Leucine transporter mutant with reduced methionine transport activity and use thereof

Abstract

The application discloses a leucine transporter mutant with reduced methionine transport activity and application thereof, and belongs to the technical field of bioengineering. The mutant provided by the application contains amino acid mutations at positions 30, 108 or 123, i.e. F30K, F30P, S108F, S108Y or L123R, relative to a wild-type leucine transporter with an amino acid sequence as shown in SEQ ID NO. 1. In order to improve the ACC transport efficiency, the application simultaneously provides a double mutation of S108F / L160Y. Experiments show that, compared with the wild type, the E. coli strain of the leucine transporter mutant has significantly improved sensitivity to methionine analogs, and can significantly improve the methionine concentration in the strain. Meanwhile, the double mutant can effectively improve the tolerance of E. coli to ACC. Therefore, the application has a good application prospect in the field of ACC fermentation preparation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and more particularly relates to a leucine transporter mutant with reduced methionine transport activity and application thereof. BACKGROUND

[0002] 1-aminocyclopropane-1-carboxylic acid (ACC) is a natural ethylene biosynthesis precursor in plants, and is a non-protein amino acid ACC, which has physiological functions such as regulating fruit ripening, senescence and stress response. Studies have shown that ACC shows therapeutic potential such as anti-tumor and neuroprotective effect in the medical field.

[0003] With the in-depth application research of ACC, the market demand gradually increases, and the development of ACC large-scale production technology has also attracted increasing attention. Microbial fermentation method has become an important way for industrial production of ACC due to its green and efficient characteristics. ACC has certain antibacterial potential, which can easily lead to inhibition of bacterial growth, and reduce activity and tolerance. It is known that the leucine transporter LeuE is involved in regulating the transport activity of ACC in Escherichia coli, and the tolerance of recombinant Escherichia coli to ACC is significantly improved. However, LeuE also has methionine transport activity, which causes the intracellular methionine concentration to decrease. Methionine, as a precursor of ACC synthesis, the decrease of intracellular concentration will affect the synthesis efficiency of the end product ACC.

[0004] Therefore, it is a technical problem to be solved in the field of microbial synthesis of ACC to develop a more efficient and specific bacterial ACC transporter, and to improve the tolerance of the bacterial cell to ACC and reduce the outward transport of the precursor methionine to increase the intracellular concentration of methionine through engineering modification. SUMMARY

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present application is to provide a leucine transporter mutant with reduced methionine transport activity, which has a lower affinity for methionine. The technical problem to be solved by the present application is to provide the application of the aforementioned leucine transporter mutant with reduced methionine transport activity, specifically to apply the leucine transporter mutant gene to ACC synthesis, which can effectively improve the binding force of intracellular methionine and ACC without reducing the intracellular methionine concentration, and promote the efflux of ACC.

[0006] To solve the above technical problems, the technical solutions provided by the present application are as follows:

[0007] A leucine transporter mutant with reduced methionine transport activity, said mutant comprising an amino acid substitution selected from position 30, 108 or 123 relative to the wild-type leucine transporter shown in SEQ ID NO. 1; said substitution is selected from any one of:

[0008] (a) the phenylalanine at position 30 is mutated to lysine;

[0009] (b) the phenylalanine at position 30 is mutated to proline;

[0010] (c) the serine at position 108 is mutated to phenylalanine;

[0011] (d) the serine at position 108 is mutated to tyrosine;

[0012] (e) the leucine at position 123 is mutated to arginine.

[0013] In some embodiments, said mutant comprises the serine at position 108 is mutated to phenylalanine relative to the wild-type leucine transporter shown in SEQ ID NO. 1.

[0014] In some embodiments, said leucine transporter mutant with reduced methionine transport activity further comprises the leucine at position 160 is mutated to tyrosine.

[0015] A nucleic acid molecule encoding any one of said leucine transporter mutants.

[0016] An expression cassette comprising said nucleic acid molecule.

[0017] A recombinant vector comprising said nucleic acid molecule or said expression cassette.

[0018] A recombinant host cell comprising any one of said leucine transporter mutants, said nucleic acid molecule, said expression cassette or said recombinant vector.

[0019] In some embodiments, said recombinant host cell is a bacterium.

[0020] Use of any one of said leucine transporter mutants, said nucleic acid molecule, said expression cassette, said recombinant vector or any one of said recombinant host cells in the production of ACC.

[0021] A method for producing ACC, comprising: fermenting said recombinant host cell, and harvesting ACC from the culture.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application obtains multiple leucine transporter mutants with reduced methionine transport activity through bioinformatics analysis of Escherichia coli leucine transporter. The leucine transporter mutants of the present application can improve the sensitivity of Escherichia coli to methionine analogs and thus reduce the precursor efflux. On this basis, in order to improve the ACC transport efficiency, the present application simultaneously provides a double mutation of S108F / L160Y. Experiments show that the Escherichia coli strain overexpressing the leucine transporter mutant with reduced methionine transport activity has significantly improved sensitivity to methionine analogs compared with the wild type, and can significantly improve the intracellular methionine concentration. At the same time, the double mutant can effectively improve the tolerance of Escherichia coli to ACC, and the strain grows better. Therefore, the present application is beneficial to improve the biosynthesis efficiency of ACC, and has good application prospect in the field of ACC fermentation preparation. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a molecular docking simulation diagram of LeuE and methionine, the left figure is a schematic diagram of the relative position of LeuE protein and methionine (green is methionine), and the right figure is a schematic diagram of amino acid residues within a distance of 5 Å from methionine;

[0025] Figure 2 is a tolerance verification diagram of recombinant Escherichia coli overexpressing LeuE mutant to methionine analogs;

[0026] Figure 3 is a tolerance verification diagram of recombinant Escherichia coli overexpressing LeuE double mutant to methionine analogs. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described below in combination with specific examples. In the following examples, if no detailed description is given, the technical means used are all conventional means familiar to those skilled in the art. Or according to the kit and product instructions. The materials, reagents, etc. used in the following examples, if not specially specified, can be obtained from commercial channels.

[0028] Example 1

[0029] Bioinformatics analysis of gene structure-Discovery Studio molecular docking

[0030] The Discovery Studio was used to perform molecular docking simulation of LeuE (amino acid sequence as shown in SEQ ID NO. 1) with methionine, and virtual amino acid mutation was performed to reduce the affinity, and the most possible virtual mutation active site was selected for further experimental verification. First, single-point mutation was performed on the amino acid residues within 10 Å from the ligand, the key amino acids for predicting the interaction between the receptor and the ligand were calculated, and saturation mutation was performed. However, the transport effect of the obtained strain on methionine was not reduced. Then, single-point mutation was performed on the amino acid residues within 5 Å from the ligand, the key amino acids for predicting the interaction between the receptor and the ligand were calculated, and virtual active mutation sites were selected according to the key sites on the molecular docking simulation diagram, and saturation mutation was continued. The results are shown in Figure 1 As shown in the table, methionine forms a hydrogen bond with the residues at positions F30, S108 and L123 of the LeuE transporter protein, and the above residues are the key residues of the active pocket. Therefore, in order to obtain a strain more sensitive to methionine, the present application performs point mutation on the following key residues of LeuE, which are F30K, F30P, S108F, S108P, S108Y, L123R, and the sequences of the mutants are shown in SEQ ID NO. 3-8, respectively. In addition, one point mutation is performed on the key residue of LeuE: L160Y. The sequences of the double mutants F30K / L160Y, F30P / L160Y, S108F / L160Y, S108Y / L160Y, L123R / L160Y obtained therefrom are shown in SEQ ID NO. 9-13, respectively.

[0031] Example 2

[0032] Construction of LeuE and mutant recombinant strains

[0033] The pTrcHis2A-leuE plasmid was mutated by the overlapping PCR method using LeuE mutant primers to construct mutants M1 (pTrcHis2A-leuE-F30K), M2 (pTrcHis2A-leuE-F30P), M3 (pTrcHis2A-leuE-S108F), M4 (pTrcHis2A-leuE-S108P), M5 (pTrcHis2A-leuE-S108Y), M6 (pTrcHis2A-leuE-L123R), A6M1 (pTrcHis2A-leuE-F30K / L160Y), A6M2 (pTrcHis2A-leuE-F30P / L160Y), A6M3 (pTrcHis2A-leuE-S108F / L160Y), A6M5 (pTrcHis2A-leuE-S108Y / L160Y), and A6M6 (pTrcHis2A-leuE-L123R / L160Y), as follows:

[0034] The genomic DNA of E. coli MG1655 was used as a template, and LeuE-F / LeuE-R was used as primers to amplify the target gene fragment LeuE (the nucleotide sequence of which is shown in SEQ ID NO. 2, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 1) using Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozyme Biotech Co., Ltd.). The obtained fragment was purified by an agarose gel recovery kit, and the vector pTrcHis2A was digested using EcoRI / BamHI. The fragment and the vector were connected by a homologous recombination kit (Nanjing Novozyme Biotech Co., Ltd.), and the DH5α competent cells were transformed. The ampicillin-resistant clones were picked, and the inserted fragment was identified by sequencing to be correct. The recombinant plasmid was named pTrcHis2A-LeuE. The corresponding full-length primers and corresponding single-point mutant primers (Table 1) were used to amplify the sequences before and after the mutation site, respectively, and the gel was recovered.

[0035] Table 1 Primer sequences (SEQ ID NO. 14-29)

[0036]

[0037] The two PCR products were mixed at a ratio of 1:1 and used as the template for the second round of PCR. The full-length primers (leuE-F and leuE-R) were used for PCR to obtain the gene fragment with single-point mutation and chimeric construction. Double mutants were introduced with a second mutation site based on the single mutants. The constructed mutant fragments were ligated with the linearized pTrcHis2A vector fragments, and then transformed into DH5a. After overnight culture, single colonies were selected for colony PCR. After agarose gel electrophoresis detection, positive strains were selected for sequencing. The strains with correct sequencing results were cultured and preserved in a -80 °C refrigerator. The strains were E. coli M1 (pTrcHis2A-leuE-F30K), M2 (pTrcHis2A-leuE-F30P), M3 (pTrcHis2A-leuE-S108F), M4 (pTrcHis2A-leuE-S108P), M5 (pTrcHis2A-leuE-S108Y), and M6 (pTrcHis2A-leuE-L123R).

[0038] Example 3

[0039] Tolerance of strains containing wild-type leuE or leuE mutants to methionine analogs and ACC

[0040] According to the virtual mutation results, six other mutants targeting methionine were constructed: E. coli M1 (pTrcHis2A-leuE-F30K), M2 (pTrcHis2A-leuE-F30P), M3 (pTrcHis2A-leuE-S108F), M4 (pTrcHis2A-leuE-S108P), M5 (pTrcHis2A-leuE-S108Y), and M6 (pTrcHis2A-leuE-L123R). The tolerance of the above strains was tested on plates with added methionine analogs norleucine (NLEU) and ethionine (ETH). Compared with the control NC (pTrcHis2A-leuE), the growth of M1, M2, M3, M5, and M6 strains was significantly inhibited (Fig. 2), and the number and area of colonies were reduced, indicating that the methionine transport capacity was weakened. Figure 2

[0041] ​Next, we aimed to obtain strains with ACC resistance while simultaneously reducing methionine resistance and enhancing the specificity of LeuE for ACC transport. The constructed strains A6M1 (pTrcHis2A-leuE-F30K / L160Y), A6M2 (pTrcHis2A-leuE-F30P / L160Y), A6M3 (pTrcHis2A-leuE-S108F / L160Y), A6M5 (pTrcHis2A-leuE-S108Y / L160Y), and A6M6 (pTrcHis2A-leuE-L123R / L160Y) were tested for ACC and methionine transport on LB agar plates containing ACC or leucine and ethionine. The results showed that, at an ACC concentration of 0.1 mM, the double mutant strains A6M3 and A6M5 exhibited better growth compared to the control. Figure 3 A6M3 was more sensitive to ethionine than the control. Figure 3 Based on the results of the ACC tolerance experiment, the double mutant A6M3 exhibits both a strong ability to transport ACC outwards and a weak ability to transport methionine outwards.

[0042] Example 4

[0043] Determination of intracellular methionine content in strains containing wild-type leuE or leuE mutant

[0044] To determine the methionine export rate, cells were cultured in LB medium for 8 hours, then transferred to basal medium for overnight culture. The overnight culture was then re-seeded into basal medium. Cells were collected at mid-logarithmic growth phase, washed three times with ice-cold basal medium, and resuspended in pre-warmed basal medium (37°C) to allow for measurement of the optical density at 600 nm. The concentration reached 2.0 for amino acid uptake assay. After pre-incubation at 37°C for 10 minutes, a methionine-methionine (Met-Met) dipeptide was added to initiate the reaction, while the culture was stirred at 750 rpm using a magnetic stirrer. Intracellular and extracellular components were separated using a modified silicone oil method. Extracellular components were recovered from the remaining cell suspension above the silicone oil layer. The cell pellet was sonicated and centrifuged, and the intracellular components were neutralized with 3M sodium carbonate. Amino acids in the extracellular and intracellular components were quantitatively analyzed by high-performance liquid chromatography (HPLC).

[0045] When the cells were cultured in the medium containing 5 mM Met-Met (methionine-methionine dipeptide), the intracellular methionine level of the strain containing wild-type leuE was about 70 mM, which was significantly lower than that of the strains M1, M2, M3, M5 and M6 overexpressing the leuE mutants (120, 83, 102, 93, 110 mM, respectively). The methionine export rate of each strain was also determined in the presence of Met-Met dipeptide. The extracellular methionine level of the strains containing the wild-type leuE mutant and strains M1, M2, M3, M5 and M6 was significantly decreased, and the export rate ranged from 89 to 111 nmol / min / mg (dry weight) (98, 102, 111, 89, 108 nmol / min / mg (dry weight), respectively; in comparison, the export rate of the strain containing the wild-type leuE was 175 nmol / min / mg (dry weight).

[0046] Although the present application has been disclosed with reference to preferred embodiments, it is not intended to limit the application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application, and therefore the scope of protection of the present application should be defined by the claims.

Claims

1. A leucine transporter mutant that reduces methionine transport activity, characterized in that, the mutant has a mutation of serine at position 108 to phenylalanine and a mutation of leucine at position 160 to tyrosine, relative to the wild-type leucine transporter shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the mutant leucine transporter of claim 1.

3. An expression cassette comprising the nucleic acid molecule of claim 2.

4. A recombinant vector comprising the nucleic acid molecule of claim 2 or the expression cassette of claim 3.

5. A recombinant host cell comprising the mutant leucine transporter of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, or the recombinant vector of claim 4.

6. The recombinant host cell of claim 5, wherein, the recombinant host cell is a bacterium.

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