SK279719B6 - Dna fragment, recombinant dna, process for the production of l-lysine - Google Patents

Abstract

A DNA fragment has a nucleotide sequence with 2.1 kb downstream of the Pst I site and Xho I site. The said DNA fragment has a genetic sequence coding the production of proteins which lead to an aspartyl-beta-semialdehyde dehydrogenase (asd) activity or to the deregulation of aspartate kinase (lysC) and is contained in the pCS2 plasmide, in Corynobacterium glutamicum, under No. DSM 5086. Here disclosed recombinant DNA is derived from the said pCS2 plasmide. A process for the production of the L-lysine, obtained by Corynebacterium or Brevibacterium genus fermentation, resides in the fact that production microorganisms contain a plasmide or some of the stated recombinant DNA. Corynobacterium glutamicum DMS 5086 is preferably used as a microorganism.

Description

Technical field

The invention relates to a DNA fragment present in a plasmid, to recombinant DNA derived from this plasmid, and to a method for producing L-lysine using a microorganism comprising said plasmid or recombinant DNA derived therefrom.

BACKGROUND OF THE INVENTION

Corynobacterium glutamicum and related genera such as Brevibacterium lactofermentum and Brevibacterium flavum are known as amino acid-forming microorganisms.

To increase productivity, artificial mutations are made.

Examples of artificial mutants thus prepared are, for example, lysine-producing strains of Corynobacterium ghitamicum which, in addition to AEC resistance (AEC = S-2-aminoethylcysteine), show homoserine and leucine auxotrophy associated therewith (U.S. Pat. No. 3,708,395) or are sensitive to U.S. Patent No. 3,871,960).

In addition to these classical methods, vector systems have been discovered which allow the transformation of microorganisms of the genera Corynobacterium and Brevibacterium (DE-OS No. 37 37 719, DE-OS No. 38 41 453, Thierbach G., Schwarzer A., Piihlcr A., Appl. Microbiol Biotechnol 29 (1988) 356-362).

EP-A-0219 027 discloses a process for the production of various amino acids, in which the genera Corynobacterium and Brevibacterium are transformed with recombinant DNA, thereby increasing the amounts of amino acids produced.

The recombinant DNA comprises a DNA fragment encoding the synthesis of aspartate semialdehyde dehydrogenase or aspartate aminotransferase.

U.S. Pat. No. 4,346,170, it is known to clone genetic information controlling lysine production in Escherichia coli which originates from a strain of the same genus with resistance to L-lysine analogs such as AEC.

The subject of U.S. Pat. No. 4,560,654 relates to the same field. In this case, however, genetic information from an AEC-resistant strain of the same genus is cloned in the lysine-auxotrophic strain Corynobacterium glutamicum in order to secrete lysine.

The identity of the cloned DNA fragment is not obvious.

Also from EP no. 88166, it can only be understood that the Corynobacterium glutamicum strain secretes lysine when transformed to obtain the phenotype of AEC resistance.

The recombinant plasmid pAec5 used for this purpose contains a 3.9 kb fragment of chromosomal DNA inserted at the BglII cleavage site of the vector pCG11.

It is an object of the present invention to alter the controllability of an important enzyme of lysine biosynthesis in a microorganism of the genus Corynobacterium or Brevibacterium so as to obtain a lysine producer or to increase the ability of lysine production.

SUMMARY OF THE INVENTION

The present invention provides a DNA fragment consisting of a 2.1 kb nucleotide sequence flanked by PstI and XhoI cleavage sites, characterized in that the amino acid sequence shown in FIG. 5 encodes the production of proteins leading to aspartyl-β-semial dehyddehydrogenase (asd) activity and aspartic kinase deregulation (lysC) is contained in the plasmid pCS2, the deletion map of which is shown in FIG. 3 deposited with Corynobacterium glutamicum under number DSM 5086.

The present invention also provides recombinant DNA derived from plasmid pCS2, characterized by a deletion map corresponding to the designation pCS26 shown in FIG. Third

The invention also provides recombinant DNA derived from plasmid pCS2, characterized by deletion maps corresponding to the designations pCS23 or pCS233 in FIG. Third

The present invention further provides a process for the production of L-lysine by culturing microorganisms of the genus Corynobacterium or Brevibacterium producing this amino acid, wherein the microorganisms comprise said plasmid or one of said recombinant DNAs.

In a preferred embodiment of the method described, Corynobacterium DMS 5086 is used.

Accordingly, the invention relates, inter alia, to a process for the production of L-lysine, characterized in that the recombinant DNA is derived from a DNA fragment having a coded genetic sequence for the production of proteins which lead to aspartyl-β-semialdehyde dehydrogenase (asd) activity and / or to deregulate aspartic kinase and consists of a DNA vector, inserted into a lysine-producing microorganism of the genus Corynobacterium or Brevibacterium, the transformants thus obtained are cultured in a suitable medium known per se, and the lysine formed is isolated by known methods.

Donor strains can be all strains, preferably Brevibacterium and Corynobacterium lysine producing bacteria, which contain the respective DNA sequences, in particular Corynobacterium glutamicum DM 58-1, which has been developed by mutagenesis of Corynobacterium ATCC 13032 with ethyl methanesulfate and demonstrates AEC resistance.

This strain is deposited under the number DSM 4697 and serves as a host bacterium for the plasmid pDM6. This can be separated by a person skilled in the art by known methods to obtain strain DM58-1 (FEMS Microbiology Review 32 (1986) 149-157)

Chromosomal DNA is extracted from the donor in a known manner and treated with a restriction endonuclease.

After construction of the recombinant DNA by introducing the chromosomal DNA fragment into the vector, the microorganism is transformed with the plasmid thus obtained, for example pCS2 according to the present invention, the restriction scheme of which is shown in FIG. 2 and which is deposited in the Corynobacterium glutamicum DM2-1 / pCS2 strain under the Budapest Treaty under the number DSM 5086 in the German Collection of Microorganisms and Cell Structures.

A preferred vector system is pZ1 (deposited in Corynobacterium glutamicum DM 274-2 under DSM 4241) or even pCV34, pCVX4, pCVX10, pCVX15, pZ9 and pZ8-1 (DE-OS 38 41 454.6) or pCV35, pECM3 and pECM1 (DE -OS 38 41 453.8).

However, composite plasmids, known from EP-A-93 611, are also useful when they replicate themselves in the genera Corynobacterium or Brevibacterium, in particular pAJ 655, pAJ 611, p AJ 440, p AJ 1844 and pAJ 3148 or also pCG 11, pCE 54 ( see EP-A 0 233 581) as well as pUL33O (Santamaria, RI et al., J. Bacteriology 162, (1958) 463-467).

The present invention also relates to microorganisms of the genus Corynobacterium or Brevibacterium containing recombinant DNA and their use for the production of L-lysine by fermentation.

The cloned DNA fragment (see FIG. 2) contains only a portion of the aspartate kinase (LysC) gene as well as the complete aspartyl-β-semialdehyde dehydrogenase (asd) gene, as determined by sequence analysis.

Part of this aspartic kinase gene has a homologous DNA sequence to the β-subunit of aspartic kinase II from Bacillus substilis.

All transformants whose plasmid has this sequence (pCS2, pCS22, pCS23, pCS24, pCS26 and pCS233) contain a substantially desensitized aspartate kinase relative to the feed-back inhibitor L-lysine and L-threonine compared to the chromosome encoded enzyme from ATCC 13032 and have AEC resistance.

Conclusions derived from a homology comparison that a fragment of the PstI-XhoI gene from DM58-1 contains only part of the LysC (AK) gene but the full asd gene can be unequivocally confirmed by enzyme measurements.

No Corynobacterium glutamicum ATCC 13032 strain transformed with a pCS2 or pCS2 derivative contains surprisingly increased aspartate kinase activity compared to the parent strain (Table 4, column 3).

In contrast, in all transformants whose plasmids contain the structural gene asd, strong superexpression of aspartyl-β-semialdehyde dehydrogenase (ASA-DH) can be detected (Table 4, column 2, Figures 3 and 4). As expected, pCS23 plasmids and pCS23 derivatives do not result in superexpression of ASA-DH.

The strong superexpression of ASA-DH resulting from the cloning of the DNA fragment according to the invention with pCS2 and derivatives derived therefrom guarantees an efficient reaction of the aspartate kinase reaction product, β-aspartyl phosphate, thereby accelerating the no-inhibited aspartate kinase reaction.

Due to the high lability of ASA-DH, the factors of superexpression, calculated from specific activities range from 31 to 65.

Compared to the current state of the art, there is a substantial simplification in that, firstly, only a portion of the LysC gene that leads to deregulation of aspartate kinase must be isolated to achieve or improve lysine secretion, and secondly based on LysC and asd in one operon. isolate the asd gene without the additional experimental costs associated with the AEC-resistance occurring with the mutation of the LysC gene, together with the LysC gene. Conversely, a DNA fragment containing LysC + asd can be isolated using the asd mutants, regardless of whether the LysC is mutated or not.

1. Characteristics of the donor DM58-1 gene and the ATCC 13032 recipient

1.1 Acquisition and phenotype of strain DM58-1

The DM58-1 strain was obtained by mutagenesis of the Corynobacterium glutamicum ATCC 13032 strain with a conventional concentration of ethyl methanesulfonate.

Selection is carried out by culturing the mixture of mutants thus obtained on minimal agar with the following composition:

glucose 20 g ammonium sulfate 10 grams urea 2.5 g potassium dihydrogen phosphate 1 g magnesium sulfate heptahydrate 0.4 g ferrous sulfate heptahydrate 2 mg manganese sulphate monohydrate 1.5 mg biotin 300 pg thiamine 900 pg agar 20 g

distilled water 1 liter (ph 7.0) containing an appropriate concentration of S-aminoethyl-D, L-cysteine (AEC). A clone isolated from this selection medium and capable of dividing on this medium, later designated DM58-1, bears no other genetic traits apart from its AEC resistance.

1.2 Enzymatic contents of aspartic kinase and aspartyl-β-semialdehyde dehydrogenase in ATCC 13032 aDM58-1

The strains ATCC 13032 and DM58-1 are cultured under directly comparable conditions in Standard I Bouillon (Merck Art. Nr. 7882) with the addition of 4 g / l glucose and 1 mM magnesium chloride at 30 ° C and 150 rpm until reaching an early time. of the stationary phase and centrifuged to separate them from the culture medium. Wash three times with 100 mM Tris / HCl (pH 7.5); 1 mM DTT and wet cell mass are suspended in one volume of the same buffer.

The suspended cells are then disintegrated in a ball mill (B. Braun Melsungen-MSK-Homogenisator, IMA-Disintegrator S) by mixing with an appropriate amount of glass beads on a glass filter and centrifuged for 30 minutes at 30,000 x g until clear.

After 15 hours of dialysis in enzyme stabilizing buffer, enzyme activity was determined in the following test mixtures:

Aspartate Kinase Test:

Tris / HCl (pH 7.5) 100 mM DTT 1 mM ammonium sulfate 400 mM magnesium chloride 20 mM NH ₂ OH.HCl 400 mM L-aspartate 300 mM ATP 40 mM a different amounts of enzyme preparations.

Addition of 150 μΐ of 10% ferric chloride hexahydrate solution; 3.3% TCA; With 0.7 N HCl per 500 μΐ of the test enzyme mixture, the enzyme reaction is stopped after a thirty minute incubation at 37 ° C. The enzymatic activity in pmol / mg.min (U / mg) is calculated from the concentration of aspartyl β-hydroxamate determined by photometric (54Ε ₅₄ ο J by calibration curve). The respective protein concentrations were determined according to the Lowry et al. Method (Lowry et al. J. Biol. Chem. 193, 265 (1951) or Bradford (Bradford Anal. Biochem. 72, 248 (1976)).

Aspartyl β-semialdehyde dehydrogenase test:

diethanolamine (ph 9.0) 120 mM NaAsO4 40 mM NADP + 1 mM L-threonine 5 mM The aspartyl-semialdehyde 1.3 mM a

different amounts of enzyme preparations in a total volume of 1 ml.

The activity reported in pmol / mg.min (U / mg) is calculated via a photometric determined (AE _{540 mn} ) NADPH synthesis rate.

Table 1 contains the specific enzyme activities of both enzymes in crude extracts of identically grown and processed Corynobacterium glutamicum ATCC 13032 and DM58-1 cells. In addition to comparable aspartate kinase contents of both strains, the AEC-resistant mutant DM58-1 contains approximately five-fold increased aspartyl-β-semialdehyde dehydrogenase activity compared to wild type.

SK 279719 Β6

1.3 In vitro Inhibition of Aspartate Kinase from Corynobacterium glutamicum ATCC 13032 aDM58-1

Table 1 shows that K. Nakayam et al. (K. Nakayama et al., Agr. Biol. Chem. 30, 611 (1966)) and S. N. Kara-Murz et al. (S.N. Kara-Murza Prikladnaya Biokhimiya; Microbiology 14, 345 (1978)) more specifically investigated inhibition of wild-type enzymes of Corynobacterium glutamicum is capable of reproduction. In contrast, the enzyme character of the AEC-resistant mutant DM58-1, whose aspartate kinase is no longer concentrically inhibited by L-lysine + L-threonine, is quite different. The enzyme S-aminoethyl-D, L-cysteine, acting on the enzyme of ATCC 13032 as a lysine analog, is also only slightly influenced by the enzyme mutants.

Table 1

Enzyme content and properties of aspartic kinase (AK) and aspartyl β-semialdehyde dehydrogenase (ASA-DH) from Corynobacterium glutamicum ATCC 13032 and DM58-1

tribe ATCC 13032 DM58-1 AK (U / me) 0016 0,011 ASA - DH (U / rnoi 12:06 12:33 combinations inhibition inhibitors AK W 10 mM L-Lγ * i mM 99 1 2 10 eiH L-Lys 10 mM L-Thr 95 2 100 mM L-Lys 10 mM L-Thr 99 21 10 mM AEC 1 nifl L-Thr 1 2 0 10 mM AEC 10 mM L-Thr 61 0 100 mM AEC 10mM L-Thr ---- 95 T

AEC: S- (aminoethyl) -D, L-cysteine

2. Cloning of DNA fragment of Corynobacterium glutamicum DM58-1 which encodes feed-back resistant aspartate kinase

3. Cloning

Total DNA was isolated from Corynobacterium glutamicum DM58-1 as described by Charter et al. (Charter et al. Curr. Topics Microb. Immunol. 96, 69 (1982)) and partially digested with the restriction enzyme PstI. The vector pZ1 (FIG. 1), which is described in German patent application no. 37 37 729.9, is linearized with PstI and dephosphorylated by alkaline phosphatase treatment. The DNA vector and the DM58-1 DNA were mixed and treated with T4 DNA ligase as described by Maniatis et al. (Maniatis T. et al., Molecular Cloning A. Laboratory Manual, Cold Spring Harbor Laboratory 1982).

Transformation of Corynobacterium glutamicum ATCC 13032 with the ligation mixture is performed as described by Thierbach et al. (Thierbach G. et al., Applied Microbiology and Biotechnology 29, 356 (1988)).

In FIG. 1 is a restriction diagram of plasmid pZ1. The thick line represents the proportion of pHM1519 and the thin line represents the proportion of pACYC177. The designation Ap ^R = ampicillin resistance gene and Km ³ = kanamycin resistance gene.

The transformation mixture was seeded on RCG / E agar plates with 300 µg / ml kanamycin and incubated for one week at 30 ° C. Thereafter, these agar plates are resolved into MM agar plates (Katasumata, R. et al., J. Bact. 159, 306 (1984)) with 50 mM AEC and 50 mM L-threonine and cultured for one day at temperature. Deň: 29 ° C. Colonies that could grow on this agar were inoculated on MM agar which additionally contained AEC, L-threonine and 10 µg / ml kanamycin to obtain single cell colonies. Plasmid DNA was isolated from such a clone, designated pCS2, and used to transform Corynobacterium glutamicum ATCC 13032. 59 out of 62 kanamycin-resistant transformants tested appeared resistant to inhibition by 50 mM AEC and 50 mM L-threionine. Plasmid pCS2 was further characterized by restriction analysis. It contains an approximately 9.9 kb insertion at the PstI cleavage site of the vector pZ1, which is 6.9 kb in length.

In FIG. 2 is a restriction diagram of the plasmid pCS2 in linearized form.

In the upper part of FIG. 2 shows the positions of the various restriction cleavage sites. Different regions of the plasmid pCS2 are shown in the lower part of the figure. The insertion of DM58-1 DNA is shown as an empty band. The ampicillin resistance gene pZ1 is shown as a black field, the kanamycin resistance gene is shown as a dotted field. Other proportions of pZ1 of plasmid pCS2 are shaded. The abbreviations used are as follows:

BamHI-B; Beli-C; Sali-S; Sca-A; Smal-M; Xhol - X.

Characteristics of aspartic kinase activity

Aspartate kinase activity was measured in strain ATCC 13032 / pCS2, as a positive control in strain DM58-1 and as a negative control in strain ATCC 13032. The strains were cultured on Standard I Boillon supplemented with 4 g / l glucose, 10 μΐ / ml of kanamycin and 1 mM magnesium chloride. Culture conditions, cell isolation, cell lysis and aspartate kinase assays are described and performed according to section 1.2. The effectors L-lysine, L-threonine and AEC are always added as stock solutions in 100 mM Tris / HCl buffer, pH 7.5.

Aspartate kinase content and enzyme inhibition from ATCC 13032 / pCS2 are shown in Table 4. Although the strain did not show any increase in specific activity, a clear desensitization against these inhibitory substances could be detected but whose degree of enzyme deregulation from the DM58-1 gene provider was not achieved ( partial deregulation).

2.3 Determination of L-lysine secretion

The lysine secretion capacity was determined for the ATCC 13032 / pCS2 strain and as a negative control for the ATCC 13032 / pZ1 strain. Following the addition of 10 µg / ml kanamycin, the culture was performed as described and the results are summarized in Table 2.

Table 2

Excretion of L-lysine by various strains of Corynobacterium glutamicum

Kmeft C. glutamicum Concentration of excreted

L-lysine.HCI (g / 1)

ATCC 13032 / pZ1 0.0

ATCC 13032 / pCS2 (= DM 2-1 / pCS2) 7.1

Erlenmeyer flask of 100 ml culture medium, composed of:

ammonium sulphate 12 g / l molasses 240 g / l

276 soya hydrolyzate 60 ml / l calcium carbonate 10 g / l.

After seeding, the culture is incubated for 72 hours at 30 ° C and 300 rpm. The lysine determination is performed in the liquid after centrifugation using an amino acid analyzer.

3. Overview of cleavage of the pCS2 DNA fragment that encodes feed-back resistant aspartate kinase

By total or partial cleavage of pCS2 with various restriction enzymes and subsequent treatment with T4 DNA ligase at low DNA concentrations, various cleavage derivatives are constructed. The production of the various cleavage derivatives is summarized in the following Table 3, and the cleavage position in the different derivatives is shown in FIG. 3. In FIG. 3 also shows the ability of the AEC to resist strains derived from Corynobacterium glutamicum ATCC 13032. In this way, the DNA region mediating AEC-resistance could be flanked to a DNA fragment of approximately 1.5 kb, which is restricted in plasmid pCS233 by the cloning PstI cleavage site and EcoRl cleavage site.

Aspartate kinase activity and enzyme activity inhibition by mixtures of lysine, optionally AEC and threonine, were determined in engineered clones. Cultivation, isolation and activity determination were performed as described. In addition, the ability of different clones to secrete L-lysine was examined. A diffusion test on L-lysine-auxotrophic indicator strain Corynobacterium glutamicum was used for this. It can be seen from Table 4 that all AEC-resistant strains have partially deregulated aspartic kinase activity and are capable of secreting L-lysine.

Table 3

Production and AEC-phenotype of various cleavage derivatives of plasmid pCS2

Plasmid Construction of AEC ^{k /} phenotype

PCS21A produced by digestion of pCS2 with BamH1 R PCS22A produced by digestion of pCS2 with BamHI and BclI R pCS2 3 produced by partial cleavage of pCS2 with SalI R pCS24 made by partial cleavage. pCS2 using XhoI R pCS26 produced by digestion of pCS2 with Scal R pCS231 made by partial cleavage of pCS23 using PstI WITH PCS232 produced by partial digestion of pCS23 with DraI with PCS233 produced by partial cleavage of pCS23 with EcoR1 R

Explanatory notes: R = resistance

S = sensitivity

In FIG. 3 shows the cleavage scheme of plasmid pCS2. The upper part of the figure depicts derivatives derived from pCS2 and the lower part of the figure depicts derivatives derived from pCS23. The ampicillin resistance limits of pZ1 are shown in black; the kanamycin resistance boundaries are dotted and the other parts of the pZ1 plasmid pCS2 are shaded. The insertions of DM58-1 DNA are shown as free bands. Cleavages are indicated by a line. Explanations of abbreviations used:

B-BamHl, G-Beli, D-Dral, E-EcoRl, S-Sali,

A-Scal, M-Smal, X-Xhol.

Table 4

Microbiological and biochemical characteristics of recombinant strains of Corynobacterium glutamicum

tribe

ASA-DH AK Reactivity of AK in pritoonosti (Ϊ) aec ^r Elimination of Lysine (11 / (U / 10mH 10mM 100mM 1 OOmH mg 1 mg) Lyi Lys Ly · AEC 1mM 10mH 10mM 10 mM rnr Thr Thr Thr

ATCC13032 0 (pZl J 06 0016 9 WITH 7 - ATCC13032 1pCS21 3 9 0.013 55 55 28 56 • - ATCC 13032 IpCS21) ⁿ 0016 46 50 24 · • - ATCC13032 PCS2Z n 0.015 4 0 41 22 n. - - ATCC13032 (pCS23) 0 03 0.014 5 1 57 30 56 • • ATCC13O3Z <pCS24> 2 07 0.011 65 55 40 62 • • ATCC13O32 (DCSZ3) 1 aa 0,015 64 53 39 60 • • ATCC13032 lPCS231) 0 060 0,013 1 1 14 10 - - ATCC 13032 1pCS232) n. n. n. · - - ATCC13D32 1PCS233) n 0.011 56 62 36 57 - DM5Í-1 IpZI) 0 330 0.009 83 100 79 93 • •

Abbreviations used: ASA-DH: aspartyl-β-semialdehyde dehydrogenase

AK: aspartate kinase

n .: not found.

4. Sequencing of the DNA fragment of plasmid pCS24, which mediates the AEC-resistance phenotype

4.1 Sequencing method

The 2.1 kb nucleotide sequence of PstI-XhoI-fragment DNA was determined by the method of Maxam and Gilbert (Maxam, AM et al., Proc. Natl. Acad. Sci. USA 74, 560-564 (1977)) with Arnold and Puhler modification. (Arnold W. et al., Gene, 70, 171 ff (1988)). Subcloning for sequencing was based on plasmid pCS24 (see FIG. 4). This is transformed according to Escherichia coli MM 294 (Merelson, M. et al., Nature 217, 1110-1114 (1968)) and the corresponding fragments cloned into the sequence vectors pSVB21, 25 and 26 (Arnold W. et al., Gene, 70 171 (1988). In the Escherichia coli JM83 strain (Mesing J., Recombinant DNA Technical Bulletin NIH Publication No. 79-99, 2, 43-48 (1979)), it is possible to demonstrate the activation of insertion by the Xgal (5-bromo-4-chloroindolyl-pD-) assay. galaktipyranozid).

The sequencing strategy is shown in FIG. 4. The nucleotide sequence was determined on both strands of DNA with overlapping clones.

In FIG. 4 shows the cleavage analysis and sequencing strategy of the chromosomal fragment of plasmid pCS2. Plasmid pCS23 and pCS24 also mediate AEC resistance and lysine production as a pCS2 clone. The hatched bands represent the proportion of vector in the plasmid.

SK 279719 Β6

Sequencing strategy: The 2.1 kb PstI-XhoI fragment of plasmid pCS24 was subcloned with the restriction cleavage sites depicted. The arrows always indicate the sequential range and reading direction. The restriction cleavage sites of the enzymes DraI (D), EcoRI (E), BglII (O), HindIII (H), Nael (A), PstI (P), SalI (S), and XhoI (X) are indicated. Shown below are two reading frames that code for aspartate kinase subunits and for aspartate-β-semialdehyde dehydrogenase (see text).

4.2 DNA sequence of 2.1 kb PstI-XhoI-fragment DNA

The DNA sequence sequenced is 2112 bp long. It carries restriction cleavage sites for BglII, DraI, EcoRI, HindIII, Nael, PstI, SalI and XhoI, with which subclones were also produced.

The nucleotide sequence was processed by sequence analysis according to the ANALYSEQ program bundle (Staden, R. et al., Nucl. Acids Res. 14, 217-323 (1986)).

There are two open reading frames (ORFs) on the sequenced DNA. Both are arranged from the PstI cleavage site to the XhoI cleavage site. There is only a small 26 bp region between the two. The ribosome binding site (RBS) is located upstream of the 2nd ORF (806-809 AGGA followed by the ATC codon). One RBS (AGGA, 268-271 with GTG start codon) was also located within the 1st ORF.

ORF 1 length 264 amino acids (AS), counting from the PstI site and 172 amino acids (corresponding to 18.6 k Dals) from internal RBS. ORF 2 is 342 amino acids in length (36.1 k Dals).

Directly beyond ORF 2 is a possible transcriptional termination structure, the so-called hair needle loop extending from several thymine residues (1864-1900). This arrangement is characteristic of β-independent termination signals in Escherichia coli and other bacterial species (Ahyda et al., Ann. Rev. Biochem. 47, 967-996 (1978)). The terminator presented herein has a stability of more than -40 kcal / mol at 30 ° C.

A possible promoter for ORF 2 was found within ORF 1 (409-437), (TTGACA-17 bp-TATTCT). Area -35 and distance to area -10 correspond exactly to E. coliConsensus-Promotor (Hawley, DK et al., Nucl. Acids Res. 11, 2237-2255 (1983)), area -10 is very similar to E. coli-Consensusregion (TATAAT).

The DNA sequence and the deduced amino acid sequence of 2.1 kb PstI-XhoI-fragment are shown below.

u á S

Str li

Ii ^s * o <nuw »

<3. Network

O cn

About 00 r — t

O r * what

O XD «-r-4

About CM

WHAT

The amino acid sequences of ORF 1 (1-794) and ORF 2 (821-1846) are given in three letter code. The numbering below the line refers to the DNA sequence. The names of the cloning cleavage sites used in sequencing are also listed below the line. Ribosome binding sites are indicated by an asterisk. The start codon is indicated by an arrow and the terminator structure is a solid line.

4.3 Amino acid sequence analysis

The amino acid sequence transmitted by ORF 1 and ORF 2 was compared with the known AK I aspartate kinase sequences (Cassan, M. et al., J. Biol. Chem. 261, 1052-1057 (1986)) from Escherichia coli and AK II from Bacillus subtilis (Chen, NY et al., J. Biol. Chem. 262, 8737-2255 (1987)), optionally with the amino acid sequences of aspartate semialdehyde dehydrogenase (ASA-DH) from Escherichia coli (Haziza C. et al., Embo J. 1, 379-384 (1982)) and Streptococcus mutans (Cardineau GA et al., J. Biol. Chem. 262, 7, 3344-3353 (1987)). For this, MALIGN (Sobel E. et al., Nucl. Acids Res. 14, 363-374, (1986)) and DIAGON (Staden R. et al., Nucl. Acid Res. 14, 217-232 ( 1986)). Significant identity between ORF 1 and the AK sequence on the one hand and between ORF 2 and the ASA-DH sequence from Streptococcus mutans on the other hand was shown. Escherichia coli has shown little homology to ASA-DH, especially in the active center region (Haziza, C. et al., Embo J. 1, 379-384 (1982)).

Computer analysis shows the following:

ORF 1 corresponds to the C-terminus of aspartate kinase, i.e., about 160 amino acids of the N-terminus are missing, as well as the complete promoter region.

- ORF 2 corresponds to aspartate semialdehyde dehydrogenase.

The homology of ORF 1 and AK II of Bacillus subtilis is apparent to those skilled in the art. AK II consists of overlapping subunits (Chen, N.Y. et al., J. Biol. Chem. 262, 8787-8798 (1987)). The β-subunit corresponds to the C-terminus of the α-subunit. Since the RBS found in ORF 1 coincides in its position exactly with the RBS of this aspartate kinase, it can be inferred by analogy that there is a cloned Corynobacterium glutamicum β-subunit aspartate kinase.

5. Examination of expression

5.1 Complementation of asd- and lysC-negative Escherichia coli strains

The identity of ORF 2 with the asd gene can be demonstrated by complementing the asd-negative Escherichia coli strain RASA 6 (Richaud, F. et al., C. R. Acad. Sc. Paris, 293, 507-512 (1981)) using plasmids pCS2 and pCS24. Plasmid pCS23, which lacks about 30 amino acids of the C-terminus of ASA-DH, is not complementary. None of these plasmids are able to complement the AKI-III negative strain of Escherichia coli Gif 106 M1 (Boy, E. et al., Biochemistry 61, 1151-1160 (1979)).

5.2. Determination of specific aspartic kinase (AK) and aspartyl beta-semialdehyde dehydrogenase (ASA-DH) in ATCC 13032 transformants with various cleavage derivatives of pCS2

The analogous conclusions drawn in section 4.3 from the homology comparison that a fragment of the Pst1-XhoI gene from DM58-1 contains only part of the lysC (AK) gene but the full asd gene can be unequivocally confirmed by enzyme measurements.

No strain of Corynobacterium glutamicum transformed with pCS2 or a pCS2 derivative contains increased aspartate kinase activity against the parental strain (Table 4, column 3).

SK 279719 Β6

In contrast, in all cases of transformants whose plasmids contained the asd-structural gene, strong superexpression of aspartyl β-scmialdchyddehyde hydrogenase was demonstrated (Table 4, column 2, Figures 3 and 4). Plasmids pCS23 and derivatives of pCS23 do not result in superexpression of aspartyl β-semialdehyde dehydrogenase. Due to the high lability of aspartyl β-semialdehyde dehydrogenase, the superexpression factors, which can be calculated from specific activity, vary between values of 31 to 65.

6. Enzymatic properties and secretion of L-lysine

The exclusion of L-lysine at 7.1 g / L over 72 hours demonstrated for ATCC 13032 pCS2 (see Table 2) can be derived from two genetically engineered changes.

a) Cloning of the aspartate kinase regulatory subunit of DM58-1 without increasing the cellular content of the enzyme.

b) Cloning of aspartyl β-semialdehyde dehydrogenase from DM58-1, resulting in a 31- to 65-fold increase in cellular enzyme content.

Claims (5)

A DNA fragment consisting of a 2.1 kb nucleotide sequence flanked by PstI- and XhoI-cleavage sites, characterized in that it encodes the amino acid sequence shown in FIG. 5, thereby ensuring the production of proteins leading to aspartyl-β-semialdehyde dehydrogenase (asd) activity and aspartic kinase (lysC) deregulation, and is contained in the plasmid pCS2, the deletion map of which is shown in FIG. 3 deposited with Corynobacterium glutamicum under number DSM 5086. Recombinant DNA derived from the plasmid pCS2 according to claim 1, characterized by a deletion map corresponding to the designation pCS26 shown in FIG. Third Recombinant DNA derived from the plasmid pCS2 according to claim 1, characterized by deletion maps corresponding to pCS23 or pCS233 in FIG. Third A method for producing L-lysine by fermentation of microorganisms of the genus Corynobacterium or Brevibacterium producing this amino acid, characterized in that the microorganisms comprise the plasmid according to claim 1 or the recombinant DNA derived therefrom according to claims 2 and 3. The method of claim 4, wherein the microorganism is Corynobacterium glutamicum DMS 5086.