WO2008072891A1

WO2008072891A1 - Microorganism whose enzyme activity for nrfe is inactivated and process for producing l-tryptophan using the same

Info

Publication number: WO2008072891A1
Application number: PCT/KR2007/006462
Authority: WO
Inventors: Jong-Il Choi; Jae-Yeong Ju; Hyang Choi; Ji-Sun Lee; Young-Lyeol Yang; Jin-Ho Lee; Young-Hoon Park
Original assignee: Cj Cheiljedang Corporation
Priority date: 2006-12-13
Filing date: 2007-12-12
Publication date: 2008-06-19
Also published as: KR100850853B1; KR20080054481A

Abstract

The present invention relates to a microorganism producing L-tryptophan and a method for producing L-tryptophan using the same, more particularly a recombinant microorganism with improved L-tryptophan productivity by inactivating its nrffi gene encoding formate-dependent nitrite reductase originated from E. coli and a method for producing L-tryptophan using the same.

Description

MICROORGANISM WHOSE ENZYME ACTIVITY FOR NRFE

IS INACTIVATED AND PROCESS FOR PRODUCING L-

TRYPTOPHAN USING THE SAME

Technical Field

[1] The present invention relates to a microorganism producing L-tryptophan and a method for producing L-tryptophan using the same, more particularly a recombinant microorganism with improved L-tryptophan productivity by inactivating its nrffi gene encoding formate-dependent nitrite reductase originated from E. coli and a method for producing L-tryptophan using the same. Background Art

[2] L-tryptophan is one of essential amino acids, which has been used as a feed additive or a raw material for medicines including injections and health foods owing to its hypnotic or tranquilizing effect.

[3] A method for producing L-tryptophan is exemplified by chemical synthesis, enzyme reaction and microorganism fermentation. For chemical synthesis, high temperature and high pressure reaction is required and as a result both D type and L type are included in the reaction product, which makes the purification process difficult. Enzyme reaction has problems of high price of indole and serine used as substrates and of instability of the enzyme, as shown in the patent description of Mitsui Toatsu (Korean Patent Publication No. 90-005773).

[4] In the meantime, L-tryptophan has been produced by fermentation of various auxotrophs and variants of E. coli and Corynebacterium. In 1980s, excellent recombinant strains were developed by genetic recombination techniques, which brought significant increase of productivity. Korean patents describing the production of L- tryptophan by direct fermentation using a microorganism can be generally divided by two major trends, which are the method using an L-tryptophan resistant strain or auxotrophic mutant strain (Korean Patent Publication Nos. 87-1813, 90-8251, 92-7405) and the method using a recombinant strain (Korean Patent Publication Nos. 90-5772, 91-5627). The main object of the method using an L-tryptophan analog resistant strain is to overcome the feed-back inhibition of tryptophan synthesis mediated by metabolites. And the main object of the method using a recombinant strain is to increase the productivity/yield of tryptophan by amplifying major enzymes, which was in fact quite successful.

[5] However, the conventional method for producing L-tryptophan using E. coli mutant has a problem of low productivity. So, the present inventors felt the necessity to increase L-tryptophan productivity by using genetic recombination technique. Disclosure of Invention

Technical Problem

[6] Thus, the present inventors continued to study to increase L-tryptophan productivity and discovered that the L-tryptophan productivity was increased by inactivating nrffi gene in E. coli, based on which the inventors completed this invention.

[7] It is an object of the present invention to provide a recombinant microorganism producing L-tryptophan whose nrffi gene is inactivated.

[8] It is another object of the present invention to provide a method for producing L- tryptophan with high yield by culturing the recombinant microorganism producing L- tryptophan.

[9] The above objects of the present invention can be achieved by the following embodiments of the present invention. Technical Solution

[10] To achieve the above objects, the present invention provides a recombinant microorganism producing L-tryptophan, preferably a microorganism with improved L- tryptophan productivity resulted from the inactivation of nrffi gene therein.

[11] The present invention is described in detail hereinafter.

[12] In this invention, the microorganism producing L-tryptophan can be any microorganism of E. coli, Corynebacterium sp., Serratia sp. or Providencia sp. as long as it can produce L-tryptophan, and preferably E. coli and more preferably E. coli CJ285 (Korean Patent Publication No. 10-2005-0059685) producing L-tryptophan having resistance against tryptophan hydroxamate, the tryptophan analog.

[13] The nrffi gene (NCBI gene ID: 16131900, SEQ. ID. NO: 7) of the present invention is known to be involved in the synthesis, binding and secretion of cytochrome c under anaerobic condition in E. coli. The nrffi gene is also confirmed to be required for formate-dependent nitrite reduction along with nrfG gene.

[14] In this invention, "inactivation" indicates the deficiency of nrffi gene having intracellular activity or the mutation of nrffi to reduce the protein level encoded by the gene inside the cell. So, once nrffi gene is inactivated, nrffi expression is decreased.

[15] The microorganism of the present invention is prepared by inactivating nrffi gene existing in chromosome of a microorganism having L-tryptophan productivity. To inactivate nrffi gene, mutation is induced by using a ray such as UV or a chemical. And from the obtained mutants, a strain having inactivated nrffi gene is selected. Also, the inactivation can be performed by DNA recombination technique. For example, the inactivation by DNA recombination technique can be achieved by inserting nucleotide sequence or the vector containing the nucleotide sequence having homology with the gene encoding nrffi into a target microorganism to induce homologous recombination. Wherein, the above nucleotide sequence or vector above can include a dominant selection marker.

[16] In this invention, the inactivated nrffi gene or its DNA fragment contains polynucleotide sequence having sequence homology with nrffi gene of a host, but this polynucleotide sequence has to have such mutation as truncation of double strand, deletion of base, substitution or insertion of base and inversion of the end so as to be incapable of expressing the protein encoded by nrffi gene.

[17] The insertion of the inactivated nrffi gene or its DNA fragment into a host cell can be achieved by transformation, conjugation, transduction or electroporation, but not always limited thereto.

[18] When the inactivated nrffi gene or its DNA fragment is introduced into a host cell by transformation, the inactivation is induced by mixing the DNA fragment with the strain culture. At this time, the strain can be transformed due to naturally suitable for the insertion of the DNA, but it is preferable for the strain to be appropriate for the DNA insertion in advance (LeBlanc et al, Plasmid 28, 130-145, 1992; Pozzi et al, J. Bacteriol. 178, 6087-6090, 1996). Particularly, the strain is cultured in LB medium until the early logarithmic phase. The cultured strain is washed with distilled water and 10% glycerol and then concentrated to provide appropriate condition for DNA insertion. For homologous recombination, nrffi gene or its DNA fragment is mutated by deletion of a part of nrffi of the genomic DNA or insertion of a foreign DNA fragment and the wild type chromosome copy of the sequence is inactivated and substituted. The nrffi inactivated DNA fragment can inactivate the wild type genome sequence with the tail sequence of the genome DNA by homologous recombination. The inactivation can be confirmed by Southern blotting or more easily by PCR.

[19] The present invention also provides a method for producing L-tryptophan with high yield by culturing the microorganism whose nrffi is inactivated.

[20] In this method for producing L-tryptophan of the present invention, the culture process of the microorganism can be performed in a proper medium and conditions known to those in the art. This culture process can be properly regulated according to the strain selected by those in the art.

[21] It is well understood by those in the art that the culture method can be selected appropriately. For example, the culture method can be selected from the group consisting of batch, fed-batch and continuous cultures, but not always limited thereto. A variety of culture methods are described in the following reference: "Biochemical Engineering" by James M. Lee, Prentice-Hall International Editions, pp 138-176.

[22] The medium for culture has to meet the culture conditions for a target strain. The medium includes various carbon sources, nitrogen sources and trace elements. The carbon source is exemplified by carbohydrate such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose; fat such as soybean oil, sunflower oil, castor oil and coconut oil; fatty acid such as palmitic acid, stearic acid, and linoleic acid; alcohol such as glycerol and ethanol; and organic acid such as acetic acid. One of these compounds or a mixture thereof can be used as a carbon source. The nitrogen source is exemplified by such organic nitrogen source as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and bean flour and such inorganic nitrogen source as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. One of these compounds or a mixture thereof can be used as a nitrogen source. The medium herein can additionally include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium- containing salts as a phosphate source. The medium also can include a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins and proper precursors can be added as well. The medium or the precursor can be added to the culture by batch-type or continuously.

[23] PH of the culture can be adjusted during the cultivation by adding such a compound as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid. The generation of air bubbles can be inhibited during the cultivation by using an antifoaming agent such as fatty acid poly glycol ester. To maintain aerobic condition of the culture, oxygen or oxygen-containing gas (ex, air) can be injected into the culture. The temperature of the culture is preferably 20 - 45⁰C, more preferably 25 - 4O⁰C. The cultivation can be continued until the production of L-tryptophan reaches a wanted level, and the preferable culture time is 10 - 160 hours.

[24] L-tryptophan can be easily separated from the culture by the conventional method well known to those in the art, which is exemplified by centrifugation, filtration, ion exchange chromatography and crystallization. For example, the culture proceeds to low speed centrifugation to eliminate biomass and the obtained supernatant proceeds to ion exchange chromatography for the separation. Best Mode for Carrying Out the Invention

[25] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

[26] However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

[27]

[28] Example 1 : Construction of a recombinant microorganism producing L-trvptophan whose nrfE is inactivated [29] In this example, nrffi gene of E. coli was inactivated by homologous recombination.

The nrffi gene (NCBI gene ID: 1631900) (SEQ. ID. NO: 7) is known to be involved in the synthesis, binding and secretion of cytochrome c in anaerobic condition. It was presumably not necessary for the culture condition for producing L-tryptophan, so that this gene was selected as a target for inactivation to minimize energy loss by unnecessary protein synthesis. The nrffi gene is represented by SEQ. ID. NO: 7.

[30] To inactivate the nrffi gene, one step inactivation, which is a method inducing mutation using lambda Red recombinase developed by Datsenko KA et al (One-step inactivation of chromosomal genes in Escherichia coli K- 12 using PCR products, Datsenko KA, Wanner BL., Proc Natl Acad Sci USA. 2000 Jun 6;97(12):6640-5), was used. To confirm the insertion into the gene, chloramphenicol resistant gene of pKD3 was used as a marker. PCR was performed by using pKD3 as a template with primer 1 and primer 2 shown in Table 1 comprising a part of nrffi gene and a part of the sequence of chloramphenicol resistant gene of pKD3, resulting in the amplification of approximately 1100 bp gene fragment [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] (PCR condition: Denaturing=94°C, 30sec/Annealing=55°C, 30 sec/Polymerization=72°C, 1 minute, 30 cycles).

[31] Table 1

[32] To obtain 5'-DNA fragment of E. coli nrffi gene, PCR was performed by using

E. coli W3110 as a template with primer 3 and primer 4, resulting in the amplification of approximately 500 bp gene fragment [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] (PCR condition: De- naturation=94°C, 30 sec/Annealing=55°C, 30 sec/Polymerization=72°C, 30 seconds, 30 cycles).

[33] Table 2

[34] To obtain 3'-DNA fragment of E. coli nrffi gene, PCR was performed by using E.coli W3110 as a template with primer 5 and primer 6, resulting in the amplification of approximately 500 bp gene fragment [Sambrook et al, Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories] (PCR condition: De- naturation=94°C, 30 sec/Annealing=55°C, 30 sec/Polymerization=72°C, 30 seconds, 30 cycles). [35] Table 3

[36] Herein, 20 pairs of nucleotide sequences of primer 1 and primer 4 were complementary, and 20 pairs of nucleotide sequences of primer 2 and primer 5 were complementary. Thus, the fragment obtained by PCR using primers 1 and 2, the fragment obtained by PCR using primers 3 and 4 and the fragment obtained by PCR using primers 5 and 6 could be linked as one fragment. The PCR products were amplified by PCR five cycles without primers. Primer 3 and primer 6 were added thereto, followed by PCR 25 cycles. As a result, approximately 2100 bp gene fragment was amplified (PCR condition: Denaturation=94°C, 30sec/Annealing=55°C, 30sec/Polymerization=72°C, 2 minutes).

[37] E. coli CJ285 producing L-tryptophan (Korean Patent Publication No.

10-2005-0059685) transformed with pKD46 by the method of Datsenko KA et al (One-step inactivation of chromosomal genes in Escherichia coli K- 12 using PCR products, Datsenko KA, Wanner BL., Proc Natl Acad Sci USA. 2000 Jun 6;97(12):6640-5) was generated as a competent strain, followed by transformation using the 2100 bp sized gene fragment obtained by PCR. The strain was confirmed to have inactivated nrffi gene by the 2100 bp sized gene fragment obtained by PCR using primer 3 and primer 6. The resultant recombinant strain was named as " E. coli COOl-0013", which was deposited at KCCM (Korean Culture Center of Microorganisms) of KFCC (Korean Federation of Culture Collection), the International Depository Authority located at 361-221, Hongje-1-Dong, Seodaemungu-Gu, Seoul, Korea, on November 28, 2006 (Accession No: KCCM 10805P).

[38]

[39] Example 2: Comparison of L-tryptophan productivity of the recombinant mi- croorganism whose nrfE is inactivated

[40] In this example, the recombinant strain CO01-0013 (KCCM 10805P) prepared in example 1 was cultured in L-tryptophan flask titer medium comprising the composition shown in Table 4 at 3O⁰C for 48 hours with stirring at 200 rpm. The con- centration of tryptophan obtained from the culture and the concentration of tryptophan obtained from the culture of E.coli CJ285 were compared. The L-tryptophan concentrations were mean values of three flasks.

[41] Table 4

[42] As a result, as shown in Table 5, the L-tryptophan concentration in the transformant COOl-0013 (KCCM 10805P) culture was 15.2% increased, compared with that in E. coli CJ285.

[43] Table 5

Industrial Applicability

[44] As explained hereinbefore, the L-tryptophan productivity can be increased by inactivating nrffi gene of the L-tryptophan producing microorganism according to the method of the present invention. More particularly, the recombinant E. coli CO01-0013 (KCCM 10805P) whose nrffi is inactivated can be prepared from E. coli CJ285 producing L-tryptophan. And the productivity of L-tryptophan can be increased by culturing the recombinant microorganism.

Claims

[1] A microorganism producing L- tryptophan whose nrffi gene is inactivated.

[2] The microorganism producing L- tryptophan according to claim 1, wherein the nrffi gene is the gene encoding formate-dependent nitrite reductase involved in nitrite reduction function. [3] The microorganism producing L- tryptophan according to claim 1, wherein the microorganism is selected from the group consisting of Escherichia coli,

Corynebacterium sp., Serratia sp. and Providencia sp. microorganisms. [4] The microorganism producing L- tryptophan according to claim 1, wherein the microorganism whose nrffi gene is inactivated is E. coli COO 1-0013 (KCCM

10805P). [5] A method for producing L-tryptophan by using one of the microorganisms of claim 1 to claim 4.