Method for improving tolerance of Escherichia coli to geraniol and use thereof

Abstract

The present invention belongs to the field of bioengineering, and proposes a method based on a double-stage reinforcement strategy of mutagenesis and efflux engineering to improve the tolerance of Escherichia coli to geraniol. Specifically, the present invention utilizes atmospheric pressure room temperature plasma (ARTP) mutagenesis technology to induce Escherichia coli, and screens and obtains an evolved strain TS with improved geraniol tolerance; further, through efflux system regulation engineering, transcription factors marA and rob are overexpressed in TS to construct an engineered strain TS-pMR, whose geraniol tolerance concentration reaches 11 g / L, which is 11 times higher than that of the original strain, breaking through the product toxicity tolerance threshold in microbial synthesis. The present invention adopts a hierarchical reinforcement strategy, avoids complex acclimation steps, greatly shortens the strain construction cycle, and improves the genetic stability of the strain. The geraniol-tolerant Escherichia coli finally obtained is more superior in tolerance than the starting strain.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and more particularly relates to a method for improving the tolerance of Escherichia coli to geraniol and a use thereof. Background Art

[0002] Geraniol is a monoterpene alcohol compound extracted from aromatic plants. It has a rich rose aroma and is widely used in spices, essential oils, and fragrance cosmetics. In addition, geraniol has a variety of pharmacological activities, including anti-cancer, anti-inflammatory, antioxidant, and antibacterial properties, and is therefore considered a potential drug candidate. Currently, the main method for commercial production of geraniol is to extract it from plant essential oils through processes such as pressing and distillation. However, this traditional production method not only occupies a large amount of arable land but also has a long production cycle, making it difficult to meet the growing market demand.

[0003] In contrast, microbial synthesis, using low-value substrates as raw materials, produces geraniol in a green and sustainable manner, becoming an important alternative to traditional plant extraction. With the advancement of metabolic engineering and synthetic biology, the microbial biosynthesis pathway of geraniol has been elucidated. This biosynthesis follows a typical terpene metabolic pathway: isoprenyl pyrophosphate (IPP), generated via the mevalonate (MVA) pathway or the 2-methyl-D-erythritol-4-phosphate (MEP) pathway, is used as a precursor to form dimethylallyl pyrophosphate (DMAPP) catalyzed by isopentenyl pyrophosphate isomerase (IDI). Geranyl diphosphate synthase then converts DMAPP to geranyl pyrophosphate (GPP), which is then hydrolyzed by geraniol synthase (GES) to yield the target product, geraniol. This synthetic pathway not only increases the yield of geraniol but also lays the foundation for its wider use in drug development and industrial applications.

[0004] While traditional metabolic engineering strategies (such as pathway optimization and enzyme modification) have made significant progress in increasing geraniol production, their industrial application still faces a fundamental technical bottleneck: geraniol's inherently potent cytotoxicity, which irreversibly damages microbial fermentation systems. In fact, the combined metabolic pressures of cell growth inhibition and product toxicity result in a common problem for conventional strains during fermentation: a sudden drop in survival and limited product synthesis. However, existing tolerance-enhancing technologies all suffer from significant drawbacks: While ARTP mutagenesis can generate a broad spectrum of mutations, its random nature results in a screening efficiency of less than 0.2% for forward mutants, necessitating the integration of large-scale screening platforms. While adaptive laboratory evolution (ALE) strategies can selectively screen for tolerant phenotypes, they require a long acclimatization cycle and the risk of phenotypic degeneration. While overexpression of efflux proteins can enhance E. coli's tolerance to specific products, it does not fundamentally address the toxicity of these products to cells. Notably, current research is largely limited to improvements at a single technical level, and a multi-level synergistic enhancement mechanism has yet to be established. Therefore, breaking the growth inhibition of bacteria caused by geraniol accumulation and solving the problem of poor tolerance of Escherichia coli to geraniol have become the key to improving the biosynthesis of compounds. Summary of the Invention

[0005] To address the problems of poor tolerance of existing Escherichia coli to geraniol and limited improvement of single technology, the present invention provides a method for constructing high-geraniol-tolerant Escherichia coli through a rapid synergistic enhancement strategy of ARTP (Atmospheric and Room Temperature Plasma) mutagenesis and efflux engineering.

[0006] First, the present invention uses atmospheric pressure room temperature plasma (ARTP) mutagenesis technology to induce Escherichia coli Trans1T1, and screens out an evolved strain TS with increased geraniol tolerance to 10 g / L, with the deposit number being: CGMCC No. 34069. Furthermore, through efflux system regulation engineering, the transcription factors marA (GeneID: 947613) and rob (GeneID: 948916) are overexpressed in TS to construct an engineered strain TS-pMR, whose geraniol tolerance concentration reaches 11 g / L, which is 11 times higher than that of the original strain, breaking through the product toxicity tolerance threshold in microbial synthesis. The present invention adopts a graded enhancement strategy, avoiding complex acclimation steps, significantly shortening the strain construction cycle, and improving the genetic stability of the strain. The resulting geraniol-tolerant Escherichia coli is superior in tolerance to the starting strain.

[0007] Therefore, in one aspect, the present invention provides an Escherichia coli strain with improved geraniol tolerance, the deposit number of which is CGMCC No. 34069.

[0008] Another aspect of the present invention also provides a method for improving the geraniol tolerance of Escherichia coli, which comprises the following steps: step A. using atmospheric pressure and room temperature plasma to treat wild-type Escherichia coli for mutagenesis to screen and obtain geraniol-tolerant mutants; and step B. overexpressing the transcriptional regulatory factors marA and rob in the mutant obtained in step A to construct a geraniol-tolerant engineered strain that regulates the efflux system.

[0009] In one embodiment, the wild-type Escherichia coli is Escherichia coli Trans1T1.

[0010] In a specific embodiment, the amino acid sequence of the transcription regulatory factor marA is shown in SEQ ID NO. 1, and / or the encoding nucleotide sequence of the transcription regulatory factor marA is shown in SEQ ID NO. 2; and / or the amino acid sequence of the transcription regulatory factor rob is shown in SEQ ID NO. 3, and / or the encoding nucleotide sequence of the transcription regulatory factor rob is shown in SEQ ID NO. 4.

[0011] In one embodiment, the step A comprises: (1) collecting the wild-type Escherichia coli cultured to the logarithmic growth phase by centrifugation, resuspending and preparing a bacterial suspension, and controlling the OD 600 is 0.6 to 0.8; (2) dropping the bacterial suspension onto a slide, moving the slide to a normal pressure room temperature plasma breeding instrument operating room for mutagenesis; (3) using helium as a working gas for mutagenesis; (4) placing the mutagenized slide into physiological saline, shaking and mixing to obtain a mixed bacterial solution; (5) diluting the mixed bacterial solution, spreading it on a solid plate containing geraniol, culturing overnight, and selecting colonies that grow well in the high concentration area and inoculating them into a solid culture medium; (6) picking strain cells from the solid culture medium for culture screening; and (7) measuring OD after the culture is completed. 600 Evaluate cell growth performance and screen evolved strains with good growth performance from positive growth strains.

[0012] In one embodiment, step B comprises: (1) constructing a recombinant plasmid that overexpresses both the transcriptional regulatory factors marA and rob; and (2) introducing the recombinant plasmid obtained in (1) into the mutant strain obtained in step A to obtain an engineered strain with further improved geraniol tolerance.

[0013] In one embodiment, the geraniol-tolerant mutant obtained in step A has a preservation number of CGMCC No. 34069, and / or can tolerate a maximum geraniol concentration of 10 g / L.

[0014] In one embodiment, the maximum geraniol concentration that the geraniol-tolerant engineered strain obtained in step B can tolerate is 11 g / L.

[0015] Another aspect of the present invention also provides a geraniol-tolerant engineered strain obtained by the above method of the present invention.

[0016] Another aspect of the present invention further provides the use of the above-mentioned preserved Escherichia coli strain or the geraniol-tolerant engineered strain of the present invention in the production of geraniol. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0018] Figure 1 is the ARTP-induced lethality rate of Escherichia coli Trans1T1;

[0019] Figure 2 are the tolerance curves of the evolved E. coli strain TS and the wild-type strain Trans1T1;

[0020] Figure 3 are the growth curves of the evolved E. coli strain TS and the wild-type strain Trans1T1;

[0021] Figure 4 This is the map of the pMR plasmid that overexpresses marA+rob genes.

[0022] Description of Sequence Listing

[0023] SEQ ID NO. 1 – Amino acid sequence of marA

[0024] SEQ ID NO. 2 – Nucleotide sequence encoding marA

[0025] SEQ ID NO. 3 – Amino acid sequence of rob

[0026] SEQ ID NO. 4 – Nucleotide sequence encoding rob

[0027] SEQ ID NO. 5 – Primers marA-F

[0028] SEQ ID NO. 6 – Primer marA-R

[0029] SEQ ID NO. 7 – Primer rob-F

[0030] SEQ ID NO. 8 – Primer rob-R

[0031] SEQ ID NO. 9 – Primer pSB1C3-F

[0032] SEQ ID NO. 10 – Primer pSB1C3-R DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0035] Unless otherwise specified, the materials, reagents, instruments and methods used in the examples of the present invention are conventional materials, reagents, instruments and methods in the art and can be obtained or implemented through commercial channels.

[0036] Unless otherwise indicated, the terms used herein have their ordinary technical meanings as understood by those skilled in the art. For definitions and terms in the art, the skilled artisan is particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0037] The terms “include” or “comprising” described in the present invention are open-ended descriptions, encompassing all the specified components or steps described, as well as other specified components or steps that will not substantially affect the description. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still having the activity described in the present invention.

[0038] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0039] As used herein, term " overexpression " refers to when the strict control of gene / protein expression (transcription) is upset, and gene may not be " turned off ", or transcribes at a high speed. High-speed transcription causes a large amount of mRNA to produce. For the overexpression of transcriptional regulatory factor marA and rob of the present invention, refer to that its DNA or RNA or protein expression level in engineering Escherichia coli of the present invention is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or 300% higher than control (but not expressing transcriptional regulatory factor marA and rob according to the present invention), or even 4, 5, 6, 7, 8, 9, 10 times or more of the corresponding DNA or RNA or protein expression level in the control. The technology and reagent for detecting gene / protein expression level are well known to those skilled in the art.

[0040] As used herein, the term "efflux system" refers to a class of proteins present on the bacterial cell membrane. Bacteria use these membrane proteins to excrete toxic substances (including antimicrobial drugs, metabolites, etc.) to regulate the stability of their internal environment and protect the bacteria themselves from damage.

[0041] One object of the present invention is to provide a method for improving the tolerance of Escherichia coli to geraniol.

[0042] In a preferred embodiment, the method comprises the following steps: (1) treating wild-type Escherichia coli with ARTP mutagenesis to screen for geraniol-tolerant mutants; (2) overexpressing the transcriptional regulatory factors marA and rob in the mutants of step (1) to construct a geraniol-tolerant engineered strain that regulates the efflux system.

[0043] There is no particular limitation on the type of E. coli host in the present invention, as long as it can overexpress the transcriptional regulatory factors marA and rob. In a preferred embodiment, the E. coli host is E. coli Trans1T1.

[0044] In a preferred embodiment, 1) the regulatory factor marA is a DNA-binding and transcriptional dual regulatory factor MarA, with GeneID 947613, its amino acid sequence as shown in SEQ ID NO. 1, and its nucleotide sequence as shown in SEQ ID NO. 2; and / or 2) the regulatory factor rob is a DNA-binding and transcriptional dual regulatory factor Rob, with GeneID 948916, its amino acid sequence as shown in SEQ ID NO. 3, and its nucleotide sequence as shown in SEQ ID NO. 4.

[0045] In a preferred embodiment, the method of the present invention comprises the following steps:

[0046] Step 1: ARTP mutagenesis and strain screening:

[0047] (1) Select E. coli Trans1T1 (purchased from Beijing Quanshijin Biotech) was used as the base cell. After the cells were transferred and cultured to the logarithmic growth phase, the bacteria were collected by low-temperature centrifugation, resuspended and diluted with sterile deionized water to prepare the bacterial suspension, and the OD was controlled. 600 0.6-0.8;

[0048] (2) In a clean bench, a bacterial suspension was aspirated and dropped onto a slide dedicated for mutagenesis. The slide was then moved to a sterilized breeding instrument operating room for mutagenesis. Using 99.99% helium as the working gas, the mutagenesis time was 0.5-1 min at a power supply of 120 W and a working gas flow rate of 10 SLM (Standard Liter per Minute).

[0049] (3) Place the mutagenized slide in physiological saline and shake to mix to obtain a mixed bacterial solution;

[0050] (4) After diluting the mixed bacterial solution, spread an appropriate amount on a LB solid plate containing geraniol and culture overnight at 37°C. Select the well-growing colonies in the high-concentration area and inoculate them into the solid culture medium;

[0051] (5) The strain cells were picked from the solid culture medium and cultured and screened using a 96-well plate. The geraniol concentration in each well was 3 g / L.

[0052] (6) After the culture is completed, measure the OD 600 The cell growth performance was evaluated and the strain with the best growth performance was selected from the positive growth strains as the evolved strain tolerant to geraniol and named E. coli TS. This strain E. coli TS was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postal Code: 100101) on April 2, 2025, with the corresponding accession number CGMCC No. 34069 and the taxonomic name Escherichia coli.

[0053] Step 2: Exclusion engineering further improves the tolerance of the evolved strains:

[0054] (1) The transcriptional dual regulatory factors marA and rob were ligated to the expression plasmid pSB1C3 to obtain the recombinant plasmid pMR;

[0055] (2) The recombinant plasmid pMR obtained in step 1) is introduced into the Escherichia coli host by electroporation or chemical transformation. E. coli TS, and

[0056] (3) Screen positive clones (for example, on an LB plate containing chloramphenicol) to obtain the engineered Escherichia coli TS-pMR.

[0057] In one embodiment, the expression vector pSB1C3 comprises a J23100 constitutive promoter, a lambda-t0 terminator, and a CmR resistance marker.

[0058] Furthermore, the mutagenesis treatment in step 1 (2) is plasma irradiation, the mutagenesis time of Trans1T1 is 0.5-1 min, the power of the mutagenesis device is set to 120 W, and the gas flow rate is set to 10 SLM.

[0059] Furthermore, the Trans1T1 mixed bacterial solution in step 1 (4) was diluted and spread on an LB solid plate containing 3 g / L geraniol.

[0060] Furthermore, the culture conditions of the 96-well plate in step 1 (5) are 37° C. and 1000 rpm / min.

[0061] Furthermore, the maximum geraniol concentration that the tolerant evolved strain TS can tolerate in step 1 (6) is 10 g / L.

[0062] Furthermore, in step 2 (1), the regulatory factor marA has a GeneID of 947613, an amino acid sequence as shown in SEQ ID NO. 1, and a nucleotide sequence as shown in SEQ ID NO. 2; the regulatory factor rob has a GeneID of 948916, an amino acid sequence as shown in SEQ ID NO. 3, and a nucleotide sequence as shown in SEQ ID NO. 4.

[0063] Furthermore, in step 2 (3) E. coli The highest geraniol tolerance concentration of TS-pMR was 11 g / L.

[0064] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications may be made to the present invention without departing from the spirit or scope of the present invention. The following examples further illustrate the present invention and are not to be construed as limiting the scope of the present invention or the specific methods described herein.

[0065] Example

[0066] Example 1

[0067] Investigation on the tolerance of different original strains of Escherichia coli to geraniol

[0068] Escherichia coli DH5α, Trans1T1 (purchased from TransGen Biotech, Beijing), MG1655, TOP10 and BL21 (DE3) were inoculated into LB medium and cultured overnight at 37°C and 200 rpm in a shaking incubator. The next day, the inoculation was performed at 1% (v / v) and cultured until the logarithmic growth phase (OD 600 =0.8-1). Aseptically take 100 μL of bacterial solution into a 2 mL EP tube, add 900 μL of sterile saline, shake and mix to make a 1:10 dilution. Follow the same steps to dilute the bacterial solution until the concentration is 10 -4 Use plate titration method to take an appropriate amount of 10 diluted -1 to 10 -4 The bacterial suspension was spotted onto plates containing varying concentrations of geraniol (CAS 106-24-1, commercially available from Aladdin, catalog number G107517). After overnight incubation, bacterial growth was observed. The results showed that Trans1T1 had the best tolerance to geraniol, tolerating up to 1000 mg / L, with minimal growth at this concentration. Therefore, Trans1T1 was selected as the starting strain for ARTP mutagenesis breeding.

[0069] Example 2

[0070] ARTP mutagenesis improves Escherichia coli's tolerance to geraniol

[0071] (1) ARTP-induced mutagenesis of Escherichia coli Trans1T1

[0072] After activation of the starting bacteria Trans1T1 on the plate, a single colony was picked and inoculated into a 50 mL shake tube for overnight culture. Then, the inoculum was transferred to a 100 mL shake flask at a rate of 1% (v / v) and cultured at 37°C and 200 rpm until the logarithmic growth phase. The bacteria were collected by low-temperature centrifugation and the Trans1T1 bacterial suspension was prepared with sterile deionized water to control the OD 600 The concentration of the bacterial suspension was 0.6-0.8. In the clean bench, an appropriate amount of bacterial suspension was dripped onto a slide dedicated for mutagenesis. The slide was then moved to the sterilized breeding instrument operating room for mutagenesis. In order to find the optimal mutagenesis time, the strain was subjected to plasma mutagenesis with an irradiation time of 15-120 s. The mutagenized sample was then diluted and plated, and a lethality curve of the bottom plate bacteria was drawn. Figure 1 As shown in Figure 2, the optimal mutagenesis time for Trans1T1 is 0.5-1 min. The power of the mutagenesis apparatus was set to 120 W and the gas flow rate was set to 10 SLM.

[0073] Place the mutagenized slide in an EP tube containing 1 mL of saline and shake to remove the strain from the slide. Dilute the slide 10-, 100-, and 1000-fold. Spread the Trans1T1 mutagenized bacteria at varying concentrations onto LB plates containing 3 g / L geraniol. Incubate overnight at 37°C. Select colonies with good growth in the high-concentration zone and inoculate them into solid culture medium.

[0074] (2) Screening of evolved Escherichia coli strains

[0075] The cells of each strain induced by ARTP were picked from the solid culture medium and inoculated into a 96-well plate containing LB medium. The geraniol concentration in each well was 3 g / L. The culture was shaken at 37°C and 1000 rpm / min. After 16 hours, an appropriate amount of bacterial culture was taken out to measure the OD value. 600 , through OD 600 The cell growth performance after mutagenesis was evaluated, and a total of 48 Trans1T1 evolved strains that grew well at a concentration of 3 g / L geraniol were obtained. The geraniol concentration was gradually increased, and the fermentation steps were repeated. Finally, the evolved strain with the strongest tolerance and the most stable tolerance phenotype was screened out from the above 48 evolved bacteria and named E. coli TS. This strain E. coli TS was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, 100101, China) on April 2, 2025, with the corresponding accession number CGMCC No. 34069 and the taxonomic name Escherichia coli.

[0076] E. coli TS grew well at a concentration of 3 g / L geraniol and also showed positive growth at a concentration of 10 g / L geraniol.

[0077] Example 3

[0078] Tolerance and passage stability of evolved Escherichia coli strains

[0079] Evolved strains E. coli The TS tolerance verification process and results are as follows:

[0080] Evolved strains obtained by screening in solid plate rejuvenation example 2 E. coli TS and the starting strain Trans1T1 were inoculated from single colonies into 50 mL test tubes and cultured overnight at 37°C, 200 rpm to obtain seed solution. The seed solution was transferred to a 100 mL Erlenmeyer flask containing 20 mL of LB medium at a 1% (v / v) inoculum, and geraniol was added at varying concentrations. The tubes were incubated at 37°C, 200 rpm, and a tolerance curve was plotted ( Figure 2 The experimental results showed that the starting strain trans1T1 had no positive growth in the liquid medium containing 1g / L geraniol. E. coli TS can grow at a maximum concentration of 10 g / L geraniol. Compared with the starting strain, E. coli TS has a significant tolerance to geraniol.

[0081] Evolved strains E. coli The genetic stability verification process and results of TS are as follows:

[0082] Evolved strains obtained by screening in solid plate rejuvenation example 2 E. coli TS and the starting strain Trans1T1 were inoculated from a single colony into a 50 mL shake tube and cultured overnight at 37°C, 200 rpm to obtain seed solution. The seed solution was transferred to a 100 mL Erlenmeyer flask containing 20 mL of LB medium at a 1% (v / v) inoculum and cultured in a shaker at 37°C, 200 rpm. Growth curves were plotted. Figure 3 As shown, E. coli The logarithmic growth phase of TS is 2-12 hours, similar to that of the control strain, and the growth condition is good. The bacterial solution at the 12th hour was transferred to a shake flask containing fresh LB medium at a 1% (V / V) inoculation rate and incubated at 37°C, 200 rpm for 12 hours. A portion of the bacterial solution was removed for strain preservation, and a portion was continued to be transferred and cultured, and so on, until the 20th generation. Table 1 shows the OD values ​​of the first and 20th generation strains. 600 The comparison results showed that the evolved Escherichia coli strain obtained by ARTP mutagenesis combined with passage domestication had good genetic stability.

[0083]

[0084] Example 4

[0085] Construction of efflux system engineered strains

[0086] The gene sequences of marA (GeneID: 947613) and rob (GeneID: 948916) were retrieved from NCBI. The corresponding primers were designed to amplify the target genes and the vector pSB1C3 (commercially available from iGEM) (see Table 2 for the specific nucleotide sequences). The amplified target fragment and vector were recovered using a gel recovery kit. The recovered and purified target fragment with homologous ends and vector were added to the same tube of Master Mix. The mixture was pipetted 5-10 times, and the reaction solution was then placed in a 50°C metal bath for 5-15 minutes to obtain the recombinant plasmid pMR (i.e., pSB1C3-marA-rob, Figure 4 ).

[0087] The recombinant plasmid was introduced by electroporation E. coli TS competent cells were used to obtain engineered Escherichia coli TS-pMR with further improved geraniol tolerance.

[0088] The specific nucleotide sequences of the primers used in the construction of the engineered strains are shown in Table 2.

[0089]

[0090] Example 5

[0091] Growth of engineered Escherichia coli with enhanced geraniol tolerance under stress

[0092] Example 4: Engineered Escherichia coli obtained E. coli TS-pMR was inoculated into a 50 mL shaking tube and cultured overnight at 37°C and 200 rpm to obtain seed solution. The seed solution was transferred to a 100 mL conical flask containing 20 mL LB medium at a 1% (V / V) inoculation volume and cultured at 37°C and 200 rpm until the logarithmic growth phase (OD 600 =0.8~1.0), aseptically take 100 uL of bacterial solution into a 2 mL EP tube, add 900 uL of sterile saline, shake and mix to make a 1:10 dilution. Follow the same steps to dilute the bacterial solution until the concentration is 10 -4 Use plate titration method to take the diluted 10 -1 , 10 -2 , 10 -3 , 10 -4 The researchers spotted a 100-fold increase in bacterial culture onto plates containing varying geraniol concentrations. After 12 hours of incubation, they found that the engineered E. coli overexpressing the marA+roB genes had larger, more complete, and rounder colonies than the control strain, indicating that the marA and roB genes can effectively enhance geraniol tolerance in E. coli by regulating the efflux system. The results showed that through efflux system regulation engineering, overexpressing the transcription factors marA and rob in TS, the engineered strain TS-pMR was constructed, which tolerated geraniol at a concentration of 11 g / L, an 11-fold increase compared to the original strain, exceeding the toxicity tolerance threshold for microbial biosynthesis.

[0093] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0094] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0095] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for improving the geraniol tolerance of Escherichia coli, comprising the following steps: The transcriptional regulatory factors marA and rob were overexpressed in a geraniol-tolerant Escherichia coli mutant strain with a deposit number of CGMCC No. 34069 to construct a geraniol-tolerant engineered strain with a regulated efflux system.

2. The method according to claim 1, wherein: The amino acid sequence of the transcriptional regulatory factor marA is shown in SEQ ID NO. 1, and / or the encoding nucleotide sequence of the transcriptional regulatory factor marA is shown in SEQ ID NO. 2; and / or The amino acid sequence of the transcriptional regulatory factor rob is shown in SEQ ID NO. 3, and / or the encoding nucleotide sequence of the transcriptional regulatory factor rob is shown in SEQ ID NO.

4.

3. The method according to claim 1, wherein the steps include: (1) constructing a recombinant plasmid that overexpresses the transcriptional regulatory factor marA and the transcriptional regulatory factor rob; and (2) The recombinant plasmid obtained in (1) was introduced into a geraniol-tolerant Escherichia coli mutant strain with a deposit number of CGMCC No. 34069 to obtain an engineered strain with further improved geraniol tolerance.

4. A geraniol-tolerant engineered strain obtained by the method according to any one of claims 1 to 3.

5. Use of the geraniol-tolerant engineered strain according to claim 4 in producing geraniol.

Images