical Approach,
Vol. I, IRL-Press, Oxford, Washington DC, USA). Selection of plasmid-bearing cells is undertaken by plating the transformation charge onto LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/1 kana ycin. After incubation overnight at 37°C, recombinant individual clones are selected. Plasmid DNA is isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions and is cleaved with the restriction enzymes Xbal and HindiII, in order to examine the plasmid by subsequent agarose-gel electrophoresis. The plasmid that is obtained is called pXK99Efda. It is represented in Figure 2.
Example 2
Integration of the vector pXK99Efda into the genome of the C. glutamicum strain DSM5715
The vector pXK99Efda named in Example 1 is electroporated into the strain C. glutamicum DSM5715 in accordance with the electroporation method of Tauch et al. (1989 FEMS
Microbiology Letters 123: 343-347). The vector cannot replicate independently in DSM5715 and is preserved in the cell only when it has integrated into the chromosome. Selection of clones with integrated pXK99Efda is undertaken by plating the electroporation charge onto LB agar
(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Habor, New York, 1989) that has been supplemented with 15 mg/1 kanamycin and IPTG (lmM) .
A selected kanamycin-resistant clone that has inserted the plasmid pXK99Efda named in Example 1 within the chromosomal fda gene of DSM5715 is designated as DSM5715 : :pXK99Efda.
Brief Description of the Figures :
Figure 1: map of the plasmid pXK99E,
Figure 2: map of the plasmid pXK99Efda.
The abbreviations and designations that are used have the following significance.
Kan: kanamycin-resistance gene aph(3Λ)-Ha from
Escherichia coli
Hindlll cleavage site of the restriction enzyme
Hindlll
Ncol cleavage site of the restriction enzyme Ncol
Xbal cleavage site of the restriction enzyme Xbal
Ptrc trc promoter
Tl termination region Tl
T2 termination region T2
laclq laclq repressor of the lac operon of
Escherichia coli
oriN replication origin ColEl from E. coli
fdaint cloned region of the fda gene