A leucine transporter mutant that improves ACC transport efficiency and its application

Abstract

This invention discloses a leucine transporter mutant that improves ACC transport efficiency and its applications, belonging to the field of bioengineering technology. The leucine transporter mutant provided by this invention is obtained by mutating valine at position 50 to arginine or leucine at position 160 to tyrosine in the *E. coli* leucine transporter protein (as shown in SEQ ID NO. 1). In experiments verifying overexpression on LB plates containing high concentrations of ACC, the *E. coli* strain with the leucine transporter mutant showed significantly improved tolerance to ACC compared to the wild type, indicating improved ACC transport efficiency. Therefore, this invention can effectively reduce the intracellular concentration of ACC in *E. coli* and improve *E. coli* tolerance to ACC, showing promising application prospects in the field of ACC fermentation preparation.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and more specifically, relates to a leucine transporter mutant that improves ACC transport efficiency and its application. Background Technology

[0002] 1-Aminocyclopropane-1-carboxylic acid (ACC) is a naturally occurring precursor for ethylene biosynthesis in plants. It is a non-protein amino acid with physiological functions including regulating fruit ripening, senescence, and stress responses. Studies have shown that ACC exhibits therapeutic potential in the medical field, including anti-tumor and neuroprotective effects.

[0003] As research into ACC applications deepens, market demand is gradually increasing, and the development of ACC large-scale production technology is receiving increasing attention. Microbial fermentation, due to its green and efficient characteristics, has become an important method for the industrial production of ACC. However, because ACC has a certain antibacterial potential, it can easily lead to inhibited cell growth, reduced activity and tolerance, thus significantly limiting ACC production efficiency.

[0004] The lack of efficient and specific transport proteins for efflux of ACC is a major reason why ACC accumulates in large quantities within bacteria, leading to toxic effects and severely limiting the improvement of fermentation levels. The known leucine transporter LeuE is involved in regulating the transport activity of ACC in *E. coli*, and recombinant *E. coli* shows significantly improved tolerance to ACC. However, current research on bacterial amino acid transport proteins mainly focuses on the transport mechanisms of classical amino acids (such as leucine and methionine), while the transport mechanisms of non-protein amino acids (ACC) remain unclear.

[0005] Therefore, it is necessary to study the mechanism of LeuE transport of ACC in order to develop a more efficient and specific bacterial ACC transporter protein, so as to improve the bacterial tolerance to ACC and the fermentation production efficiency of ACC, and provide strong technical support for the large-scale application of ACC in the fields of medicine and agriculture. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, the primary technical problem solved by this invention is to provide a leucine transporter mutant that improves ACC transport efficiency and exhibits high ACC affinity. The secondary technical problem to be solved by this invention is to provide a specific application of the aforementioned leucine transporter mutant that improves ACC transport efficiency. Specifically, this leucine transporter mutant gene is applied to ACC fermentation production to promote increased cell tolerance to ACC, promote efficient ACC synthesis and efflux, and reduce intracellular ACC concentration.

[0007] To solve the above-mentioned technical problems, the technical solution provided in this application is as follows:

[0008] A leucine transporter mutant that improves ACC transport efficiency, wherein, relative to the Escherichia coli leucine transporter shown in SEQ ID NO.1, the leucine transporter mutant has a valine mutation at position 50 replaced by arginine, or a leucine mutation at position 160 replaced by tyrosine.

[0009] A nucleic acid molecule encoding the leucine transporter mutant.

[0010] An expression cassette containing the nucleic acid molecule.

[0011] A recombinant vector containing the nucleic acid molecule or the expression cassette.

[0012] A recombinant host cell containing the aforementioned nucleic acid molecule, expression cassette, or recombinant vector.

[0013] In some embodiments, the recombinant host cell is a bacterium.

[0014] In some embodiments, the bacteria are Escherichia coli.

[0015] The application of the leucine transporter mutant, the nucleic acid molecule, the expression cassette, the recombinant vector, or the recombinant host cell in the production of ACC.

[0016] A method for producing ACC includes: fermenting the recombinant host cell and harvesting ACC from the fermentation culture.

[0017] In some embodiments, the fermentation culture conditions include: fermentation in a culture medium containing a carbon source, a nitrogen source, and inorganic salts at 25-40°C and pH 5.0-8.0.

[0018] Compared with the prior art, the beneficial effects of this application are as follows:

[0019] This application utilizes bioinformatics analysis of the leucine transporter in *E. coli* to design and obtain several leucine transporter mutants with high binding affinity to ACC. Based on this, the tolerance of *E. coli* strains overexpressing the leucine transporter mutants to ACC was verified using LB plates containing high concentrations of ACC. The results showed that, compared to wild-type strains, *E. coli* strains overexpressing the leucine transporter mutants exhibited improved tolerance to ACC and promoted strain growth. Furthermore, intracellular ACC assays showed that the intracellular ACC levels in *E. coli* A0 (pTrcHis2A-leuE) and strain A6 (pTrcHis2A-leuE-L160Y) decreased to 0.55 mg / g and 0.35 mg / g, respectively, representing reductions of 29% and 55% compared to *E. coli* NC (pTrcHis2A). This further validates that the transporter mutants enhance the transport function of ACC. Therefore, the leucine transporter mutants provided in this application can be used for the construction and screening of ACC-producing strains, improving the biosynthetic efficiency of ACC, and have promising application prospects. Attached Figure Description

[0020] Figure 1 This is a simulation diagram of the molecular docking between LeuE and ACC. The left image shows the relative positions of the LeuE protein and ACC, and the right image shows the amino acid residues within a 5 Å range of ACC.

[0021] Figure 2 This is a graph demonstrating the tolerance of recombinant E. coli overexpressing the LeuE mutant to ACC.

[0022] Figure 3 This is a graph validating the tolerance of a recombinant E. coli strain overexpressing the LeuE double mutation to ACC.

[0023] Figure 4 This is a diagram of the AlphaFold2-predicted LeuE and mutant protein structures;

[0024] Figure 5 This is a diagram showing the interatomic distances between the binding sites of LeuE and the mutant protein ACC.

[0025] Figure 6 This is a growth curve of different strains in LB medium containing ACC;

[0026] Figure 7 This is a comparison chart showing the intracellular ACC content of different strains after culturing in LB medium containing ACC. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0028] Example 1

[0029] Bioinformatics Analysis of Gene Structures – Discovery Studio Molecular Docking

[0030] Molecular docking simulations of LeuE (SEQ ID NO.1) and ACC were performed using Discovery Studio, and virtual amino acid mutations were conducted to enhance affinity. First, single-point mutations were performed on amino acid residues within 10 Å of the ligand to calculate and predict key amino acids for receptor-ligand interaction, followed by saturation mutations. However, the resulting mutant strain did not improve ACC transport efficiency. Next, single-point mutations were performed on amino acid residues within 5 Å of the ligand. Key amino acids that would interact with LeuE and ACC were calculated, and virtual active mutation sites were selected based on key sites on the molecular docking simulation diagram for further saturation mutations. The results are as follows: Figure 1 As shown, ACC forms hydrogen bonds with residues V21, V50, and L160 of the LeuE transporter, and these residues are key residues in the active pocket. The most likely dummy mutant active sites were selected for further experimental verification. Therefore, to obtain strains with stronger ACC tolerance, this application performed point mutations on the following key residues: LeuE mutation sites V21K, V50R, L160H, L160Q, L160W, and L160Y, with the mutant sequences shown in SEQ ID NO. 3-8, respectively.

[0031] Example 2

[0032] Construction of LeuE and mutant recombinant strains

[0033] LeuE was mutated using overlapping PCR with LeuE mutant primers to construct recombinant plasmids pTrcHis2A-leuE-V21K, pTrcHis2A-leuE-V50R, pTrcHis2A-leuE-L160H, pTrcHis2A-leuE-L160Q, pTrcHis2A-leuE-L160W, pTrcHis2A-leuE-L160Y, and pTrcHis2A-leuE-V50R / L160Y. ​​These recombinant plasmids were then transformed into E. coli. After inoculation, strains A1 (pTrcHis2A-leuE-V21K), A2 (pTrcHis2A-leuE-V50R), A3 (pTrcHis2A-leuE-L160H), A4 (pTrcHis2A-leuE-L160Q), A5 (pTrcHis2A-leuE-L160W), A6 (pTrcHis2A-leuE-L160Y), and A2+6 (pTrcHis2A-leuE-V50R / L160Y) were obtained. The specific steps are as follows:

[0034] Using Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd.), the genome of *E. coli* MG1655 was used as a template, and LeuE-F / LeuE-R primers were used to amplify the target gene fragment LeuE (its nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.1). The obtained fragment was purified using an agarose gel extraction kit. Simultaneously, the vector pTrcHis2A was digested using EcoRI / BamHI, and the fragment was ligated to the vector using a homologous recombination kit (Nanjing Novizan Biotechnology Co., Ltd.). The ligation was performed on DH5α competent cells, and ampicillin-resistant clones were selected. Sequencing confirmed the correct insertion of the fragment, and the recombinant plasmid was named pTrcHis2A-LeuE. Using the mutated amino acid as a node, the corresponding full-length primers and the corresponding single-point mutation primers (Table 1) were used to amplify the sequences before and after the mutation site, and the fragments were then extracted using a gel extraction kit.

[0035] Table 1 Primer sequences (SEQ ID NO. 9-22)

[0036]

[0037] The two PCR products were mixed 1:1 and used as templates for the second round of PCR. PCR was performed using full-length primers (leuE-F and leuE-R) to obtain gene fragments with single-point mutations and chimeras. The double mutant introduced a second mutation site based on the single mutant. The constructed fragments were ligated to the pTrcHis2A linearized vector fragment, transformed into DH5α, and cultured overnight. Single colonies were selected for colony PCR. After agarose gel electrophoresis, positive strains were selected and sent to the company for sequencing. Strains A1, A2, A3, A4, A5, A6, and the double mutant A2+6 with correct sequencing results were cultured and stored at -80℃.

[0038] Example 3

[0039] Tolerance of strains overexpressing wild-type LeuE or LeuE mutant to ACC

[0040] The following LeuE single-site mutation overexpression strains were constructed: A1 (pTrcHis2A-leuE-V21K), A2 (pTrcHis2A-leuE-V50R), A3 (pTrcHis2A-leuE-L160H), A4 (pTrcHis2A-leuE-L160Q), A5 (pTrcHis2A-leuE-L160W), and A6 (pTrcHis2A-leuE-L160Y). Tolerance experiments showed that strains A2 and A6 were superior to the wild-type LeuE-overexpressing E. coli strain A0 (pTrcHis2A-leuE). Strain A6 exhibited the highest tolerance; strain A0 tolerated ACC at 0.05 M, while strain A6 survived on LB plates containing 0.1 M ACC, demonstrating twice the tolerance of the wild-type strain. Figure 2 However, the double mutant A2+6 (pTrcHis2A-leuE-V50R / L160Y) did not show stronger ACC tolerance. Figure 3 ).

[0041] Example 4

[0042] Predicting the structure of LeuE and mutant proteins

[0043] Since the crystal structure of LeuE has not yet been resolved, the LeuE structure on UniProt is derived from AlphaFold calculations. Therefore, AlphaFold was chosen to simulate the structure of LeuE and its mutant proteins. The structural model was evaluated using SAVES v6.0 (https: / / saves.mbi.ucla.edu / ). The results showed that 93.5% of the residues in LeuE were located in the optimal region, with an overall quality factor of 100%, demonstrating the rationality of the predicted model. For strain A6, 95.7% of the residues were located in the optimal region, with an overall quality factor also of 100%. Figure 4 Protein structure prediction revealed a significant structural change in the mutant protein LeuE (L160Y). Next, the interatomic distances between the amino acid residues closest to the ACC binding site in the AlphaFold simulated protein structure were measured using ChimeraX. Before the mutation, the interatomic distances at the ACC binding site were 9.551 Å and 7.108 Å, respectively. After the mutation, these distances decreased to 6.876 Å and 5.831 Å. Figure 5 This indicates that the protein's inner diameter has decreased, increasing the affinity between ACC and LeuE proteins.

[0044] Example 5

[0045] The LeuE mutant alleviates the inhibition of E. coli growth by ACC.

[0046] To verify the effects of LeuE and its mutant on the growth of *E. coli*, strain A0 (pTrcHis2A-leuE) and mutant A6 (pTrcHis2A-leuE-L160Y) were cultured in LB medium containing different concentrations of ACC for 36 h. *E. coli* NC (empty vector pTrcHis2A) served as the control group. Samples were taken at regular intervals to measure the OD of the bacterial culture. 600 Value, result as Figure 6 As shown, in media containing 50 mM ACC and 70 mM ACC, strain A6 grew better than NC and A0. In LB medium containing 50 mM ACC, strains A0 (pTrcHis2A-leuE) and A6 (pTrcHis2A-leuE-L160Y) grew slightly slower than NC in the initial stage of growth, possibly because the overexpression of the transporter protein consumed cellular metabolic resources, leading to a decrease in the growth rate of *E. coli*. In LB medium containing 70 mM ACC, strain A6 grew faster, and strain A0 (pTrcHis2A-leuE) also grew better than the control group, further verifying the growth inhibition of *E. coli* by ACC and the transport function of the transporter protein LeuE for ACC.

[0047] Example 6

[0048] Intracellular ACC content assay

[0049] Prepare 25 mL LB liquid medium (50 μg / mL ampicillin) containing 50 mM and 70 mM ACC, respectively. Inoculate *E. coli* NC (pTrcHis2A), *E. coli* A0 (pTrcHis2A-leuE), and mutant A6 (pTrcHis2A-leuE-L160Y) into 3 mL LB medium (50 μg / mL ampicillin) and incubate overnight at 37°C and 200 rpm. Inoculate 500 μL seed cultures of *E. coli* DH5α (pTrcHis2A) and *E. coli* DH5α (pTrcHis2A-leuE) into 25 mL LB liquid medium (50 mM and 70 mM ACC), respectively, and incubate at 37°C and 200 rpm. Samples were taken at 0, 1.5 h, 3 h, 5 h, 7 h, 10 h, 13 h, 24 h, 30 h, and 36 h, and the absorbance of the bacterial culture at 600 nm was measured. After 36 h of culture, the culture was centrifuged, washed twice with 0.85% NaCl solution at 0℃ (12000 rpm, 3 min), resuspended in 30 ml of ACC extraction reagent (acetonitrile:water, 1:1, V / V), sonicated, and freeze-dried overnight at low temperature. The sample was then resuspended in 1 ml of extraction reagent, followed by pre-column derivatization and HPLC detection. Pre-column derivatization: 200 μL of sample solution was transferred to a 1.5 mL EP tube, and 100 μL of triethylamine acetonitrile (triethylamine concentration 1 M) solution and 100 μL of PITC acetonitrile solution (PITC concentration 0.1 M) were added. The reaction was carried out at room temperature for 1 h. 400 μL of n-hexane was added to the resulting solution, and the mixture was vortexed for 10 min. Centrifuge, collect the clear lower layer, filter through a 0.22 μm filter membrane, and perform high-performance liquid chromatography (HPLC) analysis. HPLC analysis conditions: Chromatograph: HPLC LC-20A (Shimadzu Corporation, Japan); Column: SHIMADZU C18 (5 μm, 250 mm × 4.6 mm); Mobile phase: Phase A was a salt solution of 1 M Na₂HPO₄ / NaH₂PO₄, each added to 1 L of ultrapure water; Phase B was acetonitrile. Flow rate: 1 mL / min; Column temperature: 40℃; Detection wavelength: 254 nm; Injection volume: 10 μL. Gradient program: From 0 to 8 min, the proportion of mobile phase A is 95%; from 8 to 30 min, the proportion of phase A gradually decreases to 67%; from 30 to 35 min, the proportion of phase A remains at 67%; from 35 to 40 min, the proportion of phase A is 10%; from 40 to 50 min, the proportion of phase A gradually increases to 95%; from 50 to 55 min, at the end of the program, the proportion of phase A remains at 95%.

[0050] like Figure 7As shown, after culturing for 36 hours in LB medium containing 50 mM and 70 mM ACC, the intracellular ACC concentrations of *E. coli* NC (pTrcHis2A) were 0.78 mg / g and 0.77 mg / g, respectively. At 50 mM, the intracellular ACC levels of *E. coli* A0 (pTrcHis2A-leuE) and strain A6 (pTrcHis2A-leuE-L160Y) decreased to 0.45 mg / g and 0.23 mg / g, respectively, representing a 42% and 71% reduction compared to *E. coli* NC (pTrcHis2A), respectively. At 70 mM, the intracellular ACC levels of *E. coli* A0 (pTrcHis2A-leuE) and strain A6 (pTrcHis2A-leuE-L160Y) decreased to 0.55 mg / g and 0.35 mg / g, respectively, representing a 29% and 55% reduction compared to *E. coli* NC (pTrcHis2A), respectively. The transport function of the transporter protein LeuE and its mutants for ACC was further verified.

[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A leucine transporter mutant, characterized in that, The amino acid sequence of the leucine transporter mutant is shown in SEQ ID NO.4 or SEQ ID NO.

8.

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

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

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

5. A recombinant host cell, characterized in that, The recombinant host cell contains 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 according to claim 5, characterized in that, The recombinant host cell is bacteria.

7. The recombinant host cell according to claim 6, characterized in that, The bacteria mentioned are Escherichia coli.

8. The use of the leucine transporter mutant of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, the recombinant vector of claim 4, or the recombinant host cell of any one of claims 5-7 in the production of ACC.

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