ATP-PRT mutants with reduced feedback inhibition by histidine and histidine-producing bacterial strains expressing these mutants

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 technology]

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

[0003] In an environment where the concentration of L-histidine is above a certain level, the activity of ATP-phosphoribsyltransferase is feedback inhibited by histidine, making it difficult to increase histidine production above a certain level.

[0004] Therefore, ATP-PRT mutants with increased histidine resistance are needed to increase histidine production in microorganisms, but no mutants capable of reducing the histidine feedback repression of ATP-PRT expressed in the hisG gene of E. coli are known. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Republic of Korea Patent Publication No. 10-2017-0098205 (2017.08.02) Summary of the Invention [Problem to be solved by the invention]

[0006] According to one embodiment, there is provided an ATP-phosphoribosyltransferase mutant that has reduced feedback inhibition by histidine. [Means for solving the problem]

[0007] One aspect provides an ATP-phosphoribosyltransferase mutant in which the threonine at position 252 in an ATP-phosphoribosyltransferase having the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, leucine, glycine, valine, or isoleucine.

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

[0009] In one embodiment, the ATP-phosphoribosyltransferase may be expressed from the hisG gene of E. coli.

[0010] According to one embodiment, the mutant may have reduced feedback inhibition by histidine. According to one embodiment, a strain into which an ATP-PRT containing the T252A or T252L mutation has been introduced exhibits increased histidine production compared to the wild-type strain.

[0011] According to one embodiment, the mutant may further include one or more of the following: (a) a substitution of histidine (H) at position 232 with lysine (K) or threonine (T); (b) a substitution of arginine at position 250 with histidine; (c) a substitution of glutamic acid at position 271 with lysine; or (d) a substitution of serine at position 288 with proline. According to one embodiment, it was confirmed that the inclusion of any of the mutations (a) to (d) in addition to the threonine mutation at position 252 increases histidine production.

[0012] The mutant can retain activity even at histidine concentrations of 5 mM to 25 mM.

[0013] Another aspect provides a polynucleotide encoding the ATP-phosphoribosyltransferase mutant or a vector comprising 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. The transformed strain maintains ATP-phosphoribosyltransferase activity even when the histidine concentration is increased, thereby increasing histidine production.

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

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

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

[0018] Yet another aspect provides a method for producing histidine, comprising culturing the transformed strain in a medium, and recovering histidine from the strain or the medium.

[0019] The medium may contain a carbon source, a nitrogen source, and inorganic salts. Carbon sources include, but are not limited to, sugars and carbohydrates such as glucose, sugar, citrate, fructose, lactose, maltose, or molasses; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; glycerol; alcohols such as ethanol; and organic acids such as acetic acid, all of which may be used individually or in combination. Preferably, the medium for the E. coli mutant strain contains glucose. Nitrogen sources include, but are not limited to, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean cake, urea, thiourea, ammonium salts, nitrates, and other organic or inorganic nitrogen-containing compounds. Inorganic salts include, but are not limited to, magnesium, manganese, potassium, calcium, iron, zinc, and cobalt.

[0020] To adjust the pH of the medium, basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acid compounds such as phosphoric acid or sulfuric acid, can be used in an appropriate manner. Also, antifoaming agents such as fatty acid polyglycol esters can be used to suppress foam formation, and oxygen or oxygen-containing gases (e.g., air) can be injected into the medium to maintain an aerobic state.

[0021] The culturing refers to growing microorganisms in an artificially controlled environment, and can be carried out by a culturing method widely known in the art. The culturing temperature may be 20 to 45°C, and the culturing time may be, but is not limited to, 10 to 200 hours.

[0022] The histidine recovery step can be carried out by a variety of methods well known in the art, including, but not limited to, centrifugation, filtration, anion exchange chromatography, crystallization, or HPLC. [Effects of the Invention]

[0023] According to one embodiment, the ATP-phosphoribosyltransferase mutant can maintain activity even in an environment with a high concentration of histidine.

[0024] A strain expressing an ATP-phosphoribosyltransferase mutant according to one embodiment can increase histidine production. [Brief explanation of the drawings]

[0025] [Figure 1] The results show that the enzyme activity of E. coli hisG_WT and its mutants (hisG_SDM4, hisG_SDM7) changes depending on the histidine concentration. [Figure 2] This figure shows the results of a computer simulation of the binding mode between the hisG hexamer and histidine, demonstrating that H232, S288, T252, R250, A248, E271, and E240 of hisG interact with histidine. DETAILED DESCRIPTION OF THE INVENTION

[0026] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. [Example]

[0027] Example 1: Selection of mutant strains resistant to TRA (1,2,4-triazole-3-alanine) To generate mutants with reduced resistance to L-histidine negative feedback, we used the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to generate mutants resistant to 1,2,4-triazole-3-alanine (TRA), an L-histidine derivative.

[0028] E. coli MG1655 (KCTC14419BP) was cultured in LB medium for 16 hours at 37°C and 200 rpm. After incubation, the cells were centrifuged at 4500 rpm for 10 minutes and suspended in saline / TM buffer. After resuspending the cells in the buffer, 100 μg / ml NTG was added and mutagenesis was induced at 37°C and 200 rpm for 30 minutes.

[0029] After repeating the mutagenesis process, the cells were suspended in 3 ml of DW and plated onto plate medium (composition: 8% glucose, 0.6% sodium monohydrogen phosphate, 0.2% ammonium sulfate, 0.02% magnesium sulfate, 0.001% calcium nitrate, 10 ppm ferrous sulfate, 1% TRA) for primary culture at 37°C for 2 days. Strains that formed single colonies were isolated and then secondary cultured on plate medium supplemented with 1% TRA in the same manner as the primary culture to select mutant strains. The selected mutant strains were grown on plates containing 0%, 0.5%, 1.0%, or 2.0% TRA to measure their growth (increase in cell number) and compare their resistance to TRA (see Table 1 below).

[0030] [Table 1]

[0031] Example 2: Amino acid sequence analysis of ATP-PRT enzyme of TRA-resistant mutant strains The amino acid sequences of the ATP-PRT (ATP-phosphoribosyltransferase, hisG) enzymes of mutant strains H-1 and H-2, which have increased resistance to TRA, were compared and analyzed. The sequence analysis was conducted by Macrogen, and the sequences were confirmed using the primers listed in Table 2 below.

[0032] [Table 2] As a result of confirmation, it was confirmed that some of the amino acids located at the C-terminal portion of the ATP-PRT enzyme had been substituted.

[0033] In addition, using a molecular binding mode prediction program, we analyzed the 3D structure of the E. coli hisG hexamer when docking with a histidine molecule. Based on the results of the docking analysis, we analyzed the amino acids located in the histidine entry and binding sites of ATP-PRT expressed from E. coli hisG. The simulation results revealed that H232, S288, T252, R250, A248, E271, and E240 of hisG are likely to interact with histidine (see Figure 2).

[0034] Based on the results of 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) were selected as candidates, which are likely to reduce the negative feedback loop by histidine and increase histidine production.

[0035] Example 3: Construction of a strain expressing an ATP-PRT mutant with one mutation and evaluation of its histidine productivity A one-step inactivation method was used to introduce the point mutation hisG_H232K into the chromosome of E. coli DS9H (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 E. coli DS9H genomic DNA as a template with the primer pairs hisG_HF-F / hisG_HF-R and hisG_HR-F / hisG_HR-R, respectively. Next, to obtain a cassette containing a kanamycin antibiotic marker and FRT, a cassette fragment was amplified from the pKD13 plasmid using FR(hisG)-F / FR(hisG)-R. Finally, to obtain hisG_H232K, two fragments were isolated from E. coli DS9H genomic DNA using the hisG+FR-F / 232K-R and 232K-F / hisG+HR-R primer pairs, respectively. The resulting two fragments were then ligated into a single fragment using hisG+FR-F / hisG+HR-R primers to obtain the hisG_H232K fragment. Finally, these four amplified PCR fragments were used as templates for overlapping PCR with the hisG_HF-F / hisG_HR-R primer pair to ligate the fragments into a single fragment. The ligated DNA fragments were electroporated into E. coli DS9H strains containing the pKD46 plasmid. Subsequently, PCR was performed using the hisGW-CF / hisGW-CR primers on kanamycin-resistant cell lines to identify strains with hisG_H232K introduced. The kanamycin marker, an antibiotic resistance gene, was removed from the strains in which the hisG_H232K gene was confirmed to have been introduced. The pCP20 plasmid was then introduced into the strains in which the hisG_H232K gene had been confirmed to have been introduced to induce FLP recombination, and antibiotic removal was confirmed by measuring growth on LB plates with and without the antibiotic (kanamycin). This was confirmed by the fact that antibiotic-free strains grew on LB plates but were unable to grow on LB plates containing the antibiotic (kanamycin).Finally, the sequence was confirmed using the hisGW-CF / hisGW-CR primer pair. The 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 introduced into E. coli DS9H using a similar method to that described above.

[0036] The primers used in the experiment are as shown in Table 3 below.

[0037] [Table 3]

[0038] According to Table 4 below, the hisG_H232K or hisG_H232T-introduced strains showed an increase in histidine production of approximately 22% to 26% compared to the control group. The hisG_T252A or T252L-introduced strains showed an increase in histidine production of approximately 35% to 39% compared to the control group. The hisG_E271K-introduced strain showed an increase in histidine production of approximately 34% compared to the control group. In particular, the hisG_S288P-introduced strain showed an increase in histidine production of approximately 46% compared to the control group, and the hisG_R250H-introduced strain showed an increase in histidine production of approximately 67% compared to the control group, the largest increase.

[0039] However, the H232E, E240K, and A248F mutants rather reduced the amount of histidine produced, and the R250E, T252P, T252Q, and S288K mutants did not significantly increase the amount of histidine produced.

[0040] [Table 4]

[0041] Based on these results, the seven mutants (H232T, H232K, R250H, T252A, T252L, E271K, and S288P) increased histidine production, likely due to reduced feedback inhibition by histidine. We then investigated whether combining these mutations could further improve histidine production. Example 4: Construction of a plasmid incorporating hisG_SDM4 (H232K, T252A, E271K, and S288P) Overlapping PCR was performed to generate plasmids capable of expressing mutants of the E. coli hisG ATP-PRT enzyme with amino acid substitutions H232K, T252A, E271K, and S288P. First, the gene was amplified using three primer pairs: hisG-F / 232K-R, 232K-F / 252A-R, and 252A-F / hisG-R, in pfu premix (Bioneer). The three amplified fragments were then used as templates in a second PCR with the hisG-F / hisG-R primer pair to ligate the three fragments into a single fragment (hereafter referred to as the SDM3 fragment). The SDM3 fragment and pTRC99A plasmid were then digested with EcoRI and HindIII (NEB), respectively, and the SDM3 fragment was then inserted into the pTRC99A plasmid using T4 ligase. (pTRC99A-hisG_SDM3) PCR was performed using the pTRC99A-hisG_SDM3 template and the hisG-F / 271K-R2 primer pair to obtain an SDM4 fragment containing four mutations: H232K, T252A, E271K, and S288P.

[0042] The SDM4 fragment and pTRC99A-hisG_SDM3 plasmid were then digested with EcoRI and AfeI (NEB), respectively, 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 designated hisG_SDM4.

[0043] [Table 5]

[0044] Example 5: Preparation of a plasmid incorporating hisG_SDM7 (H232T, R250H, T252L, E271K, and S288P) We created hisG_SDM7, a mutant of hisG_SDM4, by substituting a portion of its amino acid sequence with other amino acids. This was then introduced into a 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, while maintaining two mutations (E271K and S288P). (Compared to hisG_WT, the mutation positions in hisG_SDM7 are H232T, R250H, T252L, E271K, and S288P.)

[0045] First, the gene was amplified using three primer pairs: hisG-F / 232T-R, 232T-F / 250H+252L-R, and 250H+252L-F / hisG-R, using pfu premix (Bioneer). The three amplified fragments were then used as templates for another PCR reaction with the hisG-F / hisG-R primer pair to ligate the three fragments into a single fragment. The PCR fragment and pTRC99A plasmid were then digested with EcoRI and HindIII (NEB), respectively, and ligated with T4 ligase (Takara) to create pTRC99A-hisG_SDM7. Finally, the sequence was confirmed using hisG-CF / hisG-CR primers. (See Table 6 below.) The ATP-PRT mutant containing the H232T, R250H, T252L, E271K, and S288P mutations was designated hisG_SDM7.

[0046] [Table 6]

[0047] Example 6: Construction of mutant strains into which the hisG_SDM4 or hisG_SDM7 gene has been introduced 6-1. Construction of a mutant strain carrying the hisG_SDM4 gene To introduce hisG_SDM4 into the chromosome of E. coli DS9H, we used a one-step inactivation method (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 E. coli DS9H genomic DNA as a template with the primer pairs hisG_HF-F / hisG_HF-R and hisG_HR-F / hisG_HR-R, respectively. Next, to obtain a cassette containing a kanamycin antibiotic marker and FRT, a cassette fragment was amplified from the pKD13 plasmid using FR(hisG)-F / FR(hisG)-R. Finally, to obtain hisG_SDM4, the hisG_SDM4 fragment was isolated from the pTRC99A-hisG_SDM4 plasmid using the hisG+FR-F / hisG+HR-R primers. Finally, these four amplified PCR fragments were used as templates to ligate into a single fragment using overlapping PCR with the hisG_HF-F / hisG_HR-R primer pair. The ligated DNA fragments were then electroporated into E. coli DS9H strains carrying the pKD46 plasmid. Subsequently, PCR was performed using the hisGW-CF / hisGW-CR primers on kanamycin-resistant cell lines to confirm the presence of hisG_SDM4. The kanamycin marker, an antibiotic resistance gene, was then removed from the strains confirmed to have the gene. After introducing the pCP20 plasmid into the strain in which hisG_SDM4 was confirmed to be introduced and inducing FLP recombination, antibiotic removal was confirmed by measuring growth on LB plates with and without the antibiotic (kanamycin). Antibiotic-free strains were confirmed by growing on LB plates but not on LB plates containing the antibiotic (kanamycin). 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.

[0048] [Table 7]

[0049] 6-2. Construction of mutant strains carrying the hisG_SDM7 gene To introduce hisG_SDM7 into the chromosome of E. coli DS9H, we used a one-step inactivation method (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 E. coli DS9H genomic DNA as a template with the primer pairs hisG_HF-F / hisG_HF-R and hisG_HR-F / hisG_HR-R, respectively. Next, to obtain a cassette containing a kanamycin antibiotic marker and FRT, a cassette fragment was amplified from the pKD13 plasmid using FR(hisG)-F / FR(hisG)-R. Finally, to obtain hisG_SDM7, the hisG_SDM7 fragment was isolated from the pTRC99A-hisG_SDM7 plasmid using the hisG+FR-F / hisG+HR-R primers. Finally, these four amplified PCR fragments were used as templates to ligate into a single fragment using overlapping PCR with the hisG_HF-F / hisG_HR-R primer pair. The ligated DNA fragments were then electroporated into E. coli DS9H strains carrying the pKD46 plasmid. Subsequently, PCR was performed using the hisGW-CF / hisGW-CR primers on kanamycin-resistant cell lines to confirm the presence of hisG_SDM7. The kanamycin marker, an antibiotic resistance gene, was then removed from the strains confirmed to have the gene. After the pCP20 plasmid was introduced into the strain in which hisG_SDM7 was confirmed to be introduced, FLP recombination was induced, and antibiotic removal was confirmed by measuring growth on LB plates with and without the antibiotic (kanamycin). Antibiotic-free strains were confirmed by growing on LB plates but not on LB plates containing the antibiotic (kanamycin). Finally, the sequence was confirmed using the hisGW-CF / hisGW-CR primer pair. The primer sequences used to generate mutant strains containing the hisG_SDM7 gene are the same as those in Table 6.

[0050] Example 7: Determination of histidine negative feedback resistance of mutant enzymes expressed from the hisG_SDM4 or hisG_SDM7 genes The resistance to histidine-mediated negative feedback was compared between the ATP-PRT wild type (hisG_WT) and ATP-PRT mutants (hisG_SDM4 and hisG_SDM7).

[0051] LB medium was dispensed into 500 ml flasks, each containing 50 ml, and each of the three strains, DS9H, DS9H_△hisG::hisG_SDM4, or DS9H_△hisG::hisG_SDM7, was inoculated at 1%. The culture conditions were 30°C and 180 rpm. OD 600 When the ATP-PRT expression level reached 0.6, 1 mM IPTG (final concentration) was added to induce ATP-PRT expression, and the cells were cultured for approximately 4 hours. After culturing, the cells were sonicated and centrifuged. The resulting supernatant was used to evaluate ATP phosphoribosyltransferase activity. The reaction conditions for evaluating enzyme activity were determined based on existing literature (Microb Cell Fact. 2018. Mar. 17:42). The supernatant was subjected to protein quantification to ensure uniform concentration. The reactants were mixed according to the reaction composition in Table 8 below, and enzyme activity was measured.

[0052] [Table 8]

[0053] To confirm the resistance of histidine to inhibition, the histidine concentrations were adjusted to 0 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, and 50 mM, respectively. Activity was measured at 30°C with UV light at 290 nm at 2-minute intervals for 30 minutes.

[0054] As shown in Figure 1, the ATP-PRT activity of the hisG_WT enzyme decreased rapidly at histidine concentrations of 5 mM and above. However, the enzyme activity of hisG_SDM4 (H232K, T252A, E271K, S288P) decreased at histidine concentrations of 25 mM and above. Similarly to hisG_SDM4, the enzyme activity of hisG_SDM7 (H232T, R250H, T252L, E271K, S288P) decreased at histidine concentrations of 25 mM and above, but its enzyme activity was higher than that of hisG_SDM4 at each histidine concentration. Consequently, hisG_SDM7 was most resistant to histidine-induced inhibition.

[0055] Example 8: Evaluation of histidine productivity of strains expressing ATP-PRT mutant enzymes The histidine productivity of the strains carrying hisG_SDM4 or hisG_SDM7 was examined. 10 ml of medium with the composition shown in Table 9 below was dispensed into each flask, and 1% each of the DS9H, DS9H_△hisG::hisG_SDM4, or DS9H_△hisG::hisG_SDM7 strains was inoculated and cultured at 34°C and 200 rpm for 72 hours. After culture, the histidine production in each flask was compared and analyzed.

[0056] [Table 9]

[0057] The hisG_SDM4-expressing strain produced approximately 53% more histidine than the control, and the hisG_SDM7-expressing strain produced approximately 92% more histidine than the control (see Table 10).

[0058] [Table 10]

[0059] These results suggest that hisG_SDM4 and hisG_SDM7 increased histidine productivity due to reduced feedback inhibition by histidine compared to his_WT. In particular, the histidine productivity of the hisG_SDM7-expressing strain was higher than that of the hisG_SDM4-expressing strain.

[0060] Furthermore, summarizing the results of Tables 4 and 8, when any one of the amino acids at positions 232, 250, 252, 271, and 288 of E. coli hisG was mutated, the amount of histidine produced increased, and when multiple amino acids were mutated, the amount of histidine produced increased more than when only one amino acid was mutated.

[0061] [Accession number] Depository institution name: Korea Institute of Bioscience and Biotechnology Accession number: KCTC14419BP Date of acceptance: 20201228

Claims

1. In the amino acid sequence of SEQ ID NO: 1, a substitution of threonine at position 252 with alanine or leucine; or In the amino acid sequence of SEQ ID NO: 1, Substitution of threonine at position 252 with alanine or leucine, and (c) and (d) below: (c) a glutamic acid to lysine substitution at position 271; and (d) Substitution of serine at position 288 with proline At least one of the amino acid substitutions An ATP-phosphoribosyltransferase mutant comprising: The ATP-phosphoribosyltransferase mutant is an ATP-phosphoribosyltransferase mutant that does not contain any mutations other than the substitution of threonine at position 252 with alanine or leucine, the substitution of glutamic acid at position 271 with lysine, and the substitution of serine at position 288 with proline in SEQ ID NO:

1.

2. 2. The mutant of claim 1, wherein the ATP-phosphoribosyltransferase is expressed from the hisG gene of E. coli.

3. The mutant of claim 1 , wherein the mutant exhibits reduced feedback inhibition by histidine.

4. A transformed strain expressing the ATP-phosphoribosyltransferase mutant of claim 1.

5. The transformed strain of claim 4 , wherein the strain is Escherichia coli.

6. A method for producing histidine, comprising culturing the strain of claim 4.

Images