Method for regulating and controlling transport activity of strain to 1-aminocyclopropane-1-carboxylic acid

Abstract

The invention discloses a method for regulating the transport activity of a strain to 1-aminocyclopropane-1-carboxylic acid, and relates to the technical field of bioengineering. The method for regulating and controlling the transport activity of the strain to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues and the tolerance of the strain to the 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues, disclosed by the invention, comprises the step of transferring a LeuE gene into escherichia coli to obtain an escherichia coli recombinant strain with corresponding functions. The invention discloses the application of the LeuE protein and the LeuE gene in regulating and controlling the transport activity of escherichia coli to the 1-aminocyclopropane-1-carboxylic acid and / or methionine analogue and the tolerance of escherichia coli to the 1-aminocyclopropane-1-carboxylic acid and / or methionine analogue for the first time. The invention discloses that LeuE is an optimal candidate gene for constructing ACC synthetic bacteria for the first time, and the substrate specific transport of the LeuE meets the aim of enhancing the ACC tolerance of cells without influencing precursor supply.

Description

Technical Field

[0001] The 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 a non-protein amino acid. It has functions such as regulating plant development and stress resistance, and also shows good potential application value in the medical field such as anti-tumor and neuroprotection. At present, ACC can be produced greenly through microbial fermentation, but the poor tolerance of bacteria to ACC and low transport efficiency limit its efficient 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 (BCAA). The production of leucine in Escherichia coli can be significantly increased by knocking out or mutating genes related to branched-chain amino acid transport and enhancing the expression of the LeuE gene. 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 it may also affect the synthesis or degradation process of other amino acids. The study of these interactions helps to reveal the regulatory mechanism of the Escherichia coli metabolic pathway and provide a theoretical basis for metabolic engineering transformation.

[0004] Currently, there is no direct evidence that there is a correlation between the LeuE gene and E. coli's tolerance to ACC. Regarding E. coli's tolerance to ACC, there may be a variety of complex mechanisms involved, 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 industrial production, medical health and other fields. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a transport protein LeuE of 1-aminocyclopropane-1-carboxylic acid. Another technical problem to be solved by the present invention is to provide a gene encoding a 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] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

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

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

[0009] The use of LeuE protein in regulating the transport activity of Escherichia coli for methionine analogs, the amino acid sequence of the LeuE protein is shown in SEQ ID NO.2.

[0010] The application of LeuE gene in regulating the transport activity of Escherichia coli for methionine analogs, the nucleotide sequence of the LeuE gene is shown in SEQ ID NO.1.

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

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

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

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

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

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

[0017] A method for regulating the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid comprises: transferring the LeuE gene into Escherichia coli to obtain a recombinant strain of Escherichia coli with improved 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, comprising: transferring the LeuE gene into Escherichia coli to obtain a recombinant strain of Escherichia coli 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 present invention has the following beneficial effects:

[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. coliDH5α pTrcHis2A-brnFE, E. coli DH5α pTrcHis2A-ygaZH, and 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 grew 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. coliDH5α pTrcHis2A-brnFE, E. coli DH5α pTrcHis2A-ygaZH, and E. coli DH5α pTrcHis2A-yjeH, and verified their growth in 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 DH5αpTrcHis2A-yjeH grew normally on LB plates containing high concentrations of methionine analogs (NLEU and ETH).

[0024] 3) The present invention finally screened out the recombinant strain E. coli DH5α pTrcHis2A-LeuE, which showed good ACC transport activity and low transport activity for methionine analogs, which can minimize metabolic crosstalk. The results showed that LeuE is the best candidate gene for constructing ACC-synthesizing bacteria, and its substrate-specific transport meets the purpose of enhancing cellular ACC tolerance without affecting precursor supply. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the antibacterial effect of ACC on Escherichia coli;

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

[0027] Figure 3 This is a graph verifying the tolerance of recombinant E. coli overexpressing different transporters to ACC;

[0028] Figure 4 This is a graph verifying the tolerance of recombinant Escherichia coli overexpressing different transporters to methionine analogs. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0030] Example 1

[0031] The antibacterial effect of ACC on Escherichia coli DH5α was determined by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) (2018) guideline M07-A11 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th ed. CLSI standard M07. 2018.). The minimum inhibitory concentration (MIC) is the lowest concentration of ACC required to completely inhibit visible bacterial growth. ACC stock solution (filter sterilized, 25.6 mg / mL) was prepared using ultrapure water. Escherichia coli DH5α was inoculated in LB test tube medium and cultured overnight at 37°C and 220 rpm. The concentration was adjusted to 0.5 McFarland turbidity standard (≈5×10) using a DU800 spectrophotometer. 8 CFU / mL), and then diluted 1:100 in LB to reach 5×10 6 Working concentration of CFU / mL. In a sterile 96-well plate, 2-fold serial dilutions (25.6-0.025 mg / mL) were prepared using LB, and the last column of wells did not contain ACC as a negative control. 100 μL of ACC solution and 100 μL of bacterial suspension were added to each well, followed by 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), incubated in a 30°C incubator for 20 hours, and bacterial growth was observed.

[0032] The results are as follows Figure 1 As shown in the figure, bacteria in the control wells (i.e., wells without ACC solution) grew significantly, and when the ACC concentration in the LB medium reached 6.4 g / L, no colony growth was observed by naked eye, and the medium was clear. The minimum inhibitory concentration of ACC against E. coli DH5α strain in LB medium was 6.4 g / L.

[0033] Example 2

[0034] 1. Construction of recombinant plasmid

[0035] Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozyme Biotechnology Co., Ltd.), the genome of Escherichia coli MG1655 as a template, and LeuE-F / LeuE-R as primers were used to amplify the target gene fragment LeuE (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2). The obtained fragment was purified by an agarose gel recovery kit (Suzhou Youyi Landi Biotechnology Co., Ltd.), and the vector pTrcHis2A was digested with EcoRI / BamHI. The fragment was ligated with the vector using a homologous recombination kit (Nanjing Novozyme Biotechnology Co., Ltd.), and transformed into DH5α competent cells (Shanghai Sangon Biotechnology Co., Ltd.). Ampicillin-resistant clones were picked, and sequencing (Shanghai Sangon Biotechnology Co., Ltd.) confirmed that the inserted fragment was correct ( Figure 2 ), the recombinant plasmid was named pTrcHis2A-LeuE.

[0036] The construction methods of recombinant plasmids pTrcHis2A-rhtC, pTrcHis2A-AAP, pTrcHis2A-putP, pTrcHis2A-brnFE, pTrcHis2A-ygaZH and pTrcHis2A-yjeH were the same, and the target gene fragments used were rhtC, AAP, putP, brnFE, ygaZH and yjeH (the nucleotide sequences were shown in SEQ ID NO.3, 5, 7, 9, 11 and 13, respectively, and the amino acid sequences of the expressed proteins were shown in SEQ ID NO.4, 6, 8, 10, 12 and 14), respectively, and the primers used were rhtC-F and rhtC-R; AAP-F and AAP-R; putP-F and putP-R; brnFE-F and brnFE-R; ygaZH-F and ygaZH-R; yjeH-F and yjeH-R, respectively. 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′-GGAGGAATAAACCATGGATCCCAGCTAAAGAACATTACCGATACG-3′ (SEQ ID NO.19),

[0047] AAP-R:

[0048] 5'-TCGGGCCCAAGCTTCGAATTCATGTCTAATATTTGGTCTAAAGAAAACTC-3' (SEQ IDNO.20)!

[0049] putP-F:

[0050] 5'-GGAGGAATAAACCATGGATCCAATGGCTATTAGCACACCCAT-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] pTrcHis2A-LeuE, pTrcHis2A-rhtC, pTrcHis2A-AAP, pTrcHis2A-putP, pTrcHis2A-brnFE, pTrcHis2A-ygaZH, and pTrcHis2A-yjeH were transformed into E. coli DH5α, and recombinants were selected on a selection medium containing 100 mg / mL ampicillin. The culture temperature was 37°C and the culture was inverted.

[0067] The screened transformants were amplified and sequenced to verify the obtained recombinant strains, and they were named 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, respectively.

[0068] Example 3

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

[0070] Prepare LB plates containing 0.05M ACC (Amp 50 mg / mL, IPTG 0.1 M). Inoculate the strain overexpressing the transporter into LB liquid medium with antibiotics and culture overnight at 37°C and 200 rpm. Then dilute the bacterial solution to OD 600 1, 0.1, 0.01, 0.001, use a pipette to draw The cells were dropped onto LB plates containing different concentrations of ACC and cultured overnight in a 37°C incubator.

[0071] The results are as follows Figure 3 As 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 LB plates containing 0.05 M ACC.

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

[0073] If the recombinant strain can tolerate methionine analogs after overexpressing the transporter, it means that its methionine transport capacity is improved, which will reduce the intracellular concentration of ACC precursor methionine, thereby reducing 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). Inoculate the strain overexpressing the transporter into LB liquid medium with antibiotics and culture overnight at 37°C and 200 rpm. Dilute the bacterial solution to OD 600 1, 0.1, 0.01, 0.001, use a pipette to draw The cells were dropped onto LB plates containing NLEU or ETH and cultured overnight in a 37°C incubator.

[0075] The results are as follows Figure 4 As shown, 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, NLEU is norleucine and ETH is ethionine).

[0076] In summary, the recombinant strain E. coli DH5α pTrcHis2A-LeuE showed good ACC transport activity and low transport activity for methionine analogs, which can minimize metabolic crosstalk. The results showed that LeuE is the best candidate gene for constructing ACC-synthesizing bacteria, and its substrate-specific transport is in line with the purpose of enhancing cellular ACC tolerance without affecting precursor supply.

[0077] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. The use of LeuE protein in regulating the transport activity of 1-aminocyclopropane-1-carboxylic acid in Escherichia coli, characterized in that: The amino acid sequence of the LeuE protein is shown in SEQ ID NO.

2.

2. The use of LeuE gene in regulating the transport activity of 1-aminocyclopropane-1-carboxylic acid in Escherichia coli, characterized in that: The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.

1.

3. The use of LeuE protein in regulating the transport activity of Escherichia coli for norleucine and ethionine, characterized in that: The amino acid sequence of the LeuE protein is shown in SEQ ID NO.

2.

4. The use of the LeuE gene in regulating the transport activity of Escherichia coli for norleucine and ethionine, characterized in that: The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.

1.

5. Use of LeuE protein in regulating the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or norleucine and ethionine, characterized in that: The amino acid sequence of the LeuE protein is shown in SEQ ID NO.

2.

6. Use of the LeuE gene in regulating the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or norleucine and ethionine, characterized in that: The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.

1.

7. A method for regulating the transport activity of Escherichia coli for 1-aminocyclopropane-1-carboxylic acid, characterized in that: The LeuE gene was transferred into Escherichia coli to obtain a recombinant strain of Escherichia coli with improved 1-aminocyclopropane-1-carboxylic acid transport activity; The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.

1.

8. A method for regulating the transport activity of Escherichia coli for norleucine and ethionine, characterized in that: The LeuE gene was transferred into Escherichia coli to obtain a recombinant strain of Escherichia coli with reduced transport activity for norleucine and ethionine; The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.

1.

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

1.

10. A method for regulating the tolerance of Escherichia coli to norleucine and ethionine, characterized in that: The LeuE gene was transferred into Escherichia coli to obtain a recombinant strain of Escherichia coli with reduced tolerance to norleucine and ethionine; The nucleotide sequence of the LeuE gene is shown in SEQ ID NO.1.

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