AU670767B2 - Process for increasing the efficiency of coryneform bacteria which secrete L-lysine - Google Patents

Description

S F Ref: 215194

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

c c Name and Address of Applicant: o D Degussa Aktiengesellschaft 9, Weissfrauenstrasse 0-6000 Frankfurt am Main

GERMANY

Manfred Kircher and Bernd Bachmann Actual Inventor(s): Address for Service: Invention Title: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Process for Increasing the Efficiency of Coryneform Bacteria Which Secrete L-lysine The following statement is a full description of this invention, including the best method of performing it known tu me/us:- 5845/3 92 106 BT Process for increasing the efficiency of coryneform bacteria which secrete L-lysine The invention relates to a process for increasing the efficiency of coryneform bacteria which secrete L-lysine.

The essential amino-acid L-lysine is of great industrial importance as a food and animal-feed additive, as well as 10 being an active substance and a constituent of pharmaceutical The most important process for the production of L-lysine is fermentation. Utilised above all in production are coryneform bacteria of the genera Corynebacterium and Brevibacterium. As a result of mutations, regulation of the biosynthesis of lysine by these strains is altered in such a way that they produce lysine in amounts over and above what 'they themselves require and secrete it into the medium.

Such over-producers are obtained by seeking mutants in which 20 certain steps of amino-acid metabolism are blocked (eg, hse or thr auxotroph) and which are resistant to one or several analogues of lysine or which contain additional mutations.

Strains which are high producers generally possess several auxotrophs, resistances to analogues or a combination of mutations. A summary account of the development of lysine-producers is given by 0. Tosaka and K. Takinami (Progr.

Ind. Microbiol. (Biotechnol. Amino Acids) 24 (1986), 152-172; M. Hilliger, Biotec 2 (1991) 40-44.

The search for mutants which produce L-lysine is described as screening.

In screening, chance mutations are induced in an initial strain by means of conventional chemical or physical mutagens (eg, MNNG or UV) and mutants are selected by customary microbiological methods. Crucial for the success of the screening at this point is the choice of the means of selection and the appropriate application thereof.

For the selection of lysine-producers use is frequently made of structural analogues of lysine. The effect which these analogues have of inhibiting growth is neutralised by L-lysine. Among mutants which are resistant to the analogue one therefore also finds those with enhanced L-lysine production.

A well-known example of such a structural analogue of L-lysine is AEC (S-[2-aminoethyl]-cysteine). AEC differs from L-lysine only in that the carbon atom in position 4 has been replaced by a sulphur atom. This analogue has long been known, and AEC-resistant lysine-producers are described in 15 the literature Kase, K. Nakayama; Agric. Biol. Chem. 38 (1974), 993 to 1000; S. N. Kara-Murza et al.; Prikladnaya Biokhimiya Mikrobiologiya 16 (1980) 868 to 875; US-PS 3,707,441).

The efficiency of these mutants can be increased by introducing further mutations. Well-known is the combination with auxotrophs which can be easily induced by the method familiar to one skilled in the art (US-PS 3,708,395; US-PS 3,825,472; J. Plachy, Acta Biotechnol. 9 (1989) 3, 291-293; 25 A. Sassi et al.; Biotechnol. Letter 12 (1990) 4, 295-298).

Furthermore, combination with other resistances is well-known. Resistance is described, for example, to antibiotics (DE-OS 27 30964).

Objects of the invention are an increase in the efficiency of coryneform bacteria which secrete amino-acids, in particular L-lysine, by appropriate mutations, and the identification and characterisation of suitable strains by screening.

An object of the invention is a process for increasing the efficiency of coryneform strains of micro-organisms which 3 secrete L-lysine, characterised in that resistance to L-asparaginic acid B-methylester (AME) is induced in these strains.

This is effected in such a way that the initial strain is exposed to conventional chemical or physical mutagens, eg, MNNG: N-methyl-N' -nitro-N-nitrosoguanidine or UV radiation.

Selection of suitable coryneform bacteria, which preferably belong to the genera Corynebacterium and Brevibacterium, in particular Corynebacterium glutamicum, is carried out in accordance with generally known microbiological methods. The strains produced and discovered in this way are also an object of this application.

15 In contrast to other analogues of L-asparaginic acid, AME inhibits the growth of, the wild-type strain of Corynebacterium glutamicum (ATCC13032) and also mutants derived therefrom.

The strains used can, in addition, exhibit other resistances r auxotrophs.

Fermentation to produce L-lysine is effected according to generally known processes.

The fact that mutants which already produce lysine and therefore synthesise an increased amount of asparaginic acid, equivalent to over-production of lysine, are inhibited by AME, is surprising in itself.

All the more surprising in the present case is the fact that mutants selected for resistance to AME additionally exhibit increased lysine production in comparison with the initial strain. Particularly suitable are AME-resistant mutants produced according to the invention which in comparison with the parent strains have a reduced citrate-synthase content.

According to the literature, this strain characteristic is advantageous for improving the secretion of aspartate 4 amino-acids such as L-Iysine YOTOKTA, J. SHIIO, Agric.

Biol. Chem. 52 455-463 (1988)). Citrate-synthase activity is determined according to P. A. SRERE et al. (Acta Chem. Scan.

17, 129 (1963).

Examples The Examples refer to mutants of Corynebacterium glutamicum (ATCC13032) produced by treatment with MNNG.

Example 1 DM290-2 (hse-, AECr) is grown overnight in Standard 1 broth (Merck Art. 7882), washed with physiological cooking-salt solution NaCI), treated with mutagen and spread out onto plates containing AME. The plates contain the medium BMCG (Liebl et al., Appl. Microbiol. Biotechnol. (1989) 32: 205-210), supplemented w'th 200 pg/ml D,L-homoserine and 2 to 24, preferably 4 to 8, g/l AME. After five days' incubation at 30 0 C resistant colonies are inoculated.

15 To test the production of lysine, resistant colonies are incubated in CASO broth (Merck Art. 5459) for 16 h (300 rpm, 0 This suspension is diluted 1:10 in 9 ml of a medium '.containing 240 g/l molasses, 100 ml/l soybean protein hydrolysate, 12 g/l ammonium sulphate, 10 g/l calcium carbonate (pH 7) in 100 ml Erlenmeyer flasks equipped with baffle and incubated for 48 h (30 0 C, 300 rpm). After 48 h the fermentation sludge is centrifugated and the lysine concentration is determined in the supernatant by amino-acid analysis.

25 To determine the specific citrate-synthase activity the strains are cultured in Standard 1 broth (Merck Art. 7881) and 4 g/l glucose. Harvesting of the cells and production of the enzyme preparation is carried out according to a described method (G THIERBACH et al., Appl. Microbiol.

Biotechnol. 32, 443-448 (1990)).

Strain Phenotype Lys*HCl Citrate synthase [U/mg] DM290-2 hse- AECr 36.5 0.156 DM599 hse- AECr, AMEr 40.0 0.126 DM601 hse- AECr, AMEr 42.8 0.105 Example 2 DM282-2 (leu-, AECr) is grown overnight in Standard 1 broth (Merck Art. 7882), washed with physiological cooking-salt solution NaCI), treated with mutagen and spread out onto plates containing AME. The plates contain the medium BMCG, supplemented with 100 pg/ml L-leucine and AME as in Example 1.

After five days' incubation at 30 0 C resistant colonies are inoculated.

Testing of lysine production is carried out as in Example 1 in a medium containing molasses 30 g/l, sucrose 85 g/l, soybean protein hydrolysate 158 g/l, L-leu 100 mg/l, ammonium sulphate 25 potassium hydrogen phosphate 0.5 g/l, 15 magnesium sulphate 0.4 g/l, calcium chloride 10 mg/l, iron sulphate 12 mg/1, manganese sulphate 11 mg/1, citrate 0.6 g/l, biotin 0.3 mg/l, thiamine 0.2 mg/l, calcium carbonate 25 g/l.

The specific citrate-synthase activity is determined as described in Example 1.

Strain Phenotype Lys*HC1 Citrate synthase [U/mg] DM282-2 leu- AECr 29.9 1.02 25 DM597 leu- AECr, AMEr 34.4 0.957 DM596 leu AECr, AMEr 33.2 0.983 Example 3 DM286-1 (hse-, leu-, Penr, AECr) is grown overnight in Standard 1 broth (Merck Art. 7882), washed with physiological cooking-salt solution NaC1), treated with mutagen and spread out onto plates containing AME. The plates contain the medium BMCG, supplemented with 100 pg/ml L-leucine and 160 pg/ml DL-homoserine and AME as in Example 1. After five days' incubation at 30 0 C resistant colonies are inoculated.

The production of lysine is tested and the specific citrate-synthase activity is determined as in Example 2: Strain Phenotype Lys*HCl Citrate synthase [U/mg] DM286-1 hser, leu- Pen AECr 36.1 0.968 DM608 hse r leu- Pen r AECr, AMEr 37.0 0.098 DM607 hser, leu- Penr, AECr, AMEr 39.5 0.120 Example 4 Inhibition zone of Corynebacterium glutamicum (ATCC13032) as a function of AME Conc. 0 10 20 40 60 80 100 120 [g/1] 15 (AME) L-asparaginic acid B-methylester Inhib. 0 0 0 0.5 1.4 1.6 1.9 2.4 [cm] zone c ear A cell suspension is poured into soft agar (BCMG). After solidification has taken place, 0.15 ml of an AME solution in MOPS buffer (0.1 M, pH 7) in a steel cylinder (d 0.5 cm) is dripped onto the agar. After three days' incubation 25 (30 0 C) the inhibition zone is measured and assessed.

Claims (6)

1. Process for increasing the efficiency of coryneform strains of micro-organisms which secrete L-lysine, characterised in that a resistance to L-asparagine acid 3-methyl ester is induced in these strains, wherein mutants stemming from these strains additionally have lower citrate-synthase activity than the parent strains.

2. Process according to Claim 1, characterised in that strains of the genera Corynebacterium or Brevibacterium are used.

3. Process for increasing the efficiency of coryneform strains of micro-organisms which secrete L-lysine, substantially as hereinbefore described with reference to any one of the examples.

4. Strains of the genera Corynebacterium or Brevibacterium secreting L-lysine, produced according to any one of Claims 1 to 3, which exhibit a resistance to L-lysine aspraginic acid p-methyl ester.

Strains produced according to any one of Claims 1 to 3, which secrete L- 15 lysine and additionally have lower citrate-synthase activity than the parent strains.

6. Use of strains which secrete L-lysine according to Claim 4 or 5 for the fermentative production of L-lysine. Dated 4 December, 1995 Degussa Aktiengesellschaft 20 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:NLIBUU100637:SAK Process for Increasing the Efficiency of Coryneform Bacteria which secrete L-lysine ABSTRACT The invention relates to a process for increasing the efficiency of coryneform bacteria which secrete L-lysine, in which bacteria a resistance to L-asparaginic acid B-methylester has been induced and which in some cases exhibit diminished citrate-synthase activity in comparison with the parent strains. 4 9 e**oo 0 9 4 9 o