JPS58149689A - Preparation of l-lysine by fermentation - Google Patents

Abstract

PURPOSE:To prepare L-lysine in high yield, by culturing microorganism belonging to Bacillus genus and containing a vector integrated with a gene fraction participating in the lysine antagonist resistance. CONSTITUTION:A microbial strain belonging to Bacillus genus and containing a vector integrated with a gene fraction participating in the lysine antagonist resistance and obtained from the chromosome of a mutant of Bacillus genus resistant to lysine antagonist, e.g. Bacillus subtilis AJ11781 (FERM No.6360), is inoculated in a nutrient medium and cultured under aerobic condition. L-lysine can be separated from the cultured liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-') gin by fermentation. Regarding the production method of lysine using conventional fermentation method,
Methods using artificial mutant strains of Brevibacterium, Zinidomonas, Satucharomyces, Bacillus, etc. are known.

In contrast to the conventional method for producing lysine antagonists as described above, the present inventors have developed a method for producing lysine antagonists, which is obtained from the chromosome of a mutant strain of Bacillus having lysine antagonist resistance. It has been found that bacteria of the genus Bacillus containing a vector into which the region has been integrated accumulate L-lysine at a high yield. The present invention was completed based on this knowledge.

The lysine antagonist as used in the present invention is one that suppresses the growth of microorganisms belonging to the genus Bacillus, and that suppression can be completely or partially relieved by adding L-lysine to the culture medium. For example, S-(2-aminoethyl)
- cysteine (hereinafter referred to as ABC), oxalidine,
Examples include lysine hydroxamate, γ-methyllysine, α-ri p-rocaprolactam, and the like.

The bacteria to which the gene exhibiting resistance to lysine antagonists can be provided may be any mutant strain of the genus Bacillus that is resistant to lysine antagonists, but those with higher resistance are preferable. Furthermore, in many cases, better results can be obtained if a gene with a higher monolysine production ability is used as the gene I4-fM.

A method for extracting chromosomal DNA from genetic fungi is described, for example, in J. Bacteriol, + 89 + 1
066 (1966) C.

Vector DNA may be any plasmid or phage that replicates in Bacillus organisms. For example, p from microorganisms of the genus Staphylococcus
T12? , pci+114, pC221, p・223,
pL]B112 (Proc.

Natl, Acad, Sci, U, S, A, +74
．． 1680 (197?)), p U B 110
(J, Bacteriol, 1134, 318(
1978)), pTP4.1) TP5 (see above, M
icrobiol Letters+ top, 55(1
97B)), pLs16, pLS2 derived from B. subtilis
8 (see above, J, Bacteriol, + Uniniji 699 (197?)), pLs13 (J, Bac
teriol, s 129* 1487 (197?
), pPL1%pPL2 (see above, J, Bac
teriol, +124, 484 (1976))
, rhol 1, known as the temperate phage
(Gene, +589 (1979), phi 10
5 (Gene, 1, 87 (1979), SPO2 (
'Gjne, +1.61 (IQ 79)).

Furthermore, a complex plasmid constructed based on the above plasmid can of course also be used as vector DNA.

Chromosomal DNA and vector DNA are each cut using restriction endonucleases. There is a suitable restriction endonuclease for each vector, which is indicated in the literature that describes the above-mentioned vector. For chromosomal DNA, many types of restriction enzymes can be used as long as the reaction conditions are adjusted so that partial cleavage by restriction endonucleases occurs.

The chromosomal DNA fragment thus obtained and the cut vector DNA are ligated to each other by a conventional method using ligase. On the other hand, vector D was cleaved into a chromosomal DNA fragment using terminal transferase.
It is also possible to use a method in which deoxyazonylic acid and deoxycytidylic acid are added to NA, mixed, and then annealed to link them.

The recipient bacterium for the thus obtained conjugate of the chromosomal DNA fragment and the vector may be any microorganism belonging to the genus Bacillus, but it is preferable to use a strain that does not have resistance to lysine 7 antagonists or an aspartokase deletion mutant. This is convenient when selecting transformed strains.

The combination of the chromosomal DNA fragment and the vector is transferred to the DNA receptor
For example, Molec, Gen.

Genet, 68. 111 (1979) r
- Conventional transformation methods can be used as described.

t, -v To select a transformant strain containing a vector having the ability to produce lysine 7 and into which a gene region related to lysine 7 antagonist resistance has been integrated, use e.g.
A. As a recipient (■), a strain that is not resistant to lysine antagonists may be used for transformation, and a strain that grows in a medium containing the lysine antagonist may be selected. In addition, if a medium is used that allows selection of strains that also have the properties of vector DPJA, such as antibiotic resistance, selection will be easier. In this way, a recombinant vector incorporating a gene region that confers resistance to a lysine antagonist that has been screened for lysine is extracted from a transformed strain, and then
A receptor bacteria, such as L-! It is possible to further increase the amount of L-') resin accumulated by introducing it into a strain capable of producing j resin.

The method for producing L-lysine using the thus obtained lysine-producing bacteria is not particularly different from the conventional culture method for lysine-producing bacteria. That is, the medium is a normal medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, amino acids, nucleic acids, vitamins, etc. As a carbon source,
Glucose, sucrose, lactose, xylose, etc. and starch hydrolysates containing these are used.

As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others can be used. Cultivation is carried out under aerobic conditions while adjusting the pH and temperature of the medium as appropriate until accumulation of L-lysine substantially stops. In this way, a significant amount of L-lysine is produced and accumulated in the culture solution. Conventional methods can be applied to collect L-11 gin from the culture solution.

Example Bacillus subtilis AJ 1315 (FERM-P
635B) r Bact , -Penaasay
1 using Broth J (product name: manufactured by Djfco)
After culturing with shaking for 8 hours, the bacterial cells were collected, and this was transformed into N-methyl-N
'-2) Gl-N-nitrosoguanidine 500μt/
0, 1M phosphate buffer (pH 7,0) containing wl r-
The suspension was suspended and shaken for 20 minutes in ao rc to carry out mutation treatment. Next, the bacterial cells were washed twice with the same buffer solution, and a tryptophan auxotroph and an arginine auxotroph were obtained by the usual replica method. Bacillus subtilis AJ1177 was obtained by further repeating the above mutation treatment with this amino acid auxotrophic strain.
8 () a liptophan, methionine, and thymine polyauxotroph and Bacillus subtilis AJ11771 (arginine and leucine polyauxotroph) were obtained.

AJ11778 was mutated using the above mutagenesis procedure, transferred to the next medium, and cultured for 31.5 r for 5 days.The resulting colonies were harvested as ABC-resistant bacteria.

Medium Nigel = + -su 0.5 t/di, ammonium sulfate o,
lt/d7! , sodium citrate o, o s t /
dl, KH, PO40, 85t/dt, KOHO
,23t/d11MgSO4・7H200,02t/
di, FeSO4-7H200, 11197dl.

MnSO4II? H,OO,1mgl di,
L-) Liptofan 10mg/(
17! , L-)f Onine 10m9/di, L-f Mitsu 5 Q/dl, AECIIHCl 100mg/
d11mgl9/dl, pH 7.0°, and 300 colonies were inoculated into a medium for lysine production assay, and cultured for 31.5r196 hours to assay L-lysine production ability. , AJ11?79 (FERM-P6359') was selected.

fll Chromosomal DNA Preparation AJ+1779 Itr Bact-Penass
ayBroth J (product name manufactured by Di fco) medium, 3
After culturing with shaking at 0r for about 2 hours and collecting the cells in the logarithmic growth phase, the usual DNA preparation method (J, Bacterio
2.3*9 chromosomal DNA was obtained from 1, +89, +oas(t9as))c.

(2) Insertion of a chromosomal DNA fragment into a vector In order to clone the gene region governing ABC resistance and L-lysine production ability, an autonomously replicating plasmid pUB110 (expressing kanamycin and neomycin resistance) was used as the vector. Chromosomal DNA5 obtained in (1)
μt and 5 μt of plasmid pUB110 were each treated with restriction endonuclease EcoR1 for 37C160 minutes to cleave the DNA strands. After heat treatment with 65C for 10 minutes, both reaction solutions were mixed and treated with T4 phage-derived DNA ligase at 10C in the presence of ATP and dithiothreitol.
The DNA strand ligation reaction was carried out for 24 hours.

[Transformation of Bacillus subtilis AJ11711 (arginine and leucine double auxotrophic mutant strain) with a plasmid carrying AEC-resistant and lysine-producing genes was transformed into r Pennassay Btoth J (Djfc
o) The cells were inoculated and cultured overnight at 30C with shaking, and the first culture medium (glucose 0.5 v/di, (N
H4), 5o40.2t/di, KH, Po, 0
．． 6 t/d1%, HPO41.4t/dl, Mg5
O1・7H, OO, 02f/di, sodium citrate 0.1t/di1 yeast extract 0.217dl, L-
Arginine 25 mg/d7! , L-leucine 51
After inoculating the first culture medium (containing Ig/dt) and culturing with shaking for 4 hours using a 37C1 trowel, the first culture medium (Gk)-
7,0,5t/di, (NH4), 5o40.2
t 7dl, KH, PO40, 6f/dl, K, HPO
41.4t/rte.

MgSO4・7H, OO, + 2 f/di1 Sodium citrate 0.1 f//dl, yeast extract 0.02
f/d11L-Arginine 51g/d7! and L-leucine 0.5 mg/di) and 37 t
So-called competent cells (having the ability to take up DNA) were prepared by culturing with shaking for 1.5 hours (Reference: J, Bacteriol).
,+8 on -74B+961)). Add the DNA solution obtained in (2) to this competent cell suspension and incubate at 37C.
After completing the transformation reaction by further shaking culture for 2 hours, the cell suspension was treated with kanamycin at 51.4 t/d.
1% Mg5O, fist 7H, OO, 02f/di, sodium phosphate 0.1 f/d1. L-arginine 1
0 mg/d1%L-R Ishi710 wg/di%
A E CI o @y/di, agar 29/di%I
) H7,2) and cultured in 37r. After 3 days, 5 colonies appeared on the medium, which were harvested and each clone was isolated.

The properties of the obtained transformants all showed arginine, leucine multi-auxotrophy, ABC resistance, and kanamycin resistance.

Extraction of pUBllo carrying the f41AEc resistance region (3)
Among the clones obtained, AJ1178-0 (FERM
-P6361) using the method of C.I. Kada et al. (J, Bacteriol, Eng. 45.1).
365 (1981)), plasmid DNA and chromosomal DNA were separated by agarose gel electrophoresis, and the plasmid DNA fraction was collected and purified.

The new plasmid thus obtained was transformed into the original L-lysine producing strain AJ11?79 using the same method as described in (3). Kanamycin-resistant strain AJ11781 (FERM-P6360
) was obtained.

+51 Lysine production by new ML lysine-producing bacteria J11?79, AJ 1
Table 1 shows the results of culturing 1780 and AJ11781. Culture is carried out in 500-capacity MgSO4・7H
,OO,04f/di1 yeast extract 0.3f/d
l 1 F e SO4” 4Hs OI T11
9/dl s MnSO4' 4H,.

189/di1CaCO85f/dl, pH7
.
.

The culture was performed for 96 hours.

Claims (1)

[Claims] An L-lysine-producing microorganism is cultured in which a microorganism belonging to the genus Bacillus contains a vector incorporating a gene region that is resistant to lysine antagonists obtained from a mutant strain resistant to lysine antagonists of the genus Bacillus. A method for producing lysine, which comprises collecting lysine accumulated in a medium.