A method for regulating the transport activity of a strain to 1-aminocyclopropane-1-carboxylic acid

Abstract

The present invention discloses a method for regulating the transport activity of a strain to 1-aminocyclopropane-1-carboxylic acid, which relates to the technical field of bioengineering. The method for regulating the transport activity of a strain to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues and the tolerance of the strain to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues is to transfer the LeuE gene into Escherichia coli to obtain a recombinant Escherichia coli strain with corresponding functions. The present invention discloses for the first time the application of LeuE protein and LeuE gene in regulating the transport activity of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues and the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues. The present invention discloses for the first time that LeuE is the best candidate gene for constructing ACC-producing bacteria, and its substrate-specific transport meets the purpose of enhancing cell ACC tolerance without affecting precursor supply.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and more specifically, relates to a method for regulating the transport activity of a strain to 1-aminocyclopropane-1-carboxylic acid. Background Art

[0002] 1-aminocyclopropane-1-carboxylic acid (ACC) is a key precursor for plant ethylene biosynthesis and is a non-protein amino acid. It has functions such as regulating plant development and stress resistance, and also shows potential application values such as good anti-tumor and neuroprotective effects in the medical field. Currently, ACC can be green-produced through microbial fermentation, but problems such as poor tolerance of the bacterial cells to ACC and low transport efficiency limit its high-efficiency industrial production.

[0003] In the field of genetic engineering, the LeuE gene is used as one of the targets for increasing the production of branched-chain amino acids (BCAAs). By knocking out or mutating genes related to branched-chain amino acid transport and simultaneously enhancing the expression of the LeuE gene, the production of leucine in Escherichia coli can be significantly increased. This genetic engineering strategy has broad application prospects in industrial production and can meet the market demand for branched-chain amino acids such as leucine. In the metabolic network of Escherichia coli, the LeuE gene has complex interactions with other genes. For example, the expression of the LeuE gene may be regulated by key enzymes in other metabolic pathways, and at the same time, it may also affect the synthesis or degradation processes of other amino acids. The study of these interaction relationships helps to reveal the regulatory mechanism of the Escherichia coli metabolic pathway and provides a theoretical basis for metabolic engineering transformation.

[0004] Currently, there is no direct evidence indicating an association between the LeuE gene and the tolerance of Escherichia coli to ACC. Regarding the tolerance of Escherichia coli to ACC, it may involve multiple complex mechanisms, including changes in cell membrane permeability, regulation of metabolic pathways, activation of stress response mechanisms, etc. These studies will help to further explore the potential of the LeuE gene and provide more theoretical support and technical means for fields such as industrial production and medical health. Summary of the Invention

[0005] Aiming at the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide the transport protein LeuE of 1-aminocyclopropane-1-carboxylic acid. Another technical problem to be solved by the present invention is to provide the coding gene of the transport protein LeuE of 1-aminocyclopropane-1-carboxylic acid. The technical problem to be solved by the present invention is to provide a new use of the transport protein LeuE of 1-aminocyclopropane-1-carboxylic acid, which is used to regulate the transport activity of 1-aminocyclopropane-1-carboxylic acid in Escherichia coli and to regulate the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid.

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

[0007] Use of LeuE protein in regulating the transport activity of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, wherein the amino acid sequence of the LeuE protein is as shown in SEQ ID NO.2.

[0008] Use of LeuE gene in regulating the transport activity of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, wherein the nucleotide sequence of the LeuE gene is as shown in SEQ ID NO.1.

[0009] Use of LeuE protein in regulating the transport activity of Escherichia coli to methionine analogs, wherein the amino acid sequence of the LeuE protein is as shown in SEQ ID NO.2.

[0010] Use of LeuE gene in regulating the transport activity of Escherichia coli to methionine analogs, wherein the nucleotide sequence of the LeuE gene is as shown in SEQ ID NO.1.

[0011] Use of LeuE protein in regulating the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogs, wherein the amino acid sequence of the LeuE protein is as shown in SEQ ID NO.2.

[0012] Use of LeuE gene in regulating the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogs, wherein the nucleotide sequence of the LeuE gene is as shown in SEQ ID NO.1.

[0013] A method for regulating the transport activity of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, which is: transferring the LeuE gene into Escherichia coli to obtain a recombinant Escherichia coli strain with enhanced transport activity to 1-aminocyclopropane-1-carboxylic acid;

[0014] The nucleotide sequence of the LeuE gene is as shown in SEQ ID NO.1.

[0015] A method for regulating the transport activity of Escherichia coli to methionine analogs, which is: transferring the LeuE gene into Escherichia coli to obtain a recombinant Escherichia coli strain with reduced transport activity to methionine analogs;

[0016] The nucleotide sequence of the LeuE gene is as shown in SEQ ID NO.1.

[0017] A method for regulating the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, which is: transferring the LeuE gene into Escherichia coli to obtain a recombinant Escherichia coli strain with enhanced tolerance to 1-aminocyclopropane-1-carboxylic acid;

[0018] The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.1.

[0019] A method for regulating the tolerance of Escherichia coli to methionine analogs is as follows: transferring the LeuE gene into Escherichia coli to obtain a recombinant Escherichia coli strain with reduced tolerance to methionine analogs;

[0020] The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.1.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) The present invention constructs recombinant strains E. coli DH5α pTrcHis2A-LeuE, E. coli DH5α pTrcHis2A-rhtC, E. coli DH5α pTrcHis2A-AAP, E. coli DH5α pTrcHis2A-putP, E. coli DH5α pTrcHis2A-brnFE, E. coli DH5α pTrcHis2A-ygaZH, E. coli DH5α pTrcHis2A-yjeH, and verifies their growth in LB plates containing ACC. The results show that the recombinant strains E. coli DH5α pTrcHis2A-LeuE, E. coli DH5α pTrcHis2A-AAP, E. coli DH5α pTrcHis2A-brnFE, and E. coli DH5α pTrcHis2A-yjeH grow normally on LB plates containing 0.05 M ACC.

[0023] 2) The present invention constructed recombinant strains E. coli DH5α pTrcHis2A-LeuE, E. coli DH5α pTrcHis2A-rhtC, E. coli DH5α pTrcHis2A-AAP, E. coli DH5α pTrcHis2A-putP, E. coli DH5α pTrcHis2A-brnFE, E. coli DH5α pTrcHis2A-ygaZH, and E. coli DH5α pTrcHis2A-yjeH, and verified their growth conditions on LB plates containing different concentrations of methionine analogs. The results showed that the recombinant strains E. coli DH5α pTrcHis2A-AAP, E. coli DH5α pTrcHis2A-brnFE, and E. coli DH5α pTrcHis2A-yjeH grew normally on LB plates containing high concentrations of methionine analogs (NLEU and ETH).

[0024] 3) The present invention finally screened out that the recombinant strain E. coli DH5α pTrcHis2A-LeuE showed good ACC transport activity and low transport activity for methionine analogs, which could minimize metabolic crosstalk. The results showed that LeuE was the best candidate gene for constructing ACC-producing bacteria, and its substrate-specific transport met the purpose of enhancing cell ACC tolerance without affecting precursor supply. Description of the Drawings

[0025] Figure 1 is the bacteriostatic effect diagram of ACC on Escherichia coli;

[0026] Figure 2 is the amplified DNA electrophoresis diagram of the transporter-encoding genes LeuE, rhtC, AAP, putP, brnFE, ygaZH, and yjeH;

[0027] Figure 3 is the verification diagram of the tolerance of recombinant Escherichia coli overexpressing different transporters to ACC;

[0028] Figure 4 is the verification diagram of the tolerance of recombinant Escherichia coli overexpressing different transporters to methionine analogs. Detailed Embodiments

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well known to those skilled in the art.

[0030] Example 1

[0031] Using the broth microdilution method, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Clinical and Institute, 2018) M07-A11 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th ed. CLSI standard M07. 2018.), the antibacterial effect of ACC against Escherichia coli DH5α was determined. The minimal inhibitory concentration (MIC) is the lowest concentration of ACC required to completely inhibit visible bacterial growth. The ACC stock solution (filtered and sterilized, 25.6 mg / mL) was prepared using ultrapure water. Escherichia coli DH5α was inoculated into LB test tube medium and cultured overnight at 37 °C and 220 rpm. Its concentration was adjusted to 0.5 McFarland turbidity standard (≈5×10 8 CFU / mL) using a DU800 spectrophotometer, and then diluted 1:100 in LB to reach a working concentration of 5×10 6 CFU / mL. In a sterile 96-well plate, 2-fold serial dilutions (25.6 - 0.025 mg / mL) were prepared using LB. The last column of wells contained no ACC and served as the negative control. 100 μL of the ACC solution and 100 μL of the bacterial suspension were added to each well, along with Amp stock solution (final concentration 50 mg / mL) and IPTG stock solution (final concentration 0.1 mM). The 96-well plate was sealed with Parafilm® M laboratory sealing film (Amcor, Zurich, Switzerland) and incubated in a 30 °C incubator for 20 hours to observe bacterial growth.

[0032] The results are as Figure 1 shown. Bacteria grew significantly in the control wells (i.e., wells without the ACC solution), and when the ACC concentration in the LB medium reached 6.4 g / L, no colonies were visibly growing and the medium was clear to the naked eye. The minimal inhibitory concentration of ACC against Escherichia coli DH5α strain in LB medium was 6.4 g / L.

[0033] Example 2

[0034] 1. Construction of recombinant plasmid

[0035] Using Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novoprotein Scientific Inc.), with the genome of Escherichia coli MG1655 as a template and LeuE-F / LeuE-R as primers, the target gene fragment LeuE (whose nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2) was amplified. The obtained fragment was purified by an agarose gel recovery kit (Suzhou Uyland Biotechnology Co., Ltd.). At the same time, the vector pTrcHis2A was digested with EcoRI / BamHI, and the fragment was ligated to the vector through a homologous recombination kit (Nanjing Novoprotein Scientific Inc.). DH5α competent cells (Shanghai Sangon Biotech Co., Ltd.) were transformed, and ampicillin-resistant clones were picked and identified by sequencing (Shanghai Sangon Biotech Co., Ltd.) to be correct ( Figure 2 ) and the recombinant plasmid was named pTrcHis2A-LeuE.

[0036] The construction methods of the recombinant plasmids pTrcHis2A-rhtC, pTrcHis2A-AAP, pTrcHis2A-putP, pTrcHis2A-brnFE, pTrcHis2A-ygaZH, and pTrcHis2A-yjeH are the same. The target gene fragments used are rhtC, AAP, putP, brnFE, ygaZH, and yjeH (the nucleotide sequences are shown in SEQ ID NO.3, 5, 7, 9, 11, and 13 respectively, and the amino acid sequences of their expressed proteins are shown in SEQ ID NO.4, 6, 8, 10, 12, and 14 respectively). The primers used are rhtC-F, rhtC-R; AAP-F, AAP-R; putP-F, putP-R; brnFE-F, brnFE-R; ygaZH-F, ygaZH-R; yjeH-F, yjeH-R. The primer sequences are as follows:

[0037] LeuE-F:

[0038] 5‘-GGAGGAATAAACCATGGATCCAATGTTCGCTGAATACGGG-3’ (SEQ ID NO.15),

[0039] LeuE-R:

[0040] 5‘-TCGGGCCCAAGCTTCGAATTCTCAGGATTGCAGCGTCG-3’ (SEQ ID NO.16);

[0041] rhtC-F:

[0042] 5'-GGAGGAATAAACCATGGATCCAATGTTGATGTTATTTCTCACCGTC-3' (SEQ ID NO.17),

[0043] rhtC-R:

[0044] 5'-TCGGGCCCAAGCTTCGAATTCTCACCGCGAAATAATCAAATGAAT-3' (SEQ ID NO.18);

[0045] AAP-F:

[0046] 5'-GGAGGAATAAACCATGGATCCATCAGCTAAAGAACATTACCGATACG-3' (SEQ ID NO.19),

[0047] AAP-R:

[0048] 5'-TCGGGCCCAAGCTTCGAATTCATGTCTAATATTTGGTCTAAAGAAGAAACTC-3' (SEQ ID NO.20);

[0049] putP-F:

[0050] 5'-GGAGGAATAAACCATGGATCCAATGGCTATTAGCACACCGAT-3' (SEQ ID NO.21),

[0051] putP-R:

[0052] 5'-TCGGGCCCAAGCTTCGAATTCTTAGCTTTCCTGCAACCGT-3' (SEQ ID NO.22);

[0053] brnFE-F:

[0054] 5'-GGAGGAATAAACCATGGATCCAATGCAAAAAACGCAAGAGATTC-3' (SEQ ID NO.23),

[0055] brnFE-R:

[0056] 5'-TCGGGCCCAAGCTTCGAATTCTTAGAAAAGATTCACCAGTCCCAC-3' (SEQ ID NO.24);

[0057] ygaZH-F:

[0058] 5'-GGAGGAATAAACCATGGATCCAATGGAAAGCCCTACTCCAC-3' (SEQ ID NO.25),

[0059] ygaZH-R:

[0060] 5'-TCGGGCCCAAGCTTCGAATTCTTATATAATCGCCATCACTTTCCAGG-3' (SEQ ID NO.26);

[0061] yjeH-F:

[0062] 5'-GGAGGAATAAACCATGGATCCAATGAGTGGACTCAAACAAGAACT-3' (SEQ ID NO.27),

[0063] yjeH-R:

[0064] 5'-TCGGGCCCAAGCTTCGAATTCTTATGTGGTTATGCCATTTTCCG-3' (SEQ ID NO.28).

[0065] 2. Construction of recombinant strains

[0066] Transfer pTrcHis2A-LeuE, pTrcHis2A-rhtC, pTrcHis2A-AAP, pTrcHis2A-putP, pTrcHis2A-brnFE, pTrcHis2A-ygaZH, pTrcHis2A-yjeH into Escherichia coli DH5α, and select recombinants on a selective medium containing 100 mg / mL ampicillin. The culture temperature is 37 °C, and the culture is incubated in an inverted position.

[0067] Amplify and sequence the obtained transformants to verify the obtained recombinant strains, and name them as strains E. coli DH5α pTrcHis2A-LeuE, E. coli DH5α pTrcHis2A-rhtC, E. coli DH5α pTrcHis2A-AAP, E. coli DH5α pTrcHis2A-putP, E. coli DH5α pTrcHis2A-brnFE, E. coli DH5α pTrcHis2A-ygaZH, E. coli DH5α pTrcHis2A-yjeH respectively.

[0068] Example 3

[0069] 1. Tolerance of recombinant Escherichia coli to ACC

[0070] Prepare LB plates (Amp 50 mg / mL, IPTG 0.1 M) containing 0.05 M ACC respectively. Inoculate the strains overexpressing the transporter into LB liquid medium supplemented with antibiotics and culture overnight at 37°C with 200 rpm. Then dilute the bacterial solutions to OD 600 values of 1, 0.1, 0.01, and 0.001 respectively, and pipette drops onto the LB plates containing different concentrations of ACC, and culture overnight in an incubator at 37°C.

[0071] The results are as Figure 3 shown. The recombinant strains E. coli DH5α pTrcHis2A-LeuE, E. coli DH5α pTrcHis2A-AAP, E. coli DH5α pTrcHis2A-brnFE, and E. coli DH5α pTrcHis2A-yjeH grew normally on the LB plates containing 0.05 M ACC.

[0072] 2. Tolerance of recombinant Escherichia coli to methionine analogs

[0073] If the recombinant strains can tolerate methionine analogs after overexpressing the transporter, it indicates that their methionine transport ability is improved, which will reduce the intracellular concentration of methionine, the precursor of ACC, and thus reduce the metabolic flux in the ACC biosynthesis pathway.

[0074] Prepare LB plates (Amp 50 mg / mL, IPTG 0.1 M) containing 15 mM DL-norleucine (NLEU) and DL-ethionine (ETH) respectively. Inoculate the strains overexpressing the transporter into LB liquid medium supplemented with antibiotics and culture overnight at 37°C with 200 rpm. Dilute the bacterial solutions to OD 600 values of 1, 0.1, 0.01, and 0.001 respectively, and pipette drops onto the LB plates containing NLEU or ETH, and culture overnight in an incubator at 37°C.

[0075] The results are as Figure 4 shown. The recombinant strains E. coli DH5α pTrcHis2A-AAP, E. coli DH5α pTrcHis2A-brnFE, and E. coli DH5α pTrcHis2A-yjeH grew normally on the LB plates containing high concentrations of methionine analogs (NLEU and ETH, NLEU is norleucine, and ETH is ethionine).

[0076] In summary, the recombinant strain E. coli DH5α pTrcHis2A-LeuE exhibited good ACC transport activity and low transport activity for methionine analogues, minimizing metabolic crosstalk. The results indicated that LeuE was the best candidate gene for constructing ACC-producing bacteria, and its substrate-specific transport met the purpose of enhancing cell ACC tolerance without affecting precursor supply.

[0077] The above description is illustrative rather than restrictive to the present invention. Those of ordinary skill in the art understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. Application of LeuE protein in improving the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, characterized in that, The application is that overexpression of the LeuE protein promotes the increased tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, and the amino acid sequence of the LeuE protein is as shown in SEQ ID NO.

2.

2. LeuE Use of a gene in enhancing the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, characterized in that, The application is overexpression LeuE gene promotes the increased tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, and the LeuE nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A method for improving the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid, characterized in that, Transfer LeuE the gene into Escherichia coli to obtain a recombinant Escherichia coli strain with improved tolerance to 1-aminocyclopropane-1-carboxylic acid; the LeuE nucleotide sequence of the gene is shown in SEQ ID NO.1.

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