SK392792A3 - Method for raising of capacity of corynoform bacteries excluding lysine - Google Patents

Abstract

The invention relates to a process for the enhancement of the performance of L-lysine secreting coryneform bacteria in which a resistance to beta -methyl L-aspartate has been induced and which, where appropriate, have a lower citrate synthase activity than the parent strains.

Description

Technical field

The invention relates to a method for increasing the performance of 1-lysine-secreting coryneform bacteria.

Previous technical conditions ’

The essential amino acid 1-lysine is of great industrial importance as an additive to animal food and feed as well as an active ingredient and a part of pharmaceutical robots. · Ť

The most important way to produce I-lysine is by fermentation. For the production of 3G, mainly coryneform bacteria of the genera Corynebacterium and Brevibacterium are used. wherein the individual amino acid metabolism steps are blocked (e.g., Ese - or Thr auxotrophs) that are resistant to one or more lysine analogs or contain other mutations. High-performance strains generally have multiple aaxotrophs, an analogue of resistance, or a combination of mutations. A summary illustration of the development of lysine proaucents is provided by T. Tosaka and K. Takinami / Progr. Ind. Microbiol. / Biotechnol. Amir-Acids (24, 1986). 152-172;

M. Hilliger, Eiotec 2 (1991) 40-44.

The screening of 1-lysine-producing mutants is referred to as screening.

·> '

For screening, they are induced in the ejected strain by common chemical and physical mutagens, such as MNNG or UV / random mutations, and selected by conventional microbiologic methods. The mutants which are decisive for the success of sereening is now the choice of the selective agent and its appropriate use.

Often used to select lysine producers! Often structural analogues. The effect of these growth inhibitory analogs is abolished by L-lysine. Therefore, mutants that are resistant to this analog include those with an increased production of 1-lysine.

A well-known example of such a structural L-lysine analog is AEC (S-) -2-aminoethyl-7-cysteine. AEC differs from 1-lysine in that; in the 4-position, the carbon atom is replaced by a sulfur atom. This analog has long been known, and ACresistant lysine producers are described in the literature / H. Kase, K. Makayama J Agric. Eiol. Chem. 33/1974 / »

993 to 1000; S.-Kara-Murza et al., Prikladnaya Eiokhimiya Mikrobiologia, 16 (1980) 888-875 (U.S. Pat. No. 3,707,441). s

The performance of these mutants can be increased by introducing additional mutations. Combinations with auxotrophies that are readily inducible by methods known to those skilled in the art are known (US-P 3,708.395 J US-P 3,325.472; J. Plachy, Acta Bioteohnol. £ / 1939/5, 291-293; A. Sassi et al .; Eiotechnol. letter 12 (1990) 4,295-298].

Further, a combination with other resistants is known. For example, antibiotic resistance (DE-OS 27 30964) is described.

SUMMARY OF THE INVENTION It is an object of the present invention to increase the performance of coryneform bacteria secreting amino acids, in particular 1-lysine, by suitable mutations and to identify and characterize corresponding strains by screening.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method of enhancing performance

-3-coryneform L-lysine-secreting microorganisms, characterized in that resistenoe is induced in these strains to 1-aspartic acid (b-methyl ester) (AME).

This is accomplished in such a way that the starting n is exposed to the usual chemical and physical mutagens, e.g.

MNEG; N-methyl-1H-nitro-1H-nitrosoguanidine or UV radiation. The selection of suitable coryneform bacteria, preferably belonging to the genera Corynebaoterium and Brevibaoterium, in particular Cornebacterium glutamicum, is carried out by generally known microbiological methods. The strains produced and found are also an object of the invention.

In contrast to other 1-aspartic acid analogues, AME prevents both growth of, for example, wild-type Corynebacterium glutamicum (ATTC13O32) and its derived tantumum. \ ' in

In addition, the strains used may exhibit additional resistance or auxotrophy.

permentation for the production of 1-lysine is carried out according to generally known methods.

The fact that mutants that already produce lysine and therefore synthesize! the amounts of aspartic acid equivalent to lysine overproduction, are inhibited by AME, is itself surprising.

all the more surprisingly in the present case, the mutants that are selected by Aitin further show lysine production increased over that of the parent strain.

Especially suitable are AME-resistant mutants obtained according to the invention containing a reduced citrate synthase reduced in size and parental strains. According to the literature, this property is advantageous for improving the secretion of aspartate amino acids, such as Xlysine / A. YOTOETA, J, Shii, Agric. Biol. Chem. $ 2, 455463 (1983) // Determination of citrate synthase activity is carried out according to P. A. SKEEE et al. / Aota Chem. Soan. 17, 129 (1983)

The examples relate to the processing of Corynebacterium glutamic mutants (ATCC13O32) produced with MOG

Example 1

ĽM29O-Z? / hse ', AEC / soak overnight in Standard I Bouillon / Merck Art. 7382), washed against saline (0.9 EaCl), mutagenized and rubbed. plates containing AME. The plates contain BT-CG / liebl et al., Appl. Microbiol. Biotechnol (1989) (32: 205-210), supplements 200 µg / ml L, 1-molar serine and 2 to 24, preferably 4 to 8 g / L AME. After 5 days incubation at 30: ‘° C, resistant colonies are inoculated.

To test for lysine production, resistant colonies are incubated for 16 h (300 rpm, 3 ° C) in CASO-Bouillon (Merck Art, 5459) .This suspension is diluted 1: 10 with 9 ml of medium with 240 g / l molasses, 100 ml / l. soy flour hydrolyzate, 12 g / l ammonium supernatant, 10 g / l calcium carbonate (pH = 7) in a 100 ml Erlenmeyer flask with Schikane and incubated for 48 h (30 ° C, 300'rpm). and the concentration of lysine is determined in excess by amino acid analysis.

For specific citrate synthase activities, strains are cultured in Standard I Bouillon / Merk Art.

The cell harvesting and preparation of the enzyme are carried out according to the described method (G). Thierbaoh et al., Appl. Microbiol. Biotechnol, 32, 443

448 (1996) //.

tribe phenotype lys x HCl g / in oitrátsyntáza / "U / mg_7 DM290-2 hse ', AEC ^r 36.5 0,156 DM599 hse, AEC ^X , .AME ^r 40.0 0,126 DM601 hse, AEC ^r , AME ^r 42.8 0,105

Example 2 _ «I *

DM232-2 (leu, AEC) was soaked overnight in Standards I Bouillon / Merck Art. 7832), washed against saline (0.9 # EaCl), mutagenized and spread on plates containing. AME. Glosses contain BMQG medium supplemented with 100 µg / ml 1-leucine and AME as in Example 1.

After 5 days of incubation at 30 ° C, resistant colonies were seeded.

Testing of lysine production is carried out as in Example 1 in medium with molasses 30 g / l, sucrose 85 g / l, soy flour hydrolyzate 153 g / l, 1-leu 100 mg / l, amrnonium sulfate 25 g / l, potassium hydrogen phosphate 3.5 g / l, magnesium sulphate 0,4 g / l, kaleium chloride 10 mg / l, iron sulphate 12 mg / l, manganese sulphate 11 mg / l, citrate 0,6 g / l, biotin 0,3 mg / 1 thiamine 0,2 mg / l, calcium carbonate 25 g / l. Detection of specific activity of citrate synthase is performed as described in Example 1.

tribe

DM282-2

DM597

DM596 phenotype lys r HG1 leu ', ABC ¹ ' 29.9 leu, AEC ^r , AME ^r 34.4 leu *, AEC ^r , AME ^r 33.2 citrate synthesized Z «/ mg /

1.02

0,957

0,983

Example 3

DM236-1 ("leu", Pen ^r , AEC ^r ) is soaked in Standard I Bouillon (Merck Art _c 73821), washed against

6 * saline (9 $ HaCl), mutagenized and spread on plates containing AME. The plates contain BMCG medium supplemented with 100 µg / ml 1-leucine, and 160 µg / ml BI-homoserine and AMfi as in Example 1.

After 5 days of incubation at 30 ° C, resistant colonies are inoculated.

- Testing of lysine production and determination of citrate synthase specific activity is carried out as in Example 2:

tribe phenotype lys atCl / g / 1 / DM236-1 hse ⁵ , leu ^ Pen ¹ , AEC ^r 36.1 DM608 hse ^r , leu ”, Pen ^r , AEC ^r , AME ^r 37.0 DM607 hsn ^x , leu, pen ^x , A.BC ^X , AME ^X 39.5

citrate synthase (U / mg)

0,963 .0,093

Example 4

Corynebacterium glutamicum inhibitory zone (ATCC13O32) depending on AME end. 0 10 20 40 60 30 t 100 120 / g / 1 / (AME) / Ô -meetyl ester of I.-asp aragic acid incase door 000 0,5 1,4 1,6 1,9 2,4 / om / / clear /

The slurry is poured into soft agar (BMCG).

After solidification, 0.15 ml of a solution of AME in MOPS buffer (0.1 M, pH = 7) in a steel cylinder (d = 0.5 cm) was dripped onto agar.

After 3 days of incubation (30 ° C), the inhibitory door is measured and roswell.

Claims (6)

»ΑΤΕ »ϊ ^; 0 YEARS 1. A way to improve the performance of corynephrons. 1-lysine-secreting microorganism strain, characterized in that xesistance to L-aspartic acid β-methyl ester is induced in these strains, 2. The method of claim 1, wherein the mutants originating from these strains further have less citrate synthase activity than the parent strains. A method according to claim 1 or 2, characterized in that strains of the genus Corynebacterium or Brevibacterium are used. ^; The strains of the genus C.orynebaoterium or Brevibacterium expressing l-lysine, verobene according to claim 1, which exhibit resistance to the 1-aspartic acid β-methyl ester. The L-lysine secreting strains produced according to claim 2, further having less citrate synthase activity than the parent strains. Use of the l-lysine secreting strain according to claim 1. 4 or 5 for the fermentative production of 1, -lysine.