KR101830001B1 - Strain overexpressing l-tryptophan by improving prpp synthesis pathway and process for producing l-tryptophan using the same - Google Patents

Abstract

The present invention relates to an Escherichia genus mutant strain which enhances productivity of L-tryptophan by strengthening a supply of phosphoribosylpyrophosphate (PRPP), and a method for producing L-tryptophan using the strain. More specifically, the present invention provides an Escherichia genus mutant strain and a method using the same for producing L-tryptophan in which activities of transaldolase and transketolase are weakened or inactivated while the activity of glucose-6-phosphate 1-dehydrogenase is strengthened to enhance the productivity of L-tryptophan.

Description

[0001] The present invention relates to a method for producing L-tryptophan by overexpressing L-tryptophan through an improved PRPP synthesis route and a method for producing L-tryptophan using the same. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to an Escherichia genus mutant strain which enhances the production of L-tryptophan by enhancing the supply of phosphoribosylpyrophosphate (PRPP), and a method for producing L-tryptophan using the same.

L-tryptophan, one of the essential amino acids, is important as a starting material for polymerisation of proteins and nicotinamide in vivo. When used in combination with lysine and methionine in vegetable protein, it can increase the protein value. It is widely used as a raw material for medicines such as amino acid enhancer, feed additive, liquid medicine, and health food material. Such L-tryptophan has been produced through chemical synthesis, enzymatic reaction, fermentation using microorganisms, and the like.

In the conventionally known pentose phosphate pathway, biosynthesis of L-tryptophan is accompanied by the formation of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (PEP) to produce precursor chorismate , E4P), and the sub-substrates PRPP, serine and glutamine. As a method for enhancing the production ability of L-tryptophan, most of strategies for enhancing PEP and E4P in order to increase the productivity of aromatic amino acids were performed, and in particular, the tktA gene or the like was overexpressed in order to enhance E4P. However, in the case of tryptophan, the supply of PRPP, which accounts for about 40% of the molecular structure as well as PEP and E4P, is considered to be a major variable (see FIG. 1) in order to enhance the productivity, The development of more advanced tryptophan-producing strains is required.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present invention relates to an Escherichia genus mutant strain which enhances the production ability of L-tryptophan by enhancing the supply of PRPP and a method for producing L-tryptophan using the Escherichia genus mutant. More particularly, the present invention relates to transalolase and transketolase Enhances the activity of glucose-6-phosphate 1-dehydrogenase by weakening or inactivating the activity of transketolase and enhancing the activity of L-tryptophan by increasing the activity of glucose-6-phosphate 1-dehydrogenase And a method for producing L-tryptophan using the same.

In one aspect of the present invention, there is provided a strain in which the activity of the prephenate dehydratase is weakened or inactivated, the activity of the transaldolase and transketolase is weakened or inactivated, and the glucose- Wherein the activity of L-tryptophan is enhanced by the activity of 6-phosphate 1-dehydrogenase.

According to one embodiment of the present invention, the trans-aldolase is a talA gene encoding a trans-aldolase, and the trans-ketolase is a gene encoding a trans-isoleucine by deleting all or a part of a tktB gene encoding a transketolase, The activity of the glucose-6-phosphate 1-dehydrogenase may be weakened or inactivated, and the activity of the glucose-6-phosphate 1-dehydrogenase may be enhanced by increasing the expression of the zwf gene encoding the glucose-6-phosphate 1-dehydrogenase.

According to one embodiment of the present invention, the Escherichia spp. Strain may be Escherichia coli.

 An aspect of the present invention is a method for producing Escherichia genus, comprising: (a) culturing the Escherichia genus mutant strain in a medium; And (b) recovering L-tryptophan from the cultured strain and the culture medium.

According to one embodiment of the present invention, the Escherichia spp. Strain may be E. coli.

The present invention provides Escherichia genus mutant strains that enhance the activity of phospholibosyl pyrophosphate, which is a core substrate required for tryptophan biosynthesis, and can produce L-tryptophan more efficiently and economically by improving the yield .

FIG. 1 shows the pathway of L-tryptophan production in Escherichia genus mutants according to one embodiment of the present invention.
Fig. 2 shows a PCR electrophoresis image (M: ladder marker, C: control strain (1,958 bp), and comparative example 1: talA and tAbA in which a DNA fragment of a tryptophan producing strain lacking the talA and tktB genes was amplified tktB disruption strain (1,067 bp)).
Fig. 3 shows a PCR electrophoresis image (M: ladder marker, C: control strain (1,958 bp), and Example 1: PCR amplification of a DNA fragment of a tryptophan producing strain in which talA and tktB genes were deleted and a zwf gene was inserted. talA, tktB disruption and zwf insertion strain (2,429 bp)).
Figure 4 shows the ability of the Escherichia genus mutant to produce tryptophan according to one embodiment of the present invention at the laboratory level (10 ml).
Figure 5 shows the ability of the Escherichia genus to produce tryptophan according to one embodiment of the present invention at the laboratory level (5 L).

In one aspect of the present invention, in the strain in which the activity of the prefenate dihydratase is weakened or inactivated, the activity of transaldolase and transketolase is weakened or inactivated, and glucose-6-phosphate 1 -Dehyde logenase activity of the Escherichia genus is increased, and L-tryptophan production ability is enhanced.

The present inventors have made efforts to develop an Escherichia sp. Strain capable of efficiently producing L-tryptophan. As a result, they have found that trans-aldolase, transketolase and glucose-6-phosphate 1-dehydrogenase It was confirmed that by controlling the activity of the agase, the supply of PRPP was regulated and consequently the production ability of L-tryptophan could be regulated.

The parent strain of the strain according to the present invention may be an Escherichia genus strain in which the activity of the prefenate dihydratase is weakened or inactivated, and it may be a pheA The activity of the enzyme may be attenuated or inactivated by deletion of all or a part of the gene.

According to one embodiment of the present invention, the ability of L-tryptophan to produce can be determined by a tal gene encoding the trans-aldolase, preferably a talA gene and a tkt gene encoding the transketolase, preferably a tktB gene The activity of each of the enzymes is weakened or inactivated due to deletion of all or a part of the enzymes, and the expression of the zwf gene encoding the glucose-6-phosphate 1-dehydrogenase is increased to increase the expression of PRPP And finally, the productivity of L-tryptophan can be improved.

As used herein, the term " attenuated activity "means that the expression level of the gene as an object is lower than that of the original expression. Such activity is attenuated by nucleotide substitution, insertion, deletion, The activity of the enzyme itself is decreased compared with the activity of the enzyme of the original microorganism and the degree of the enzyme activity in the whole cell is lower than that of the wild type strain or the strain before transformation due to the inhibition of the expression of the gene encoding it or the translation inhibition , And combinations thereof.

As used herein, the term " inactivated "means a case where the expression of a gene encoding a protein such as an enzyme is not expressed at all in comparison with a strain of a wild-type strain or strain before transformation, and a case where the expression thereof is absent.

As used herein, the term " increased expression "means that the expression level of the gene, which is the object, is increased over the original expression level. If there is no gene to increase expression in the pre- When the gene is introduced into the strain of the above strain to increase the expression level and the gene to be expressed in the pre-mutation strain is present, it is preferable to further introduce one or more genes into the strain or to increase the expression level of the existing gene .

In the present invention, a method of modifying an expression control sequence may be performed by inducing a mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof in the nucleic acid sequence of the expression control sequence, And the like. The expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating termination of transcription and translation.

In addition, the method of modifying a gene sequence on a chromosome may be carried out by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof in order to further weaken the activity of the enzyme, Or by replacing the gene sequence with an improved gene sequence or an improved gene sequence so that there is no activity.

As used herein, the term " enhanced activity "refers to the case where the activity of the enzyme itself is enhanced through nucleotide substitution, insertion, deletion, or a combination thereof encoding the gene compared to the enzyme activity of the original microorganism, Or promoting the translation of a gene involved in the expression of the gene, or a combination thereof, when the degree of the overall enzyme activity in the cell is higher than that of the wild-type strain or the strain before the transformation.

As used herein, the term " part "means not all of the sequence of the polynucleotide, and may be 1 to 300, preferably 1 to 100, more preferably 1 to 50, but is not limited thereto .

According to one embodiment of the present invention, in order to produce the Escherichia genus mutant strain of the present invention, a step of isolating and obtaining Escherichia genus transformed from Escherichia sp. Strain not transformed do.

In carrying out the step of isolating and obtaining the transformed strains, a selectively labeled vector can be used. The selection mark used in the present invention includes an antibiotic resistance gene commonly used in the art, and specifically includes ampicillin, But are not limited to, resistance genes for gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline, preferably ampicillin or kanamycin resistance genes.

According to an embodiment of the present invention, the Escherichia spp. Strain may be Escherichia coli, preferably E. coli deficient in the pheA gene, for example, a deposited strain of Accession No. KFCC11660P, but is not limited thereto .

(A) culturing Escherichia sp. Strain according to the present invention in a culture medium; And (b) recovering L-tryptophan from the cultured strain and the culture medium.

The strain used in the present invention can be cultured through a culture method known in the art. As the medium, natural medium or synthetic medium can be used. Examples of the carbon source of the medium include glucose, sucrose, dextrin, glycerol and starch. Examples of the nitrogen source include peptone, meat extract, yeast extract, dried yeast, soybean cake, urea, thiourea, ammonium salt, nitrate And other organic or inorganic nitrogen-containing compounds may be used, but are not limited to these components.

Examples of inorganic salts contained in the medium include, but are not limited to, phosphates such as magnesium, manganese, potassium, calcium and iron, nitrates, carbonates, chlorides and the like. Amino acids, vitamins, nucleic acids and related compounds may be added to the medium in addition to the carbon source, the nitrogen source and the components of the inorganic salt.

The temperature of the culture may be 27 to 40 캜, more preferably 30 to 37 캜, but is not limited thereto. The incubation period can be continued until the desired amount of the useful substance is obtained, preferably 10 to 100 hours, but is not limited thereto.

The step of recovering the L-tryptophan may be carried out by culturing the desired L-tryptophan from the culture using a suitable method known in the art according to the culture method of the microorganism of the present invention, for example, batch, continuous or fed- And the recovering step may include a purification step.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Comparative Example  One. ΔtalAΔtktB  Production of strain

The talA and tktB genes were simultaneously deleted by homologous recombination in the wild-type strain of the parent strain W0G (accession number: KFCC11660P).

For this, 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], and mutagenesis was carried out using a one-step inactivation method. The kanamycin gene of the pKD13 plasmid was used to confirm gene insertion.

Polymerase chain reaction (PCR) was performed using the parental W0G chromosome and pKD13 plasmid as a template in order to make DNA having a part of the gene of each gene and kanamycin resistance gene. The primers used were as described in Table 1 below: (i) denaturation step: 30 seconds at 95 ° C, (ii) annealing step: 30 seconds at 58 ° C, and (iii) extension ) Step: A total of 30 runs were performed at 72 캜 for 2 minutes. The PCR product was electrophoresed on 0.8% agarose gel, and a desired size band was obtained. The fragment of the PCR band was overlapped by PCR again under the following conditions to obtain the desired fragment DNA (denaturation step: 95 30 seconds at 30 ° C, 30 seconds at 58 ° C, and 3 minutes at 72 ° C for a total of 30 times).

The transformed W0G strain transformed with vector pKD46 in which the above DNA fragment was inserted was transformed into a competent state, transformed, plated on a solid medium containing kanamycin, and cultured at 37 ° C for 24 hours. The obtained colony was again confirmed to have deleted the talA and tktB genes by PCR (see FIG. 2), and a strain in which the gene was deleted was developed.

Example  One. ΔtalAΔtktB :: zwf  Production of strain

The talA and tktB genes were deleted by homologous recombination and the zwf gene was inserted into the wild type strain of the parent WOG strain (accession number: KFCC11660P).

PCR was carried out in the same manner as in Example 1, except that the primers used in the PCR were those shown in Table 2 below, and the extension step of overlap PCR was performed at 72 ° C for 4 minutes.

The above DNA fragment was transformed with pKD46 into a competent state, transformed, plated on a solid medium containing kanamycin, and cultured at 37 DEG C for 24 hours. The obtained colony was again subjected to PCR to confirm that the talA and tktB genes were deleted and the zwf gene was inserted (see FIG. 3), and a mutant strain was developed.

Experimental Example  One. ΔtalAΔtktB  :: zwf  Tryptophan of the strain Production capacity  Confirm

The W0G strain, the strain prepared in Comparative Example 1 and Example 1 were used to confirm the degree of enhancement of the productivity.

For evaluation, each strain was cultured overnight in LB solid medium and inoculated into a 10 ml flask with the composition of Table 3 below. After the strain inoculation, the cells were cultured for 72 hours, and the concentration of L-tryptophan obtained therefrom was measured (see Table 4 and FIG. 4).

As shown in Table 4, when the talA and tktB genes of the present invention were deleted and the zwf gene was overexpressed, the yield of tryptophan was about 74% as compared with that of Comparative Example 1 in which only the parent strain and the talA and tktB genes were deleted, .

Therefore, it was confirmed that the amount of PRPP increased as a result of deletion of the talA and tktB genes and overexpression of the zwf gene, resulting in an increase in L-tryptophan production capacity.

Experimental Example  2. ΔtalAΔtktB  :: zwf  Tryptophan of the strain Production capacity  Confirm

The W0G strain and the strain prepared in Example 1 were used to confirm and compare the degree of enhancement of the productivity.

For evaluation, each strain was inoculated overnight in a LB solid medium and inoculated into a 5 L Jar fermenter having a composition of 18.75% glucose in Table 3 above. After the strain inoculation, the cells were cultured for 76 hours, and the concentration of L-tryptophan obtained therefrom was measured (see Table 5 and FIG. 5).

As shown in FIG. 5, when the talA and tktB genes of the present invention were deleted and zwf gene was overexpressed, tryptophan production was improved by about 60.9%, yield was improved by 61.8%, and fermentation The time was shortened compared to the mothers.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and equivalents thereof are included in the scope of the present invention. .

<110> Daesang Incorporation <120> STRAIN OVEREXPRESSING L-TRYPTOPHAN BY IMPROVING PRPP SYNTHESIS          PATHWAY AND PROCESS FOR PRODUCING L-TRYPTOPHAN USING THE SAME <130> PN160187 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 1 tcctcaaaat cgtacccggt 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 2 ccttcttttt ccagctcttc t 21 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 3 agaagagctg gaaaaagaag gttgagcgat tgtgtaggct g 41 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 4 atggtgccag cccagttttt gaattaattc cggggatccg 40 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 5 aaaaactggg ctggcaccat 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 6 agtcgttttc tcccagtcct 20 <210> 7 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 7 gctgtttgcg ttaccgccat ggtctgtttc ctgtgtgaaa tt 42 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 8 atggcggtaa cgcaaacagc 20 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 9 atggtgccag cccagttttt cgcaagatca tgttaccggt 40

Claims (6)

In a strain in which the activity of the prephenate dehydratase is weakened or inactivated,
Transaldolase A and transketolase B are deleted because all or part of the transaldolase A gene encoding transalanase and the transketolase B gene encoding transketolase are deleted, Escherichia having the ability to produce L-tryptophan, in which the activity of glucose-6-phosphate 1-dehydrogenase is weakened or inactivated and the activity of glucose-6-phosphate 1-dehydrogenase is enhanced, A mutation strain.
delete The method according to claim 1,
Wherein the mutant strain has an increased expression of a zwf gene encoding a glucose-6-phosphate 1-dehydrogenase.
The method according to claim 1,
Wherein the Escherichia genus strain is Escherichia coli.
(a) culturing Escherichia sp. strain according to claim 1 in a medium; And
(b) recovering L-tryptophan from the cultured strain and the culture medium
&Lt; / RTI &gt;
6. The method of claim 5,
Wherein the strain Escherichia is Escherichia coli.

Images