Europäisches Patentamt *EP000913466B1* (19) European Patent Office

Office européen des brevets (11) EP 0 913 466 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.7: C12N 1/20, C12P 13/06 of the grant of the patent: // (C12N1/20, C12R1:19) 07.04.2004 Bulletin 2004/15

(21) Application number: 98120261.7

(22) Date of filing: 26.10.1998

(54) Method for producing L- Verfahren zur Herstellung von L-Leucin Procédé de préparation de L-leucine

(84) Designated Contracting States: • Arkadievich, Livshits Vitaly (VNIIGenetika) DE FR NL SE 113545, Moscow (RU) • Ivanovich, Kozlov Yury (VNIIGenetika) (30) Priority: 29.10.1997 RU 97117875 113545, Moscow (RU) • Georgievich, Debabov Vladimir (VNIIGenetika) (43) Date of publication of application: 113545, Moscow (RU) 06.05.1999 Bulletin 1999/18 (74) Representative: Strehl Schübel-Hopf & Partner (73) Proprietor: Ajinomoto Co., Inc. Maximilianstrasse 54 Tokyo (JP) 80538 München (DE)

(72) Inventors: (56) References cited: • Markovich, Gusyatiner Mikhail (VNIIGenetika) EP-A- 0 530 803 EP-A- 0 698 668 113545, Moscow (RU) US-A- 3 970 519 • Grigorievna, Lunts Maria (VNIIGenetika) 113545, Moscow (RU) • CALHOUN D H: "Threonine deaminase from • Valerievna, Ivanovskaya Lirina (VNIIGenetika) Escherichia coli: feedback- hypersensitive 113545, Moscow (RU) from a genetic regulatory mutant." • Georgievna, Rostova Yulia (VNIIGenetika) JOURNAL OF BACTERIOLOGY, (1976 APR) 126 113545, Moscow (RU) (1) 56-63. , XP002122210 • Aleksandrovna, Bachina Tatiana (VNIIGenetika) 113545, Moscow (RU) Remarks: • Zavenovich, Akhverdyan Valery (VNIIGenetika) The file contains technical information submitted 113545, Moscow (RU) after the application was filed and not included in this • Moiseevich, Khurges Evgeny (VNIIGenetika) specification 113545, Moscow (RU)

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 913 466 B1

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Description and the productivity of L-leucine. [0009] But EP-A-0 698 668 discloses a process for Technical Field producing L-leucine comprising culturing a microorgan- ism belonging to the genus Escherichia having resist- [0001] The present invention relates to a method for 5 ance to a leucine analogue and an ability to produce L- producing L-leucine, especially to a method for produc- leucine. ing L-leucine using a bacterium belonging to the genus Escherichia. L-leucine is an essential amino acid which DISCLOSURE OF THE INVENTION can be used as a nutritious additive for food or feed, reagent or material for medical treatment, pharmaceu- 10 [0010] The present invention has been made from the tical or chemical industry, or a growth factor used for pro- aforementioned viewpoint, an object of which is to im- duction of other amino acids such as lysine. prove the productivity of L-leucine of bacteria belonging to the genus Escherichia and to provide an efficient 'and Background Art cost-effective method for producing L-leucine. 15 [0011] As a result of diligent investigation in order to [0002] In the past, L-leucine has been produced by a achieve the aforementioned object, the present inven- method of fermentation primarily using microorganisms tors have found that conferring L-leucine resistance to belonging to the genus Brevibacterium, Corynebacteri- a bacterium belonging to the genus Escherichia im- um or Serratia or mutants thereof which produce L-leu- proves the productivity of L-leucine, and completed the cine (Amino acid fermentation, JAPAN SCIENTIFIC SO- 20 present invention. CIETY'S PRESS, pp.397-422, 1986). [0012] In a first aspect, the present invention provides [0003] US 3,970,519 for example discloses microor- a process for selecting an L-leucine producing bacteri- ganisms of the genus Brevibacterium or Corynebacte- um belonging to the genus of Escherichia which com- rium which resist feedback inhibition by leucine or its prises selecting a strain of a bacterium belonging to the analogs and require at least one of isoleucine, threonine 25 genus of Escherichia that is capable of producing L-leu- or methionine as a growth nutriment and which produce cine and selecting a strain of said bacterium that can L-leucine under aerobic culture conditions. grow in a medium containing 15 g/l of L-leucine. [0004] The highest level of L-leucine accumulation [0013] In a second aspect, the present invention pro- was obtained when using Brevibacterium flavum VKPM vides a bacterium obtainable by the process of the first B-2736: this strain produces L-leucine at a concentra- 30 aspect. tion up to 26 g/L on sucrose-containing media for 72 h [0014] In a third aspect, the present invention pro- of fermentation in a laboratory fermenter (USSR Author vides bacteria having the accession numbers VKPM- Certificate 1394711). And Brevibacterium lactofermen- 7386, VKPM-7387 and VKPM-7388. tum 34 produces L-leucine up to 34 g/L on a medium [0015] In a fourth aspect, the present invention pro- with glucose (Appl. Environ. Microbiol., 51, p.1024 35 vides a method for producing L-leucine, comprising the (1986)). step of culturing the bacteria of the first aspect. [0005] As described above, the productivity of L-leu- [0016] In a fifth aspect, the present invention provides cine has been improved to some extent, however, the a method for producing L-leucine, comprising the steps development of a more efficient and cost-effective meth- of: od for producing L-leucine is required in order to meet 40 increasing demand for L-leucine in the future. a) selecting a bacterium by a process as defined in [0006] On the other hand, microorganisms belonging the first aspect, to the genus Escherichia are potentially utilized as po- b) culturing the bacterium obtained in step a), and tent L-leucine-producing bacteria due to their rapid c) recovering L-leucine from the medium. growth rate, prominent data obtained from genetic anal- 45 ysis and plentiful genetic materials. However, there are [0017] Preferred embodiments of the present inven- few reports which disclose the production of L-leucine tion are defined in the independent claims. using bacteria belonging to the genus Escherichia. [0018] The present invention will be explained in de- [0007] As L-leucine-producing bacterial strains of the tail below. genus Escherichia, a strain which is resistant to β- 50 thienylalanine, strain which is resistant to β-thienyla- <1> Bacterium belonging to the genus Escherichia of lanine and β-hydroxyleucine (Japanese Patent Publica- the present invention tion No.62-34397 for two) and a strain which is resistant to 4-azaleucine or 5,5,5-trifluoroleucine (Japanese [0019] A bacterium of the present invention is a bac- Laid-Open Publication No. 8-70879) are known. 55 terium belonging to the genus Escherichia, which has [0008] However, there have been known neither L- an ability to produce L-leucine and is resistant to L-leu- leucine-resistant bacteria belonging to the genus Es- cine. The bacterium belonging to the genus Escherichia, cherichia nor a relation between L-leucine resistance may be exemplified by Escherichia coli (E. coli). A bac-

2 3 EP 0 913 466 B1 4 terium belonging to the genus Escherichia which has an pathway, which includes a common pathway among the ability to produce L-leucine is exemplified, for example, L-valine biosynthesis and L-leucine biosynthesis. by bacteria having a resistance to leucine analogs such [0022] Of above-mentioned reactions in the L-leucine as β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine biosynthetic pathway, the rate determining step is the and 5,5,5-trifluoroleucine, which are described in Japa- 5 synthetic reaction from α-ketoisovalerate to α-isopro- nese Patent Publication No. 62-34397 and Japanese pylmalate catalyzed by α-isopropylmalate synthase Patent Laid-Open Publication No. 8-70879, and by bac- which suffers feedback inhibition by L-leucine. Also, the terium which can be bred by genetic engineering tech- expression of leuABCD operon is repressed by L-leu- niques as described in WO96/06926. The bacterium be- cine. Expression of ilvBN gene encoding acetohydroxy longing to the genus Escherichia of the present inven- 10 acid synthase I suffers concerted repression by L-valine tion can be obtained by selecting a strain which is re- and L-leucine, expression of ilvGM gene encoding ace- sistant to L-leucine from bacteria belonging to the genus tohydroxy acid synthase II suffers concerted repression Escherichia having an ability to produce L-leucine. Al- by L-isoleucine, L-valine and L-leucine, and expression ternatively, the bacterium belonging to the genus Es- of ilvIH gene encoding acetohydroxy acid synthase III cherichia of the present invention can be also obtained 15 suffers repression by L-leucine. by selecting a strain which has an ability to produce L- [0023] α-Isopropylmalate synthase which is inhibited leucine from bacteria belonging to the genus Es- as well as leuABCD operon which is repressed only con- cherichia being resistant to L-leucine. The most pre- cern the biosynthesis of L-leucine. Therefore the above ferred embodiment is a bacterium belonging to the ge- inhibition and repression do not cause cutting the route nus Escherichia, which is further resistant to leucine an- 20 for supplying any nutrient substance, even if there exists alog(s). an excess amount of L-leucine. Furthermore, although [0020] In a bacterium belonging to the genus Es- expression of ilvIH gene is repressed, expression of il- cherichia, L-leucine is synthesized through biosynthetic vBN gene and ilvGM gene encoding the other isozymes pathway inherent to L-leucine which diverges from the are not effected. Therefore, it is thought that the pres- final intermediate (2-ketoisovalerate) of L-valine biosyn- 25 ence of an excess amount of L-leucine does not effect thesis system. In a bacterium belonging to the genus the growth of cells, however the present inventors un- Escherichia, the final steps of L-valine biosynthesis and expectedly discovered that cell growth was inhibited un- biosynthesis inherent to L-leucine are carried out by a der the presence of an excess amount of L-leucine. Fur- group of encoded by ilvGMEDA operon and thermore, the present inventors succeeded in improving those encoded by leuABCD operon, respectively. 30 the L-leucine productivity of a bacterium belonging to [0021] The leuABCD operon includes leuA, leuB, the genus Escherichia by conferring the L-leucine re- leuC and leuD genes. Among them, leuA encodes α- sistance. isopropylmalate synthase, leuB encodes β-isopropyl- [0024] The method of obtaining bacteria belonging to malate dehydrogenase, leuC and leuD encodes α-iso- the genus Escherichia which have L-leucine resistance propylmalate . Of these enzymes, α-isopro- 35 and bacteria belonging to the genus Escherichia which pylmalate synthase catalyzes the synthetic reaction have resistance to leucine analogs will be explained be- from α-ketoisovalerate to α-isopropylmalate, α-isopro- low. pylmalate isomerase catalyzes the isomerization reac- [0025] The bacteria belonging to the genus Es- tion from α-isopropylmalate to β-isopropylmalate and β- cherichia which have L-leucine resistance can be ob- isopropylmalate dehydrogenase catalyzes the dehydro- 40 tained by culturing bacteria belonging to the genus Es- genation reaction from β-isopropylmalate to α-ketoiso- cherichia in a minimal medium containing L-leucine at caproic acid which is the final intermediate of L-leucine the concentration that causes growth inhibition. Growth biosynthesis. The amination reaction from α-ketoisoc- inhibition herein refers to slow growth or stop of growth. aproic acid to the final , L-leucine, is mainly cat- The selection of the mutants may be performed once or alyzed by . Bacteria belonging to the ge- 45 more. The concentration of L-leucine in the medium is nus Escherichia possess four kinds of , 15 g/L. The bacteria belonging to the genus Escherichia namely, transaminase A (aspartate-glutamate ami- may be subjected to a mutation treatment prior to the notransferase) encoded by aspC gene, transaminase B selection. Mutation may be performed by ultraviolet ir- (BCAA aminotransferase) encoded by ilvE gene which radiation or by treatment with mutagen usually used for is included in ilvGMEDA operon, transaminase C 50 artificial mutagenesis such as N-methyl-N'-nitro-N-nitro- (alanine-valine aminotransferase) encoded by avtA soguanidine (NTG) or nitrous acid and the like. gene and transaminase D (tyrosine aminotransferase) [0026] The bacteria belonging to the genus Es- encoded by tyrB gene. These enzymes participate in cherichia which have L-leucine resistance, obtained as various amination reactions. Of these enzymes, mentioned above, can grow in the presence of L-leucine transaminase B and transaminase D catalyze the 55 at such a concentration that their parent strain cannot above-mentioned amination reaction from α-ketoisoc- grow. aproic acid to L-leucine. Transaminase C and transam- [0027] As mentioned above, L-leucine is concerned inase D catalyze the final step of L-valine biosynthetic with several regulation steps on L-leucine biosynthesis.

3 5 EP 0 913 466 B1 6

Therefore; single mutation which causes L-leucine re- obtained by culturing the strain in a minimal medium sistance may be effective for L-leucine productivity, containing L-valine and selecting growing strains in the however, it is preferably that more regulations are de- same manner as for L-leucine resistance or leucine an- sensitized by double or multiple mutations. A bacterium alog resistance. belonging to the genus Escherichia which has single 5 [0033] However, it is not required to confer L-valine mutation can be used as starting source for breeding of resistance to a bacterium belonging to the genus Es- L-leucine producing strains, even though its productivity cherichia unlike the case of the K-12 strain described of L-leucine is low. above, in the case of using a bacterium which belonging [0028] The bacteria belonging to the genus Es- to the genus Escherichia which possesses acetohy- cherichia which have resistance to leucine analogs can 10 droxy acid synthase which does not suffer from feed- be obtained by culturing bacteria belonging to the genus back inhibition by L-valine for breeding a bacterium be- Escherichia in a minimal medium containing a leucine longing to the genus Escherichia having L-leucine re- analog at the growth inhibitory concentration and select- sistance. ing growing strains. The leucine analog is exemplified [0034] The bacterium belonging to the genus Es- by 4-azaleucine, 3-hydroxyleucine, α-thienylalanine 15 cherichia of the present invention may be enhanced in and 5,5,5-trifluoroleucine and the like, preferably by activity of one or more enzymes of L-leucine biosynthet- 4-azaleucine and 3-hydroxyleucine. ic pathway by usual mutation treatment or genetic engi- [0029] The selection of leucine-analog-resistant mu- neering techniques. Such an enhancement of the activ- tants may be performed with one kind of leucine analog, ity of the enzyme may be performed by introduction of alternatively with more kinds of leucine analogs. The se- 20 recombinant DNA which is obtained by inserting a DNA lection of the mutants may be performed once or more fragment having an entire or a partial ilvGMEDA operon for one kind of leucine analog. and/or leuABCD operon into a plasmid, phage or trans- [0030] An amount of leucine analog which is to be poson to a bacterium belonging to the genus Es- added to the medium depends on a kind of leucine an- cherichia. alog, but preferably amounts to 0.1 g/L or more in the 25 [0035] The analysis of the nucleotide sequence of case of 4-azaleucine or 3-hydroxyleucine. The bacteria leuABCD operon was described in Nucleic Acid Res., belonging to the genus Escherichia may be subjected 20, 3305-3308 (1992). The entire sequence of leuABCD to a mutation treatment prior to the selection in the same operon has been registered in the database (DDBJ ac- manner as described above. cession no. D10483, internet address of DDBJ: http:// [0031] In the case of selecting bacteria belonging to 30 www.ddbj.nig.ac.jp). A DNA fragment having leuABCD the genus Escherichia which have leucine-analog-re- operon can be obtained by amplifying the DNA fragment sistance as well as leucine resistance, any order of se- in accordance with PCR (polymerase chain reaction, re- lection for each of resistance is acceptable and the order fer to White,T.J. et al., Trends Genet., 5,185 (1989)) in is not restricted. which oligonucleotides prepared on the basis of the [0032] In the case of using E. coli K-12 or its deriva- 35 above described sequences are used as primers and tives as the bacterium belonging to the genus Es- chromosomal DNA of a bacterium belonging to the ge- cherichia, it is preferable to confer L-valine resistance nus Escherichia is used as template for PCR. Alterna- in addition to L-leucine and/or L-leucine analog resist- tively, leuABCD operon can also be obtained by screen- ance. K-12 strain does not express an active isozyme II ing a chromosomal DNA library of a bacterium belong- of acetohydroxy acid synthase because a frameshift 40 ing to the genus Escherichia in accordance with hybrid- mutation exists in ilvG gene encoding a large subunit of ization by using an oligonucleotide probe prepared on the isozyme II which is one of acetohydroxy acid syn- the basis of the above described sequences. thase isozymes of branched-chain amino acids biosyn- [0036] The entire nucleotide sequence of ilvGMEDA thetic pathway (Proc. Natl. Acad. Sci. USA 78, 922-925, operon and the nucleotide sequence of upstream region 1981). The isozyme II does not suffer feedback inhibi- 45 of the operon are described in Nucleic Acid Res., 15, tion by L-valine, however other isozymes, isozyme I and 2137-2155 (1987) and Gene, 97, 21-27 (1991), respec- isozyme III, suffer feedback inhibition by L-valine. tively. A DNA fragment having ilvGMEDA operon can be Therefore, K-12 strain cannot grow in a minimal medium obtained by PCR or hybridization using oligonucleotide in presence of excessive amount of L-valine, because probe or primers prepared on the basis of the above de- biosynthesis of L-isoleucine, L-valine and L-leucine is 50 scribed sequence. Incidentally, in the case of using Es- inhibited. Consequently, to obtain a L-leucine producing cherichia coli K-12 or its derivative to obtain ilvGMEDA strain derived from K-12 strain, it is preferable to use a operon, it is preferable to use a strain having a reverse strain having a reverse mutation of ilvG gene in which mutation of ilvG gene in which the frame is restored so the frame is restored so as to recover the activity of the as to recover the activity of the acetohydroxy acid syn- acetohydroxy acid synthase. Such a strain having a re- 55 thase. The methods for obtaining ilvGMEDA operon and verse mutation of ilvG gene will expresses L-valine re- the method for amplifying the operon in a cell of a bac- sistance (Proc.Natl.Acad.Sci.USA 78,922-925, 1981). terium belonging to the genus Escherichia are fully de- K-12 strain which has a resistance to L-valine can be scribed in WO96/06926 and FR 2627508, respectively.

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<2> Method for producing L-leucine Example 1: Construction of the L-leucine resistant strains of Escherichia coli [0037] L-leucine can be efficiently produced by culti- vating the bacterium which can be obtained as de- <1> Selection of L-leucine resistant strains scribed above in a culture medium, producing and ac- 5 cumulating L-leucine in the medium, and recovering L- [0043] The strains of Escherichia coli having L-leu- leucine from the medium. cine resistance and leucine analog resistance were con- [0038] In the method of present invention, the cultiva- structed from the standard laboratory wild-type strain E. tion of the bacterium belonging to the genus Es- coli K-12 by stepwise selection as described below. The cherichia, the collection and purification of L-leucine 10 mutant strain having each resistance was obtained by from the liquid medium may be performed in a manner selecting spontaneous mutants having the resistance. similar to the conventional fermentation method by Concretely, E. coli K-12 or its mutant strain which was which L-leucine is produced using a bacterium. A medi- to be selected was plated on agar plate including L-leu- um used in culture may be either a synthetic medium or cine or leucine analog(s) at various concentrations indi- a natural medium, so long as the medium includes a car- 15 cated below. Then, the grown strain was selected. bon and a source and minerals and, if neces- [0044] At first, a mutant strain which was resistant to sary, a suitable amount of nutrients which the bacterium 5 g/L of L-valine was selected from E. coli K-12 prior to used requires for growth. The carbon source may in- selection of the strain having L-leucine resistance and clude one or more of various carbohydrates such as glu- leucine analog resistance and obtained the strain B-5 cose and sucrose, and various organic acids. Regarding 20 (Valr). From the strain B-5, a mutant strain which was the mode of assimilation of the used bacterium, alcohol resistant to 1 g/L of L-leucine was selected and desig- including ethanol and glycerol may be used. As the ni- nated as No.325 (Valr, Leur). Then, a mutant strain trogen source, it is possible to use various ammonium which was resistant to 0.1 g/L of 4-aza-D,L-leucine salts such as ammonia and ammonium sulfate, other (hereinafter referred to as "4-azaleucine") was selected nitrogen compounds such as amines, a natural nitrogen 25 from the strain No.325 and obtained the strain No. 244 source such as peptone, soybean hydrolysate or digest- (Valr, Leur,ALr). From the strain No. 244, a strain which ed fermentative microbe. As minerals, potassium phos- was resistant to 2 g/L of 4-azaleuicine was selected and phate, magnesium sulfate, sodium chloride, ferrous sul- obtained the strain No. 70 (Valr, Leur,ALrr). The symbol, fate, manganese sulfate, or calcium carbonate may be "Valr", "Leur" or "ALr" represents the strain which was used. 30 conferred resistance to L-valine, L-leucine or azaleu- [0039] The cultivation is performed preferably under cine, respectively. The symbol, ALrr represents the aerobic conditions such as a shaking culture, and an strain which was conferred azaleucine resistance twice. aeration and stirring culture, at a temperature of 20-40 °C, preferably between 30 and 38 °C. The pH of the cul- <2> The relationship between the resistance to L- ture is usually between 5 and 9, preferably between 6.5 35 leucine and L-leucine production and 7.2. The pH of the culture can be adjusted with am- monia, calcium carbonate, various acids, various bases, [0045] To examine the relationship between the L-leu- and buffers. Usually, cultivation for 1 to 3 days leads to cine resistance and L-leucine production, the spontane- the accumulation of the target L-leucine in the liquid me- ous mutant strains resistant to 15 g/L of L-leucine from dium. 40 the strain No.70 which was obtained as described [0040] After cultivation, insoluble substances such as above. cells are removed from the liquid medium by centrifuga- [0046] Seven colonies isolated at random from the tion and membrane filtration, and then the target L-leu- strain No. 70 and 10 mutants isolated at random from cine can be collected and purified by ion exchange, con- the L-leucine resistant mutants which were derived from centration and precipitation. 45 the strain No. 70 were examined for L-leucine produc- [0041] A bacterium belonging to the genus Es- tion. As a result, any of the leucine resistant mutants cherichia of the present invention can be utilized as L- derived from the strain No.70 were more productive than leucine producing strain or starting source for breeding the parent strain. The increase in production was 60% of L-leucine producing strain. The present invention on the average. makes it possible to produce L-leucine more efficiently 50 in comparison with a formerly known method of produc- Example 2: Breeding of L-leucine producing strains from ing L-leucine using a bacterium belonging to the genus Escherichia coli K-12 Escherichia. [0047] The L-leucine producing strains were con- Best Mode for Carrying Out the Invention 55 structed by stepwise selection of strains which had re- sistance to L-valine, azaleuicine, hydroxyleucine and L- [0042] The present invention will be more concretely leucine from E. coli K-12 as described below. Concrete- explained below with reference to Examples. ly, E. coli strains which were to be selected were plated

5 9 EP 0 913 466 B1 10 on agar plate including L-valine, leucine analog or L-leu- 15 g/l of L-leucine from a strain that is capable of cine at various concentrations indicated as follows. producing L-leucine. Then, the grown strain was selected. [0048] A mutant strain which was resistant to 5 g/L of 3. The process according to claim 1, wherein the L- L-valine was selected from E. coli K-12 and obtained the 5 leucine producing bacterium is obtained by select- strain No. 101 (Valr), which did not produce L-leucine. ing a strain that is capable of producing L-leucine From the strain No. 101, a mutant strain which was re- from a strain that can grow in a medium containing sistant to 1.3 g/L of azaleucine. The obtained strain, No. 15 g/l of L-leucine. 51 (Varr,ALr), produced about 0.05 - 0.1 g/L of L-leucine. Then, a strain having a resistance to 2 g/L of 3-hydroxy- 10 4. The process according to any one of claims 1 to 3, D,L-leucine (hereinafter referred to as hydroxyleucine) wherein the strain is further resistant to leucine an- was selected from the strain No. 51 and obtained the alog(s). strain No.4 (Varr,ALr, Hleur). The symbol, "Hleur" rep- resents the strain which was conferred hydroxyleucine 5. The process according to claim 4, wherein said leu- resistance. The strain No. 4 produced more leucine 15 cine analog is selected from the group consisting of (about 0.4 - 0.6 g/L). 4-azaleucine and 3-hydroxyleucine. [0049] The strain No. 4 was treated with NTG and mu- tants having resistance to 15 g/L of L-leucine were se- 6. A bacterium obtainable by a process of any one of lected. As a result, two mutant strains, No. 57 and No. the claims 1 to 5. 103 (Varr,ALr, Hleur, Leur) were obtained. The leucine 20 production by those mutants reached 1.5 - 1.7 g/L. 7. A bacterium having the accession number VKPM- [0050] Among above strains, No. 4 (Escherichia coli 7386. K-12,4), No. 57 (Escherichia coli K-12,57) and No. 103 (Escherichia coli K-12,103) have been deposited in 8. A bacterium having the accession number VKPM- Russian National Collection of Industrial Microorgan- 25 7387. isms (Russia 113545 Moscow 1 Dorozhny proezd, 1) based on Budapest Treaty under the accession num- 9. A bacterium having the accession number VKPM- bers of VKPM-7387, VKPM-7386 and VKPM-7388, re- 7388. spectively. 30 10. A method for producing L-leucine, comprising the Example 3: The production of L-leucine by the strains step of culturing a bacterium according to any one No 57. and No. 103 of claims 6 to 9.

[0051] Cells of the strains No. 57 and No. 103 were 11. A method for producing L-leucine, comprising the grown for 30 hours at 37°C on M9 agar plates. Each 35 steps of: loopful of cultures was inoculated into a shaker flask (250 ml), containing 15 ml of fermentation medium, con- a) selecting a bacterium by a process as de- taining (%) glucose (6), ammonium sulfate (1.5), potas- fined in claim 1, sium hydrophosphate (0.15), magnesium sulfate (0.1), b) culturing the bacterium obtained in step a), thiamine (0.00001), calcium carbonate (2). The cultiva- 40 and tion was carried out for 48 hours at 32°C on rotary shak- c) recovering L-leucine from the medium. er (250 rpm). The L-leucine production by the strain No. 57 was 1.5 g/L, by the strain No. 103 was 1.7 g/L. 12. The method according to claim 11, wherein the bac- terium is further resistant to leucine analog(s). 45 Claims 13. The method according to claim 12, wherein the leu- cine analog is selected from the group consisting of 1. A process for selecting an L-leucine producing bac- 4-azaleucine and 3-hydroxyleucine. terium belonging to the genus of Escherichia which comprises selecting a strain of a bacterium belong- 50 ing to the genus of Escherichia that is capable of Patentansprüche producing L-leucine and selecting a strain of said bacterium that can grow in a medium containing 15 1. Verfahren zur Selektion eines L-Leucin produzie- g/l of L-leucine. renden Bakteriums der Gattung Escherichia, wel- 55 ches das Selektieren eines Stammes eines Bakte- 2. The process according to claim 1, wherein the L- riums der Gattung Escherichia, welcher L-Leucin leucine producing bacterium is obtained by select- produzieren kann, und das Selektieren eines Stam- ing a strain that can grow in a medium containing mes dieses Bakteriums umfasst, welcher in einem

6 11 EP 0 913 466 B1 12

Medium wachsen kann, das 15 g/l L-Leucin enthält. genre Escherichia produisant de la L-leucine, qui comprend la sélection d'une souche de bactérie ap- 2. Verfahren nach Anspruch 1, wobei das L-Leucin partenant au genre Escherichia qui est capable de produzierende Bakterium durch Selektieren eines produire de la L-leucine et la sélection d'une souche Stammes, welcher in einem 15 g/l L-Leucin enthal- 5 de ladite bactérie qui peut croître dans un milieu tenden Medium wachsen kann, aus einem Stamm contenant 15 g/l de L-leucine. erhalten wird, der L-Leucin produzieren kann. 2. Procédé selon la revendication 1, dans lequel la 3. Verfahren nach Anspruch 1, wobei das L-Leucin bactérie produisant de la L-leucine est obtenue en produzierende Bakterium durch Selektieren eines 10 sélectionnant une souche qui peut croître dans un Stammes, welcher L-Leucin produzieren kann, aus milieu contenant 15 g/l de L-leucine à partir d'une einem Stamm erhalten wird, welcher in einem 15 g/ souche qui est capable de produire de la L-leucine. l L-Leucin enthaltenden Medium wachsen kann. 3. Procédé selon la revendication 1, dans lequel la 4. Verfahren nach einem der Ansprüche 1 bis 3, wobei 15 bactérie produisant de la L-leucine est obtenue en der Stamm ferner gegen ein oder mehrere Leu- sélectionnant une souche qui est capable de pro- cinanaloga resistent ist. duire de la L-leucine à partir d'une souche qui peut croître dans un milieu contenant 15 g/l de L-leucine. 5. Verfahren nach Anspruch 4, wobei das Leucinana- logon aus der aus 4-Azaleucin und 3-Hydroxyleucin 20 4. Procédé selon l'une quelconque des revendications bestehenden Gruppe ausgewählt ist. 1 à 3, dans lequel la souche est, en outre, résistante à un/des analogue(s) de la leucine. 6. Bakterium, erhältlich durch ein Verfahren nach ei- nem der Ansprüche 1 bis 5. 5. Procédé selon la revendication 4, dans lequel ledit 25 analogue de la leucine est choisi parmi le groupe 7. Bakterium mit der Hinterlegungsnummer VKPM- constitué par la 4-azaleucine et la 3-hydroxyleuci- 7386. ne.

8. Bakterium mit der Hinterlegungsnummer VKPM- 6. Bactérie pouvant être obtenue par un procédé se- 7387. 30 lon l'une quelconque des revendications 1 à 5.

9. Bakterium mit der Hinterlegungsnummer VKPM- 7. Bactérie ayant le numéro d'entrée VKPM-7386. 7388. 8. Bactérie ayant le numéro d'entrée VKPM-7387. 10. Verfahren zur Herstellung von L-Leucin, welches 35 die Stufe des Kultivierens eines Bakteriums nach 9. Bactérie ayant le numéro d'entrée VKPM-7388. einem der Ansprüche 6 bis 9 umfasst. 10. Procédé de production de L-leucine, comprenant 11. Verfahren zur Herstellung von L-Leucin, welches l'étape consistant à cultiver une bactérie selon l'une folgende Stufen umfasst: 40 quelconque des revendications 6 à 9.

a) Selektieren eines Bakteriums durch ein Ver- 11. Procédé de production de L-leucine, comprenant fahren nach Anspruch 1, les étapes consistant à : b) Kultivieren des Bakteriums, welches in Stufe a) erhalten wurde, und 45 a) sélectionner une bactérie par un procédé tel c) Gewinnen des L-Leucins aus dem Medium. que défini dans la revendication 1, b) cultiver la bactérie obtenue dans l'étape a) et 12. Verfahren nach Anspruch 11, wobei das Bakterium c) récupérer la L-leucine depuis le milieu. ferner gegenüber L-Leucinanaloga resistent ist. 50 12. Procédé selon la revendication 11, dans lequel la 13. Verfahren nach Anspruch 12, wobei das Leucinana- bactérie est, en outre, résistante à un/des analogue logon aus der aus 4-Azaleucin und 3-Hydroxyleucin (s) de la leucine. bestehenden Gruppe ausgewählt ist. 13. Procédé selon la revendication 12, dans lequel 55 l'analogue de la leucine est choisi parmi le groupe Revendications constitué par la 4-azaleucine et la 3-hydroxyleuci- ne. 1. Procédé de sélection d'une bactérie appartenant au

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