ATP-PRT mutants with reduced feedback inhibition by histidine and histidine-producing strains expressing the same

Abstract

The present invention relates to a mutant of ATP-phosphoribosyltransferase derived from Escherichia coli hisG that has reduced feedback inhibition by histidine, and a bacterial strain expressing the same, which maintains activity even at high histidine concentrations, thereby enabling increased histidine production.

Description

Technical Field

[0001] The present invention relates to an ATP-PRT mutant with reduced feedback inhibition by histidine and a histidine-producing strain expressing the same.

Background Art

[0002] ATP-phosphoribosyltransferase (hereinafter sometimes referred to as ATP-PRT) catalyzes the first step of histidine biosynthesis in bacteria, fungi, or plants.

[0003] In an environment where the concentration of L-histidine is present at a certain level or higher, the activity of ATP-phosphoribosyltransferase is feedback-inhibited by histidine, so it is difficult to increase the histidine production amount to a certain level or higher.

[0004] Therefore, an ATP-PRT mutant with increased histidine resistance is required to increase the histidine production amount of microorganisms. However, no mutant capable of reducing the histidine feedback inhibition of ATP-PRT expressed by the hisG gene of Escherichia coli is known.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] According to one specific example, an ATP-phosphoribosyltransferase mutant with reduced feedback inhibition by histidine is provided.

Means for Solving the Problems

[0007] One aspect provides an ATP - phosphoribosyltransferase mutant in which the serine at position 288 is substituted with proline in the amino acid sequence of SEQ ID NO: 1.

[0008] The amino acid sequence of SEQ ID NO: 1 is the sequence of ATP - phosphoribosyltransferase expressed from the wild - type hisG of Escherichia coli. ATP - phosphoribosyltransferase may be referred to as ATP - PRT. ATP - PRT catalyzes the reaction 1-(5 - phospho - D - ribosyl)-ATP + diphosphate ⇔ ATP+5 - phospho - alpha - D - ribose 1 - diphosphate, which is the first step of histidine biosynthesis. In the present application, the ATP - phosphoribosyltransferase is used interchangeably with "hisG".

[0009] According to one specific example, the ATP - phosphoribosyltransferase may be expressed from the hisG gene of Escherichia coli (E. coli).

[0010] According to one specific example, the feedback inhibition by histidine of the mutant can be reduced. According to one example, the strain introduced with ATP - PRT containing the S288P mutation had an increased histidine production compared to the wild - type.

[0011] According to one specific example, the mutant may further include one or more of the following: (a) the arginine at position 250 is substituted with histidine; (b) the histidine (H) at position 232 is substituted with lysine (K) or threonine (T); (c) the threonine at position 252 is substituted with alanine, leucine, glycine, valine, or isoleucine; (d) the glutamic acid at position 271 is substituted with lysine. According to one example, it was confirmed that in addition to the serine mutation at position 288, further including the mutations of (a) - (d) above increased the histidine production.

[0012] The mutant can have activity even at a histidine concentration of 5 mM to 25 mM.

[0013] Another aspect provides a polynucleotide encoding the ATP - phosphoribosyltransferase mutant, or a vector containing the same. The vector may be a plasmid or a phage.

[0014] Another aspect provides a transformed strain expressing the ATP - phosphoribosyltransferase mutant. The transformed strain may be a strain into which a polynucleotide encoding the ATP - phosphoribosyltransferase mutant, or a vector containing the same, has been introduced. Since the strain maintains the activity of ATP - phosphoribosyltransferase even when the histidine concentration increases, the histidine production amount can be increased.

[0015] The strain expressing the ATP - phosphoribosyltransferase mutant can increase histidine production by about 22 - 92%.

[0016] The transformation can be carried out by a known method, for example, by the electroporation method (van der Rest et al., Appl. Microbiol. Biotechnol., 52, 541 - 545, 1999).

[0017] According to one specific example, the strain may be a strain of the genus Escherichia, specifically, Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii (Escherichia hermannii), or Escherichia vulneris strain.

[0018] Still other aspects provide a method for producing histidine, which includes the step of culturing the transformed strain. The method for producing histidine can include the step of culturing the transformed strain in a medium and the step of recovering histidine from the strain or the medium.

[0019] The medium can include a carbon source, a nitrogen source, and inorganic salts. The carbon source can include, for example, sugars and carbohydrates such as glucose, sucrose, citrate, fructose, lactose, maltose, or molasses; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; fatty acids such as palmitic acid, stearic acid, linoleic acid; glycerol; alcohols such as ethanol; organic acids such as acetic acid, and is not particularly limited and can be used individually or as a mixture. Preferably, the medium for the E. coli mutant strain may contain glucose. The nitrogen source can include, for example, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean cake, urea, thiourea, ammonium salts, nitrates, and other compounds containing organic or inorganic nitrogen, and is not particularly limited. Also, for the inorganic salts, magnesium, manganese, potassium, calcium, iron, zinc, cobalt, etc. can be used, and is not limited thereto.

[0020] In addition, for adjusting the pH of the medium, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner. Also, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress bubble formation, and oxygen or an oxygen-containing gas (e.g., air) can be injected into the medium to maintain an aerobic state.

[0021] The culturing means growing the microorganism in an artificially regulated environment and can be carried out by culturing methods widely known in the art. The temperature during culturing can be 20 - 45°C, and it can be cultured for 10 - 200 hours, but is not limited thereto.

[0022] The step of recovering the histidine can use various methods well-known in the art. For example, centrifugation, filtration, anion exchange chromatography, crystallization, or HPLC can be used, but it is not limited thereto.

Advantages of the Invention

[0023] The ATP-phosphoribosyltransferase variant according to one specific example can maintain its activity even in a high-concentration histidine environment. The strain expressing the ATP-phosphoribosyltransferase variant according to one specific example can increase the histidine production amount.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0025] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for explaining one or more specific examples as examples, and the scope of the present invention is not limited to these examples.

[0026] Example 1: Selection of Mutant Strains with TRA (1,2,4-triazole-3-alanine) Resistance To create mutants with a blunted negative feedback by L-histidine, a mutant resistant to 1,2,4-triazole-3-alanine (TRA), a derivative of L-histidine, was created using N-methyl-N’-nitro-N-nitrosoguanidine (NTG), a chemical mutagen

[0027] E. coli MG1655 (KCTC14419BP) was cultured in LB medium for 16 hours (37 °C, 200 rpm). After culturing, it was centrifuged at 4500 rpm for 10 minutes and suspended in saline / TM buffer. After adding buffer to the cells and resuspending them, 100 μg / ml of NTG was added, and mutagenesis was induced at 37 °C, 200 rpm for 30 minutes

[0028] After repeating the mutagenesis process, the cells were suspended in 3 ml of D.W., and this was spread on a plate medium (composition: 8% glucose, 0.6% monosodium phosphate, 0.2% ammonium sulfate, 0.02% magnesium sulfate, 0.001% calcium nitrate, 10 ppm iron sulfate, 1% TRA) and primary cultured at 37 °C for 2 days. Strains that formed single colonies were isolated and secondary cultured in the same manner as the primary culture on a plate medium supplemented with 1% TRA to select mutants

[0029] The growth (increase in cell number) of the selected mutants on plate media supplemented with 0%, 0.5%, 1.0%, or 2.0% TRA was measured to compare the resistance to TRA. (See Table 1 below)

[0030]

Table 1

[0031] Example 2: Amino acid sequence analysis of the ATP-PRT enzyme of TRA-resistant mutants The amino acid sequences of the ATP-PRT (ATP-phosphoribosyltransferase, hisG) enzymes of mutant strains H-1 and H-2 with increased resistance to TRA were comparatively analyzed. The sequence analysis was carried out by commissioning Macrogen, and the sequences were confirmed using the primers shown in Table 2 below.

[0032]

Table 2

[0033] As a result of the confirmation, it was confirmed that a part of the amino acids located at the C-terminal portion of the ATP-PRT enzyme was substituted.

[0034] Also, using a molecular binding mode prediction program, the three-dimensional structure at the time of docking with histidine molecules of the hisG hexamer derived from E. coli was analyzed, and based on the results of the docking analysis, the amino acids located at the histidine entry and binding sites of ATP-PRT expressed from E. coli hisG were analyzed. As a result of the simulation, it was revealed that H232, S288, T252, R250, A248, E271, and E240 of hisG are highly likely to interact with histidine. (See Figure 2)

[0035] Based on the results of the ATP-PRT (hisG) amino acid mutations and docking analysis of the TRA-resistant mutant strains, 14 amino acid mutants (H232T, H232E, H232K, E240K, A248F, R250H, R250E, T252A, T252L, T252P, T252Q, E271K, S288K, and S288P) with a high possibility of reduced negative feedback by histidine and increased histidine production were selected as candidates.

[0036] Example 3: Preparation of an ATP-PRT mutant expression strain having one mutation and evaluation of its histidine productivity To introduce hisG_H232K with a point mutation into the chromosome of E. coli DS9H strain, a one-step inactivation method was used (Warner et al., PNAS, 6:6640-6645 (2000)). First, to obtain the forward and reverse fragments of the hisG gene for homologous recombination, the hisG_HF and hisG_HR fragments were amplified using primer pairs hisG_HF-F / hisG_HF-R and hisG_HR-F / hisG_HR-R, respectively, with E. coli DS9H genomic DNA as the template. Then, to obtain a cassette containing a kanamycin antibiotic marker and FRT, the cassette fragment was obtained by amplifying from the pKD13 plasmid using FR(hisG)-F / FR(hisG)-R. Finally, to obtain hisG_H232K, two fragments were obtained using primer pairs hisG+FR-F / 232K-R and 232K-F / hisG+HR-R from E. coli DS9H genomic DNA, respectively. The two obtained fragments were ligated into one fragment using the hisG+FR-F / hisG+HR-R primer again to obtain the hisG_H232K fragment. Finally, using these four amplified PCR fragments as templates, they were ligated into one fragment using overlapping PCR with the hisG_HF-F / hisG_HR-R primer pair. The ligated DNA fragment was introduced into the E. coli DS9H strain carrying the pKD46 plasmid by electroporation. Thereafter, PCR was performed using the hisGW-CF / hisGW-CR primers on the cell line showing kanamycin resistance to confirm the strain into which hisG_H232K was introduced. The process of removing the kanamycin marker, which is an antibiotic resistance gene, was carried out on the strain in which the introduction was confirmed. After introducing the pCP20 plasmid into the strain in which the introduction of hisG_H232K was confirmed to induce FLP recombination, the presence or absence of antibiotic removal was confirmed by whether it grew on LB plate media with and without the addition of the antibiotic (kanamycin). It was confirmed by taking advantage of the fact that the strain from which the antibiotic was removed grew on LB plate media but could not grow on LB plate media with the addition of the antibiotic (kanamycin).Finally, the sequence was confirmed using the hisGW-CF / hisGW-CR primer pair. By the same method as the above method, hisG_H232T, hisG_R250H, hisG_T252A, hisG_T252L, hisG_E271K, hisG_S288P, hisG_H232E, hisG_240K, hisG_A248F, hisG_R250E, hisG_T252P, hisG_T252Q, and hisG_S288K were respectively introduced into the E. coli DS9H strain.

[0037] The primers used in the above experiment are as shown in Table 3 below.

[0038] [Table 3]

[0039] According to Table 4 below, the strains introduced with hisG_H232K or hisG_H232T had about a 22% - 26% increase in histidine production compared to the control group. The strains introduced with hisG_T252A or T252L had about a 35% - 39% increase in histidine production compared to the control group. The strain introduced with hisG_E271K had about a 34% increase in histidine production compared to the control group. In particular, the strain introduced with hisG_S288P had about a 46% increase in histidine production compared to the control group, and the strain introduced with hisG_R250H had the highest increase of about 67% in histidine production compared to the control group.

[0040] However, the H232E, E240K, and A248F mutants had a decrease in histidine production instead, and the R250E, T252P, T252Q, and S288K mutants did not have a significant increase in histidine production.

[0041] [Table 4]

[0042] According to the above results, the mutants of the seven types (H232T, H232K, R250H, T252A, T252L, E271K, and S288P) have increased histidine production, which is presumably due to a decrease in feedback inhibition by histidine. Hereinafter, it was confirmed whether the production of histidine can be further improved by combining these mutations.

[0043] Example 4: Preparation of a plasmid into which hisG_SDM4 (H232K, T252A, E271K, and S288P) was introduced Overlapping PCR was performed to prepare a plasmid capable of expressing a mutant in which the amino acids of H232K, T252A, E271K, and S288P were substituted in the ATP-PRT enzyme derived from Escherichia coli hisG. First, three pairs of primers, hisG-F / 232K-R, 232K-F / 252A-R, and 252A-F / hisG-R, were used to amplify the gene with pfu premix (bioneer), respectively. Then, using the three amplified fragments as templates respectively, PCR was carried out again with the hisG-F / hisG-R primer pair to ligate the three fragments into one fragment (hereinafter sometimes referred to as the SDM3 fragment). Then, the SDM3 fragment and the pTRC99A plasmid were each treated with the restriction enzymes EcoRI and HindIII (NEB), and the SDM3 fragment was introduced into the pTRC99A plasmid using T4 ligase. (pTRC99A-hisG_SDM3) PCR was carried out with the pTRC99A-hisG_SDM3 template and the hisG-F / 271K-R2 primer pair to obtain an SDM4 fragment into which four mutations of H232K, T252A, E271K, and S288P were introduced.

[0044] Then, the SDM4 fragment and the pTRC99A-hisG_SDM3 plasmid were each treated with the restriction enzymes EcoRI and AfeI (NEB), and pTRC99A-hisG_SDM4 was constructed using T4 ligase (Takara). Finally, the sequence was confirmed using the hisG-CF / hisG-CR primer pair. (See Table 5 below) The ATP-PRT mutant containing the H232K, T252A, E271K, and S288P mutations was named hisG_SDM4.

[0045]

Table 5

[0046] Example 5: Preparation of a plasmid into which hisG_SDM7 (H232T, R250H, T252L, E271K, and S288P) was introduced hisG_SDM7 was prepared by substituting some of the amino acids in the amino acid sequence of the hisG_SDM4 enzyme with other amino acids, and this was introduced into the plasmid. Using pTRC99A-hisG_SDM4 as a template, the 232nd amino acid was substituted with T, the 250th amino acid with H, and the 252nd amino acid with L, and the two mutations (E271K and S288P) were maintained as they were. (Compared with hisG_WT, the positions of the mutations in hisG_SDM7 are H232T, R250H, T252L, E271K, and S288P)

[0047] First, genes were amplified using three pairs of primers, namely primer hisG-F / 232T-R, 232T-F / 250H+252L-R, and 250H+252L-F / hisG-R, with pfu premix (bioneer). Then, using the three amplified fragments as templates respectively, PCR was carried out again with the hisG-F / hisG-R primer pair to ligate the three fragments into one fragment. Subsequently, the PCR fragment and the pTRC99A plasmid were digested with EcoRI and HindIII (NEB) respectively and ligated with T4 ligase (Takara) to prepare pTRC99A-hisG_SDM7. Finally, the sequence was confirmed using the hisG-CF / hisG-CR primers. (See Table 6 below) The ATP-PRT mutants containing the H232T, R250H, T252L, E271K, and S288P mutations were named hisG_SDM7.

[0048]

Table 6

[0049] Example 6: Preparation of mutant strains into which the hisG_SDM4 or hisG_SDM7 gene has been introduced 6-1. Preparation of mutant strains into which the hisG_SDM4 gene has been introduced To introduce hisG_SDM4 into the chromosome of E. coli DS9H strain, a one-step inactivation method was used (Warner et al., PNAS, 6:6640-6645 (2000)). First, to obtain the forward and reverse fragments of the hisG gene for homologous recombination, the hisG_HF and hisG_HR fragments were amplified using the primer pairs hisG_HF-F / hisG_HF-R and hisG_HR-F / hisG_HR-R, respectively, with E. coli DS9H genomic DNA as the template. Then, to obtain a cassette containing a kanamycin antibiotic marker and FRT, the cassette fragment was obtained by amplifying from the pKD13 plasmid using FR(hisG)-F / FR(hisG)-R. Finally, to obtain hisG_SDM4, the hisG_SDM4 fragment was obtained from the pTRC99A-hisG_SDM4 plasmid using the hisG+FR-F / hisG+HR-R primers. Finally, using these four amplified PCR fragments as templates, they were ligated into one fragment using overlapping PCR with the hisG_HF-F / hisG_HR-R primer pair. The ligated DNA fragment was introduced into the E. coli DS9H strain carrying the pKD46 plasmid by electroporation. Subsequently, PCR was performed using the hisGW-CF / hisGW-CR primers on cell lines showing kanamycin resistance to confirm the strain into which hisG_SDM4 was introduced. The process of removing the kanamycin marker, an antibiotic resistance gene, was carried out on the strain in which the introduction was confirmed. After introducing the pCP20 plasmid into the strain in which the introduction of hisG_SDM4 was confirmed to induce FLP recombination, the presence or absence of antibiotic removal was confirmed by whether it grew on LB plate media with and without the addition of the antibiotic (kanamycin). It was confirmed by taking advantage of the fact that the strain from which the antibiotic was removed grew on LB plate media but could not grow on LB plate media with the addition of the antibiotic (kanamycin). And finally, the sequence was confirmed using the hisGW-CF / hisGW-CR primer pair. The primers used in the experiment are listed in Table 7 below.

[0050]

Table 7

[0051] 6-2. Preparation of Mutant Strains into Which the hisG_SDM7 Gene Has Been Introduced To introduce hisG_SDM7 into the chromosome of E. coli DS9H strain, a one-step inactivation method was used (Warner et al., PNAS, 6:6640-6645 (2000)). First, to obtain the forward and reverse fragments of the hisG gene for homologous recombination, the hisG_HF and hisG_HR fragments were amplified using primer pairs hisG_HF-F / hisG_HF-R and hisG_HR-F / hisG_HR-R with E. coli DS9H genomic DNA as a template, respectively. Then, to obtain a cassette containing a kanamycin antibiotic marker and FRT, the cassette fragment was obtained by amplifying from the pKD13 plasmid using FR(hisG)-F / FR(hisG)-R. Finally, to obtain hisG_SDM7, the hisG_SDM7 fragment was obtained from the pTRC99A-hisG_SDM7 plasmid using the hisG+FR-F / hisG+HR-R primers. Finally, using these four amplified PCR fragments as templates, they were ligated into one fragment using overlapping PCR with the hisG_HF-F / hisG_HR-R primer pair. The ligated DNA fragment was introduced into the E. coli DS9H strain having the pKD46 plasmid by electroporation. Thereafter, PCR was performed using the hisGW-CF / hisGW-CR primers on the cell line showing kanamycin resistance to confirm the strain into which hisG_SDM7 was introduced. The process of removing the kanamycin marker, which is an antibiotic resistance gene, was carried out on the strain in which the introduction was confirmed. After introducing the pCP20 plasmid into the strain in which the introduction of hisG_SDM7 was confirmed to induce FLP recombination, the presence or absence of antibiotic removal was confirmed by whether it grew on LB plate media with and without the addition of the antibiotic (kanamycin). It was confirmed by taking advantage of the fact that the strain from which the antibiotic was removed grew on LB plate media but could not grow on LB plate media supplemented with the antibiotic (kanamycin). Finally, the sequence was confirmed using the hisGW-CF / hisGW-CR primer pair. The primer sequences used for the preparation of the mutant strain into which the hisG_SDM7 gene was introduced are the same as those in Table 6 above.

[0052] Example 7: Measurement of the negative feedback resistance of histidine of the mutant enzyme expressed from the hisG_SDM4 or hisG_SDM7 gene The negative feedback resistance of histidine of ATP-PRT wild type (hisG_WT) and ATP-PRT mutants (hisG_SDM4 and hisG_SDM7) was compared.

[0053] 50 ml of LB medium was dispensed into 500 ml flasks, and three strains of DS9H, DS9H_ΔhisG::hisG_SDM4, or DS9H_ΔhisG::hisG_SDM7 were inoculated at 1% each. The culture conditions were 30 °C and 180 rpm. OD 600 When it reached 0.6, the expression of ATP-PRT was induced with 1 mM IPTG (final concentration), and additional culture was carried out for about 4 hours. The cells obtained after culture were sonicated and centrifuged. The resulting supernatant was used for the activity evaluation of ATP phosphoribosyltransferase. The reaction conditions for evaluating the enzyme activity were carried out with reference to existing literature. (Microb Cell Fact. 2018. Mar. 17:42) The supernatant was quantified for protein to make the concentrations consistent, and after mixing the reactants with the reaction composition shown in Table 8 below, the enzyme activity was measured.

[0054]

Table 8

[0055] In particular, to confirm the resistance to the suppression of activity by histidine, the concentrations of histidine were set to 0 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, and 50 mM, respectively. The activity measurement was carried out at 30 °C and a UV wavelength of 290 nm at 2-minute intervals for 30 minutes. According to Figure 1, the ATP-PRT activity of the hisG_WT enzyme decreased sharply when the histidine concentration was 5 mM. However, for hisG_SDM4 (H232K, T252A, E271K, S288P), the enzyme activity decreased when the histidine concentration was 25 mM. In addition, for hisG_SDM7 (H232T, R250H, T252L, E271K, S288P), similar to hisG_SDM4, the enzyme activity decreased when the histidine concentration was 25 mM, but at each histidine concentration, the enzyme activity was increased compared to hisG_SDM4. As a result, hisG_SDM7 was the most resistant to the inhibition of activity by histidine.

[0056] Example 8: Evaluation of Histidine Productivity of ATP-PRT Mutant Enzyme-Expressing Strains The histidine productivity of the strains into which hisG_SDM4 or hisG_SDM7 was introduced was confirmed. 10 ml of the medium with the composition shown in Table 9 below was dispensed into each flask, and the DS9H, DS9H_ΔhisG::hisG_SDM4, or DS9H_ΔhisG::hisG_SDM7 strain was inoculated at 1% each, and cultured at 34 °C and 200 rpm for 72 hours. After culturing, the histidine production amounts in each flask were comparatively analyzed.

[0057]

Table 9

[0058] The hisG_SDM4-expressing strain had an approximately 53% increase in histidine production compared to the control group, and the hisG_SDM7-expressing strain had an approximately 92% increase in histidine production compared to the control group. (See Table 10)

[0059]

Table 10

[0060] According to the above results, it is considered that hisG_SDM4 or hisG_SDM7 has a decreased feedback inhibition by histidine compared to his_WT and an increased histidine productivity. In particular, the histidine productivity of the hisG_SDM7-expressing strain was higher than that of the hisG_SDM4-expressing strain.

[0061] Also, summarizing the results in Table 4 and Table 8, when any one of the amino acids at positions 232, 250, 252, 271, and 288 of E. coli-derived hisG was mutated, the production amount of histidine increased, and when multiple amino acids were mutated, the production amount of histidine increased more than when one amino acid was mutated.

[0062] [Deposit number] Depositing institution name: Korea Research Institute of Bioscience and Biotechnology Deposit number: KCTC14419BP Deposit date: 20201228

Claims

1. In an ATP - phosphoribosyltransferase consisting of the amino acid sequence of SEQ ID NO: 1, an ATP - phosphoribosyltransferase variant in which the serine located at position 288 is substituted with proline, wherein the ATP - phosphoribosyltransferase variant does not contain mutations other than the substitution of serine at position 288 with proline in SEQ ID NO:

1.

2. The ATP - phosphoribosyltransferase is the variant according to Claim 1, expressed from the hisG gene of Escherichia coli (E. coli).

3. The variant is the variant according to Claim 1, in which the feedback inhibition by histidine is reduced.

4. A transformed strain expressing the ATP - phosphoribosyltransferase variant according to Claim 1.

5. The strain is Escherichia coli, the transformed strain according to Claim 4.

6. A method for producing histidine comprising the step of culturing the strain according to Claim 4.

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