Application of plant source transport protein in improving yield of escherichia coli product

Abstract

The invention provides an application of a plant-derived transport protein in improving the yield of an escherichia coli product. According to the invention, the plant-derived transporter is introduced into the escherichia coli for the first time, and a method for improving the enzyme catalysis and fermentation metabolism yield of the escherichia coli is provided. Results show that expression of the plant source transporter in escherichia coli is improved for various escherichia coli products, and a new way is provided for obtaining escherichia coli products with higher yield. Meanwhile, due to the influence of the plant source transporter on the growth condition of the escherichia coli, a new way is provided for mixed fermentation.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to the application of plant-derived transport proteins in improving the yield of Escherichia coli products. Background Art

[0002] There are many studies on the association between transporters and product yields, and the forms of biological species expressing transporters involved are also diverse. For example, the expression of Fgr transporter in tomatoes increases the ratio of fructose to glucose in tomato fruits and increases sweetness; the expression of ABC family efflux proteins in Streptomyces increases the yield of the pesticide milbemycin; the expression of Ansamitocin P-3 (AP-3) efflux protein increases the fermentation yield of AP-3 (an important drug precursor for tumor treatment); the expression of uracil permease and nucleoside transporter in Escherichia coli significantly increases the yield of cytidine; the expression of cysteine / acetylserine transporter in Escherichia coli increases the yield of L-serine; the expression of epothilone multidrug efflux protein increases the yield of epothilone (a compound used to prevent cancer cell division); the yield of erythromycin precursor 6-deoxyerythrolide B is significantly increased through efflux protein engineering technology. However, due to the recognition specificity of transporters, the application of each transporter is generally aimed at increasing the yield or efficiency of a specific product.

[0003] As one of the most widely studied and applied microorganisms in the field of biotechnology and genetic engineering, Escherichia coli (E. coli) is of great importance in scientific research and industrial applications. Due to its easy cultivation, fast growth rate, and simple genetic manipulation, E. coli is one of the most powerful protein expression tools and the most commonly used bacterial species for protein expression.

[0004] Therefore, it is necessary to screen a universal transporter for Escherichia coli, in order to use the transporter to improve the enzyme catalytic efficiency or the metabolic product production of Escherichia coli. Summary of the invention

[0005] In order to solve the problem that the transporter in the prior art is specific to a specific host and has recognition specificity, the present invention provides an application of a plant-derived transporter in improving the yield of Escherichia coli products. The results show that heterologous expression of the Arabidopsis-derived NPF / SWEET transporter gene in Escherichia coli affects cell membrane permeability and links it to related catalytic or metabolic reactions, which can further improve production efficiency and expand the scope of action of the transporter to the Escherichia coli chassis with a wider application.

[0006] The technical solution adopted by the present invention is: application of plant-derived transporter protein in improving the yield of Escherichia coli products, and the application includes: constructing a vector containing a gene encoding the plant-derived transporter protein into an Escherichia coli engineered bacterium for producing a target product; wherein the expression of the plant-derived transporter protein is induced by IPTG and / or rhamnose; the plant-derived transporter protein is selected from at least one of the NPF family and the SWEET family, preferably at least one of NPF1.2, NPF2.5, NPF8.1, SWEET1, and SWEET5, and more preferably SWEET5.

[0007] The inventor team extracted NPF family and SWEET family transporters from Arabidopsis thaliana (NPF1.2, NPF2.5, NPF8.1, SWEET1, SWEET5 amino acid sequences see Chinese patent CN 117777249A) and made a preliminary analysis of their ability to transport gibberellins, but in the process of using Escherichia coli to purify transporters, it was found that the expression of NPF / SWEET transporter genes would cause abnormal growth of Escherichia coli, mostly in filamentous growth. In order to explain this phenomenon and explore the possible application of NPF / SWEET transporter expression in Escherichia coli, by observing the bacterial growth curve and bacterial morphology of transporters at different induction concentrations, the effects of transporter expression on the cell wall, cell membrane, cell division, etc. of Escherichia coli were analyzed, and based on this potential effect, the role of transporters in enzyme catalysis and bacterial metabolism in Escherichia coli was explored, in order to use NPF / SWEET transporters to improve the enzyme catalysis efficiency or bacterial metabolite production in Escherichia coli.

[0008] Preferably, the application comprises: culturing the constructed engineered Escherichia coli until OD600 reaches 0.6-0.8, inducing culture using IPTG; wherein the working concentration of IPTG is 0.025-0.1Mmol / L, preferably 0.025-0.05Mmol / L.

[0009] Preferably, the application includes: when the constructed Escherichia coli engineered bacteria is cultured to OD600 of 0.6 to 0.8, induction culture is performed using rhamnose; wherein the working concentration of rhamnose is 0.1 v / v% to 0.3 v / v%, preferably 0.2 v / v% to 0.3 v / v%, and more preferably 0.2 v / v%

[0010] Preferably, the application includes: constructing a vector encoding a gene for a target product catalytic enzyme induced by IPTG and a vector encoding a gene for a plant-derived transporter induced by rhamnose into an Escherichia coli engineered bacterium for producing the target product. The target product catalytic enzyme may be a PPK enzyme for catalyzing ADP to generate ATP, or a deacetylase for catalyzing chloroacetamide to generate chloroamine.

[0011] Preferably, the application includes: when the constructed Escherichia coli engineered bacteria is cultured to OD600 of 0.6 to 0.8, induction culture is carried out using IPTG for 10 to 14 hours, and after replacing the culture medium, induction culture is carried out using rhamnose for 10 to 14 hours; wherein the working concentration of IPTG is 0.05 to 0.1 Mmol / L, preferably 0.1 Mmol / L; the working concentration of rhamnose is 0.2 v / v% to 0.3 v / v%, preferably 0.2 v / v%.

[0012] Preferably, the application comprises: constructing a vector of a PPK enzyme encoding gene induced by IPTG and a vector of a plant-derived transporter encoding gene induced by rhamnose into Escherichia coli; when the constructed Escherichia coli is cultured to OD600 of 0.6 to 0.8, inducing the culture for 10 to 14 hours using IPTG at a working concentration of 0.025 to 0.05 Mmol / L, preferably 0.1 Mmol / L, and after replacing the culture medium, inducing the culture for 10 to 14 hours using rhamnose at a working concentration of 0.2 v / v% to 0.3 v / v%, preferably 0.2 v / v%; constructing an ADP / AMP-ATP reaction system using the wet cells of the induced Escherichia coli as a catalyst and ADP / AMP and polyP as substrates, reacting for 5 to 10 minutes at pH 7.5 to 8.0 and 35 to 40°C to prepare ATP.

[0013] Preferably, the application comprises: constructing a vector encoding a gene for a plant transporter induced by IPTG and a vector encoding a gene for a deacetylase induced by rhamnose into Escherichia coli; when the constructed Escherichia coli is cultured to an OD600 of 0.6 to 0.8, inducing the culture for 10 to 14 hours using IPTG at a working concentration of 0.025 to 0.05 Mmol / L, preferably 0.1 Mmol / L, and after replacing the culture medium, inducing the culture for 10 to 14 hours using rhamnose at a working concentration of 0.2 to 0.3 v / v%, preferably 0.2 v / v%; constructing a chloroacetamide-chloroamine reaction system using the wet cells of the induced Escherichia coli as a catalyst and chloroacetamide as a substrate, reacting for 12 to 24 hours at pH 7.5 to 8.0 and 35 to 40° C. to prepare the chloroamine.

[0014] Preferably, the application comprises: constructing a vector encoding a gene for a plant-derived transporter induced to be expressed by rhamnose into an L-methionine-producing Escherichia coli engineered bacterium; culturing the constructed Escherichia coli engineered bacterium for 24 to 36 hours, and then inducing and culturing the constructed Escherichia coli engineered bacterium for 12 to 24 hours using rhamnose at a working concentration of 0.2 to 0.3 v / v%, preferably 0.2 v / v%, to prepare L-methionine.

[0015] Beneficial effects of the present invention: The present invention introduces plant-derived transport proteins into Escherichia coli for the first time, providing a method for increasing the enzyme catalysis and fermentation metabolism yield of Escherichia coli. The results show that the expression of plant-derived transport proteins in Escherichia coli improves a variety of Escherichia coli products, providing a new way to obtain higher yields of Escherichia coli products. At the same time, due to the influence of plant-derived transport proteins on the growth of Escherichia coli, a new way is also provided for mixed bacterial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The following are the effects of expressing different plant-derived transport proteins in E. coli under 0.1 Mmol / L IPTG induction on the growth curve of E. coli in Example 1.

[0017] Figure 2 The results of using propidium iodide (PI) to stain Escherichia coli to verify whether the cells are alive in Example 1; A is uninduced Escherichia coli, and B is induced Escherichia coli expressing the transporter protein.

[0018] Figure 3 The results of quantitative verification of Escherichia coli expressing the transporter protein after induction with 0.1 Mmol / L IPTG in Example 1; A is the control diluted 10,000 times, each experimental group was diluted 1,000 times, the bacterial solution was evenly spread on LB solid culture medium, and the colony count after overnight culture; B is the calculated resting rate of Escherichia coli after induction.

[0019] Figure 4 The growth curves of strains expressing different plant-derived transporters induced by different concentrations of IPTG in Example 1 are shown.

[0020] Figure 5 The cell morphology of the strain expressing SWEET5 induced by different concentrations of IPTG in Example 1.

[0021] Figure 6 The bacterial density of the strain expressing SWEET5 with nitrogen segment and / or carbon segment removed in Example 1.

[0022] Figure 7 The bacterial density of the strain expressing SWEET5 with different carbon segment transmembrane helices removed in Example 1; A is a schematic diagram of carbon segment transmembrane helix removal, B is a comparison of transmembrane helix removal results, and C is a comparison of transmembrane helix removal results under IPTG induction.

[0023] Figure 8 The ATP conversion rate comparison in Example 2.

[0024] Fig. 9 The figure is a comparison of the conversion rates of chloroamines in Example 3.

[0025] Fig.10 The figure is a comparison of the L-methionine fermentation yield in Example 4.

[0026] Fig.11 This is the plasmid map of IPTG-induced SWEET5 used in Example 1.

[0027] Fig.12 This is the plasmid map of rhamnose-induced SWEET5 used in Examples 2 to 4. DETAILED DESCRIPTION

[0028] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0029] The IPTG-inducible plasmid pET-28a and rhamnose-inducible plasmid pGASTON used in this experiment were from the Institute of Enzyme and Biocatalysis Engineering, Zhejiang University of Technology. The expression plasmids of PPK enzyme, deacetylase and methionine used were from the laboratory's previous construction or the Institute of Biocatalysis Engineering and Institute of Synthetic Biotechnology, Zhejiang University of Technology.

[0030] Example 1: Effect of IPTG-induced expression of transporter protein on the growth of Escherichia coli

[0031] Plasmid construction: By designing primers, the target transport protein NPF1.2, NPF2.5, NPF8.1, SWEET1, and SWEET5 gene fragments were obtained by PCR, and they were connected with the linearized vector pET-28a obtained by reverse PCR using a one-step cloning kit to obtain the corresponding recombinant plasmids.

[0032] E. coli transformation: add 10 μL of recombinant plasmid to 100 μL of E. coli BL21 (DE3) competent cells, place on ice for 30 minutes, then heat shock at 42°C for 45 seconds, add 700 μL of LB medium and incubate at 37°C in a test tube shaker for 1 hour, take 100 μL, apply to LB solid medium, and culture overnight. Pick a single colony and perform colony PCR, or inoculate a test tube and sequence verification.

[0033] Preparation of kanamycin mother solution: Weigh 1.5 g of kanamycin sulfate powder, add 30 mL of sterile ultrapure water to obtain 50 mg / mL of mother solution, filter the mixed solution through a 0.22 μm filter membrane to remove bacteria or impurities, and finally dispense into 1.5 mL sterile EP tubes and store in a -20°C refrigerator.

[0034] Seed liquid culture: The seed liquid culture process is first screened on an LB plate containing ampicillin or kanamycin according to the antibiotic resistance gene carried by Escherichia coli, and single colonies are obtained by streaking. Then, these single colonies are transferred to a test tube containing 1‰ ampicillin or kanamycin (concentration of 50g / L) in 10mL LB medium. At 37°C and 180rpm, a constant temperature shaker is used for culturing for 12 hours to obtain seed liquid for subsequent use.

[0035] Protein induction: Add 100mL of LB medium to a 500mL shake flask, then transfer the cultured seed solution to LB liquid medium at a volume fraction of 2%, and add ampicillin or kanamycin at a final concentration of 50μg / mL. Cultivate in a constant temperature shaker at 37°C and 180rpm until OD600 reaches 0.6-0.8, then add 5μL, 10μL, 15μL and 20μL IPTG, that is, the working concentrations are 0.025Mmol / L, 0.05Mmol / L, 0.075Mmol / L, and 0.1Mmol / L for induction culture, continue to culture at 20°C for 20 hours, take samples every 4h, and use a spectrophotometer to measure the corresponding OD800 value. Finally, use 8000rpm to centrifuge at 4°C for 10 minutes to collect the bacteria, and store them in a refrigerator at -20°C for use.

[0036] Dead and live cell staining identification: Take an appropriate amount of PI aqueous solution and add it to PBS to prepare a 10-50μM (6.7-33.4μg / mL) PI solution, add PI solution equivalent to one-tenth of the volume of the culture medium to the cell culture medium, and then culture the cells at 37°C for 15 to 25 minutes. After that, wash the cells twice with PBS or other appropriate buffer solutions, and finally observe them under a fluorescence microscope with an excitation wavelength of 535nm and an emission wavelength of 615nm.

[0037] E. coli expressing NPF1.2, NPF2.5, NPF8.1, SWEET1, and SWEET5 transporters were induced with 20 μL of the inducer IPTG added, and the growth curve of the bacteria was measured. Figure 1As shown, it can be found that the empty vector (control) without the target gene has no obvious effect on cell growth under the condition of 20μL IPTG induction, reaching the highest cell density at 12h, and the cell density OD800 after 20h is maintained at 1.61. In contrast, the growth of strains expressing NPF1.2, NPF2.5, NPF8.1, SWEET1, and SWEET5 was all restricted, among which the cell density decreased slightly before 4h. It is speculated that the cells are more sensitive after induction, and the toxicity of IPTG itself has an effect on the cells. After that, the cell density slowly increased, and the OD600 at 20h reached about 0.75.

[0038] It has been found that the expression of transport proteins will affect the growth of E. coli, but the life state of the induced E. coli cannot be confirmed. In order to confirm the life state of the induced E. coli, using SWEET5 as an example, propidium iodide (PI) was used to stain the induced cells. Living cells can effectively block the dye from entering the cytoplasm. The results showed that most cells did not show color, which means that most of the cells after induction are resting cells ( Figure 2 ).

[0039] Quantitative analysis of 20 μL IPTG-induced cells was performed by evenly applying the bacterial solution to LB solid medium, with the control diluted 10,000 times and each experimental group diluted 1,000 times. After overnight culture, the number of colonies was calculated and the resting cell rate after induction was obtained. Figure 3 It can be seen that the resting rate is maintained above 99%.

[0040] In order to exclude the possibility that excessive inducer concentration may affect bacterial growth, 0 μL, 5 μL, 10 μL, 15 μL, and 20 μL of IPTG were added, i.e., the working concentrations were 0 mM / L, 0.025 mM / L, 0.05 mM / L, 0.075 mM / L, and 0.1 mM / L, respectively, to induce the empty vector and six transformants, and monitor the cell growth ( Figure 4 ). Figure 4 ①-⑥ in the middle are control bacteria, NPF1.2, NPF2.5, NPF8.1, SWEET1, and SWEET5 expression bacteria, respectively. The results showed that although reducing the amount of IPTG added was helpful for cell growth, it was limited to adding 5μL and 10μL, while the growth curves of cells adding 15μL and 20μL of IPTG were almost the same. At the same time, by comparing with the control, it was found that the cell density level of the empty vector remained at a high level even if 20μL of IPTG was added, which directly shows that the expression of the transporter protein caused the cell growth to be affected, excluding other factors.

[0041] Taking the strain expressing SWEET5 as an example, the cell morphology under different induction amounts and induction times was observed using an optical microscope, such as Figure 5 , where the red arrows are cells that have become filamentous, and the blue arrows are cells that have become spherical. It can be found that the bacterial morphology is normal without the addition of inducers. With the increase of induction time and induction amount, the morphological changes of the bacteria become more obvious. It can be speculated that the cell wall or cell membrane of E. coli and its growth and division are affected by the expression of transporter proteins.

[0042] It is known that almost all SWEET transporter families have seven transmembrane structures, which are simpler than NPF structures. After SWEET5 modeling, it can also be seen that it has seven transmembrane structures. First, the carbon and nitrogen segments are cut off. The results are as follows Figure 6 As shown. It can be found that after removing the N-terminus, the bacterial density of the transporter after 20 μL IPTG induction for 20 hours is lower than that with a complete nitrogen segment, while there is no obvious difference in the bacterial density after removing the C-terminus. Therefore, it can be inferred that the N-terminus is connected to the transmembrane structure and plays a role in the formation of membrane-related channels or the increase of permeability. In order to verify this hypothesis, Figure 7 In A and B, the transmembrane helices were removed piece by piece starting from the carbon end. It was found that the bacterial density increased after removing one segment of the transmembrane helices at 20h. When only two segments of the transmembrane helices were left, the effect on bacterial growth was significantly reduced. Figure 7 It can also be seen that when three or four transmembrane structures are removed, there is still a significant decrease in bacterial growth concentration after inducing transporter expression. It is not until five to six transmembrane structures are removed that the growth is close to the control. The growth trend is as follows Figure 7 Middle C.

[0043] Example 2: Effect of plant-derived transporters on the reaction of PPK enzyme catalyzing ADP to generate ATP

[0044] The constructed plasmids for inducing SWEET5 expression with rhamnose and PPK enzyme expression with IPTG were transformed into competent BL21 (DE3). When the constructed E. coli was cultured to OD600 of 0.6-0.8, 0.1Mmol / L IPTG was used for induction culture for 12 hours. After the culture medium was replaced, 0.2v / v% rhamnose was used for induction culture for 10 hours. The wet cells of the induced E. coli were used as catalysts, and ADP / AMP and polyP were used as substrates to construct the ADP / AMP-ATP reaction system. The reaction was carried out at pH 8.0, 37°C, and 800rpm for 5 minutes to prepare ATP. Figure 8It can be seen that in the ATP generation reaction, the catalytic efficiency of Escherichia coli expressing the transporter protein SWEET and the PPK enzyme for the substrate is close to that of the organic reagent CTAB / Tweem-80, and is about 1.2 times that of the control.

[0045] ADP / AMP-ATP reaction system: The total reaction volume is 1 mL, including: 50 μL 10 mM ADP / AMP, 20 μL 20 mM Mg 2+ , 200 μL 20 mM polyP, 50 μL 5 g / L wet bacteria. After adjusting the pH, add 680 μL of 100 mM Tris-HCl (PH = 8.0) and react at 37°C and 800 rpm for 5 min.

[0046] ATP detection: After reacting for 5 minutes according to the above method, centrifuge at 12000 rpm for 1 minute, obtain the supernatant, dilute 5 times, take 100 μL and put it into the inner liner tube for later use.

[0047] Preparation of mobile phase: prepare 20 mM potassium phosphate buffer as mobile phase and adjust the pH to 7. Filter with a sterile filter membrane (including organic membrane and water membrane) to remove impurities and bacteria. Ultrasonicate for 30-40 minutes before use to remove bubbles.

[0048] High performance liquid chromatography (HPLC) conditions: potassium phosphate buffer 100%; flow rate 1 mL / min; detection wavelength 254 nm; injection volume 20 μL; column temperature 40° C.; ATP peak time is about 5 min.

[0049] Example 3: Effect of plant-derived transporters on the reaction of chloroacetamide to chloroamine catalyzed by deacetylase

[0050] The constructed plasmids for inducing SWEET5 expression with rhamnose and deacetylase expression with IPTG were transformed into competent BL21 (DE3). When the constructed E. coli was cultured to OD600 of 0.6-0.8, 0.1Mmol / L IPTG was used for induction culture for 12 hours. After the culture medium was replaced, 0.2v / v% rhamnose was used for induction culture for 10 hours. The wet cells of the induced E. coli were used as catalysts and chloroacetamide as substrate to construct a chloroacetamide-chloroamine reaction system. The reaction was carried out at pH 8.0 and 37°C for 24 hours to prepare chloroamine. Fig. 9 It can be seen that the catalytic efficiency of the transporter-expressing bacteria in the chloroamine reaction is higher than that of the organic reagent, which is about 1.8 times that of the control. The higher efficiency of the transporter-expressing bacteria in the chloroamine catalytic reaction is speculated to be because the ATP reaction only takes 5 minutes, while the chloroamine reaction takes 24 hours, and the protein-expressing bacteria are suitable for long reaction times.

[0051] Chloroacetamide-chloroamine reaction system: The total reaction volume is 10 mL, specifically including: 5 mL 25 g / L wet bacteria, 5 mL 3 g / L chloroacetamide, and the buffer is 100 mM Tris-HCl (PH=8.0). The reaction is carried out at 37°C and 180 rpm for 24 hours.

[0052] Chloroamine detection: Take 200μL sample, boil for 15 minutes, centrifuge at 12000 for 2min. Then take 250μL water, 900μL methanol, 100μL supernatant to prepare sample, pass through organic membrane and put into inner liner tube for standby use. Chloroamine detection generally starts with the detection of substrate chloroacetamide content, the initial concentration of substrate is 20mM, as long as (20mM-remaining substrate) / 20*100% is the conversion rate of the product.

[0053] Preparation of mobile phase: The mobile phase is methanol and water. The pH of water is adjusted to 3 with phosphoric acid. Impurities and bacteria are removed by filtration with a sterile filter membrane (including organic membrane and water membrane). Air bubbles are removed by ultrasonication for 30-40 minutes before use.

[0054] HPLC conditions: the ratio of pure methanol and water in the mobile phase is 3:7; the detection wavelength is 210nm; the injection volume is 20μL; the column temperature is 40℃; the flow rate is 1mL / min; the elution time of chloroacetamide is about 4.5min.

[0055] Example 4: Effect of transporter proteins on metabolic fermentation of Escherichia coli

[0056] The constructed plasmid for inducing SWEET5 expression with rhamnose was transformed into Escherichia coli that had been metabolically engineered to produce L-methionine (Huang Jian Feng, Liu Zhi Qiang, Jin Li Qun, Tang Xiao Ling, Shen Zhen Yang, Yin Huan Huan, Zheng Yu Guo. Metabolic engineering of Escherichia coli for microbial production of L-methionine [J]. BIOTECHNOLOGY AND BIOENGINEERING, 2017, 114 (4): 843-851.), and cultured in a shake flask for 48 h. After 24 h of growth, 0.2 v / v% rhamnose was added for induction. Fig.10 It can be seen that the expression of the transporter protein facilitates the excretion of E. coli metabolites, and the product is increased by 1.3 times compared with the control bacteria.

[0057] L-methionine fermentation system: Pick a single colony and inoculate it into a 5mL LB test tube, culture it at 37°C in a shaker at 180rpm overnight, transfer it to the sterilized MS fermentation medium at a transfer volume of 5%, and add 0.2g of sterilized calcium carbonate, 20μL of 0.1MIPTG (final concentration of 0.1mM), 20μL of 50g / L kanamycin (final concentration of 50mg / L), 20μL of 0.2g / LVB12 (final concentration of 0.2mg / L), and 20μL of 10g / L lysine (final concentration of 0.01g / L), and culture it at 30°C in a shaker at 180rpm for 48h.

[0058] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. Application of plant-derived transporter protein in increasing the yield of Escherichia coli products, characterized in that: The application includes: constructing a vector containing a plant-derived transporter encoding gene into an Escherichia coli engineered bacterium for producing a target product; Wherein, the expression of the plant-derived transporter is induced by IPTG and / or rhamnose; The plant-derived transporter is selected from at least one of the NPF family and the SWEET family.

2. The use according to claim 1, characterized in that The plant-derived transporter is selected from at least one of NPF1.2, NPF2.5, NPF8.1, SWEET1, and SWEET5.

3. The use according to claim 1, characterized in that The applications include: When the constructed Escherichia coli engineered bacteria is cultured to OD600 of 0.6 to 0.8, IPTG is used for induction culture; wherein the working concentration of IPTG is 0.025 to 0.1 Mmol / L; or, When the constructed Escherichia coli engineering bacteria are cultured to an OD600 value of 0.6 to 0.8, rhamnose is used for induction culture; wherein the working concentration of rhamnose is 0.1 v / v% to 0.3 v / v%.

4. The use according to claim 1, characterized in that The application includes: constructing a vector encoding a gene for a target product catalytic enzyme induced by IPTG and a vector encoding a gene for a plant-derived transporter induced by rhamnose into an engineered Escherichia coli for producing the target product.

5. The use according to claim 4, characterized in that The application includes: when the constructed Escherichia coli engineered bacteria is cultured to OD600 of 0.6 to 0.8, induction culture is carried out using IPTG for 10 to 14 hours, and after replacing the culture medium, induction culture is carried out using rhamnose for 10 to 14 hours; wherein the working concentration of IPTG is 0.05 to 0.1 Mmol / L, and the working concentration of rhamnose is 0.2 to 0.3 v / v%.

6. The use according to claim 1, characterized in that The applications include: The vector encoding the gene for the PPK enzyme induced by IPTG and the vector encoding the gene for the plant-derived transporter induced by rhamnose were co-constructed into Escherichia coli; When the constructed Escherichia coli is cultured to OD600 of 0.6 to 0.8, IPTG is used for induction culture for 10 to 14 hours, and after the culture medium is replaced, rhamnose is used for induction culture for another 10 to 14 hours; The wet cells of Escherichia coli after induction culture were used as catalysts, ADP / AMP and polyP were used as substrates, and an ADP / AMP-ATP reaction system was constructed. The reaction was carried out at pH 7.5-8.0 and 35-40°C for 5-10 minutes to prepare ATP.

7. The use according to claim 1, characterized in that The applications include: The vector encoding the deacetylase gene induced by IPTG and the vector encoding the plant-derived transporter gene induced by rhamnose were co-constructed into Escherichia coli; When the constructed Escherichia coli is cultured to OD600 of 0.6 to 0.8, IPTG is used for induction culture for 10 to 14 hours, and after the culture medium is replaced, rhamnose is used for induction culture for another 10 to 14 hours; The wet cells of Escherichia coli after induction culture are used as catalysts and chloroacetamide is used as substrate to construct a chloroacetamide-chloroamine reaction system. The reaction is carried out at pH 7.5-8.0 and 35-40° C. for 12-24 hours to prepare chloroamine.

8. The use according to any one of claims 6 or 7, characterized in that: The working concentration of the IPTG is 0.05-0.1 Mmol / L; The working concentration of rhamnose is 0.2-0.3 v / v%.

9. The use according to claim 1, characterized in that The applications include: The vector encoding the plant-derived transporter gene induced by rhamnose is constructed and introduced into the L-methionine-producing Escherichia coli engineering bacteria; The constructed Escherichia coli engineering bacteria are cultured for 24 to 36 hours, and then induced by rhamnose for 12 to 24 hours to prepare L-methionine.

10. The use according to claim 9, characterized in that The working concentration of rhamnose is 0.2-0.3 v / v%.

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