Method for improving geraniol tolerance of escherichia coli and application thereof

Abstract

The invention belongs to the field of bioengineering, and provides a method based on a mutagenesis and efflux engineering two-stage strengthening strategy so as to improve the geraniol tolerance of escherichia coli. Specifically, the method comprises the following steps: mutagenizing escherichia coli by using a normal-pressure room-temperature plasma (ARTP) mutagenesis technology, and screening to obtain an evolutionary strain TS with improved geraniol tolerance; furthermore, through excretion system regulation and control engineering, transcription factors marA and rob are over-expressed in TS, an engineering strain TS-pMR is constructed, the geraniol tolerance concentration of the engineering strain reaches 11 g / L and is 11 times higher than that of an original strain, and the product toxicity tolerance threshold value in microbial synthesis is broken through. The staged strengthening strategy is adopted, complex domestication steps are avoided, the construction period of the strain is greatly shortened, meanwhile, the genetic stability of the strain is improved, and the finally obtained geraniol-tolerant escherichia coli is more excellent in tolerance compared with an initial strain.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering. More specifically, the present invention relates to a method for improving the tolerance of Escherichia coli to geraniol and its uses. Background Art

[0002] Geraniol is a monoterpenoid alcohol compound extracted from aromatic plants, with a rich rose aroma, and is widely used in the fields of spices, essential oils, and fragrance cosmetics. In addition, geraniol also has rich pharmacological activities, including anti-cancer, anti-inflammatory, antioxidant, and antibacterial properties, and is therefore regarded as a potential candidate drug. 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 and is difficult to meet the growing market demand.

[0003] In contrast, microbial synthesis uses low-value substrates as raw materials to produce geraniol in a green and sustainable manner, becoming an important direction to replace traditional plant extraction. With the development of metabolic engineering and synthetic biology technologies, the microbial synthesis pathway of geraniol has been elucidated. This biosynthetic process follows the typical terpene metabolism pathway: using isopentenyl pyrophosphate (IPP) generated by the mevalonate (MVA) pathway or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway as a precursor, and catalyzed by isopentenyl pyrophosphate isomerase (IDI) to form dimethylallyl pyrophosphate (DMAPP). Subsequently, geranyl diphosphate synthase converts DMAPP into geranyl pyrophosphate (GPP), and finally through hydrolysis by geraniol synthase (GES), the target product geraniol is obtained. Such a synthesis pathway not only increases the yield of geraniol, but also lays a foundation for its wider use in drug development and industrial applications.

[0004] Although traditional metabolic engineering strategies (such as pathway optimization and enzyme molecular modification) have made significant progress in enhancing geraniol synthesis yield, their industrial application still faces a fundamental technical bottleneck - the inherent strong cytotoxicity of geraniol causes irreversible damage to the microbial fermentation system. In fact, the dual metabolic stress of cell growth inhibition and product toxicity superposition leads to common problems of a sharp drop in survival rate and limited product synthesis in the fermentation culture of conventional strains. However, existing tolerance improvement technologies all have significant defects: Although ARTP mutagenesis can generate a wide range of mutations, its random mutagenesis characteristics result in a positive mutant screening efficiency of less than 0.2%, and a large-scale screening platform is required; Although the adaptive laboratory evolution (ALE) strategy can screen for tolerance phenotypes directionally, the domestication cycle is long and there is a risk of phenotype degeneration; Although overexpression of efflux proteins can improve the tolerance of Escherichia coli to specific products, it does not fundamentally solve the problem of product toxicity to cells. It is worth noting that current research is mostly limited to the improvement of a single technical level, and a multi-level synergistic mechanism has not been established. Therefore, breaking the growth inhibition caused by geraniol accumulation and solving the problem of poor geraniol tolerance of Escherichia coli have become the key to enhancing compound biosynthesis. Summary of the Invention

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

[0006] First, the present invention uses the Atmospheric and Room Temperature Plasma (ARTP) mutagenesis technology to mutagenize Escherichia coli Trans1T1, and screens an evolved strain TS with geraniol tolerance increased to 10 g / L, and the preservation number is: CGMCC No. 34069. Further, through the 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 hierarchical strengthening strategy, avoiding complex domestication steps, greatly shortening the strain construction cycle, and at the same time improving the genetic stability of the strain. Finally, the obtained Escherichia coli tolerant to geraniol is superior to the starting strain in terms of tolerance ability.

[0007] Therefore, on the one hand, the present invention provides an Escherichia coli strain with improved geraniol tolerance, and its preservation number 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. Mutagenizing wild-type Escherichia coli by atmospheric and room temperature plasma mutagenesis, and screening to obtain a geraniol-tolerant mutant strain; and Step B. Overexpressing the transcriptional regulators marA and rob in the mutant strain obtained in Step A to construct a geraniol-tolerant engineering strain that regulates the efflux system.

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

[0010] In a specific embodiment, the amino acid sequence of the transcriptional regulator marA is as shown in SEQ ID NO. 1, and / or the coding nucleotide sequence of the transcriptional regulator marA is as shown in SEQ ID NO. 2; and / or the amino acid sequence of the transcriptional regulator rob is as shown in SEQ ID NO. 3, and / or the coding nucleotide sequence of the transcriptional regulator rob is as shown in SEQ ID NO. 4.

[0011] In a specific embodiment, Step A includes: (1) Centrifuging to collect wild-type Escherichia coli cultured to the logarithmic growth phase, resuspending and preparing a bacterial suspension, and controlling OD 600 to be 0.6 to 0.8; (2) Suspending the bacterial suspension in drops on a slide, moving the slide to the operation room of an atmospheric and room temperature plasma breeding instrument for mutagenesis treatment; (3) Using helium as the working gas for mutagenesis; (4) Placing the mutagenized slide into physiological saline and shaking well to obtain a mixed bacterial solution; (5) Diluting the mixed bacterial solution and spreading it on a solid plate containing geraniol, culturing overnight, and picking colonies that grow well in the high-concentration area and inoculating them into a solid medium; (6) Picking strain cells from the solid medium for culture and screening; and (7) After the culture is completed, evaluating the cell growth performance by measuring OD 600 and screening evolution strains with good growth performance from the forward-growing strains.

[0012] In a specific embodiment, Step B includes: (1) Constructing a recombinant plasmid overexpressing both the transcriptional regulators marA and rob; and (2) Introducing the recombinant plasmid obtained in (1) into the mutant strain obtained in Step A to obtain an engineering strain with further improved geraniol tolerance.

[0013] In a specific embodiment, the geraniol-tolerant mutant strain obtained in Step A has a deposit number of CGMCC No. 34069, and / or the highest geraniol concentration it can tolerate is 10 g / L.

[0014] In a specific embodiment, the highest 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 also provides the application of the above-preserved Escherichia coli strain or the geraniol-tolerant engineered strain of the present invention in the production of geraniol. Description of the Drawings

[0017] From the following detailed description in conjunction with the drawings, the above features and advantages of the present invention will become more apparent, wherein: Figure 1 is the ARTP mutagenesis lethality rate of Escherichia coli Trans1T1; Figure 2 is the tolerance curve of Escherichia coli evolved strain TS and wild-type strain Trans1T1; Figure 3 is the growth curve of Escherichia coli evolved strain TS and wild-type strain Trans1T1; Figure 4 is the plasmid map of pMR overexpressing marA + rob genes.

[0018] Description of the Sequence Listing SEQ ID NO. 1 - Amino acid sequence of marA SEQ ID NO. 2 - Coding nucleotide sequence of marA SEQ ID NO. 3 - Amino acid sequence of rob SEQ ID NO. 4 - Coding nucleotide sequence of rob SEQ ID NO. 5 - Primer marA-F SEQ ID NO. 6 - Primer marA-R SEQ ID NO. 7 - Primer rob-F SEQ ID NO. 8 - Primer rob-R SEQ ID NO. 9 - Primer pSB1C3-F SEQ ID NO. 10 - Primer pSB1C3-R Detailed Embodiments

[0019] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the examples are all conventional methods.

[0021] Unless otherwise specified, the materials, reagents, instruments and methods used in the embodiments of the present invention are all conventional materials, reagents, instruments and methods in the art, and can be obtained or implemented through commercial channels.

[0022] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For the definitions and terms in this field, it is particularly recommended that those skilled in the art refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, 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).

[0023] As used herein, "comprising" or "including" is an open-ended description that includes all specified components or steps described, as well as other specified components or steps that do not materially affect; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still has the activity described in the present invention.

[0024] As used herein, "and / or" includes all combinations of the items connected by this term, and each combination should be regarded as having been listed separately herein. For example, "A and / or B" includes "A", "A and B", and "B". 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".

[0025] As used herein, the term "overexpression" means that when the strict control of gene / protein expression (transcription) is disrupted, the gene may not be "turned off" or may be transcribed at a high rate. High-speed transcription results in the production of a large amount of mRNA. For the overexpression of the transcriptional regulators marA and rob of the present invention, it means that the DNA or RNA or protein expression level is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or 300% higher in the engineered Escherichia coli of the present invention than in the control (without overexpressing the transcriptional regulators marA and rob according to the present invention), or even 4, 5, 6, 7, 8, 9, 10 times or more the corresponding DNA or RNA or protein expression level in the control. Techniques and reagents for detecting gene / protein expression levels are well known to those skilled in the art.

[0026] As used herein, the term "efflux system" refers to a class of proteins present on the bacterial cell membrane through which bacteria excrete toxic substances (including antibacterial drugs, metabolites, etc.) to regulate the stability of their internal environment and protect the bacteria themselves from damage.

[0027] An object of the present invention is to provide a method for improving the tolerance of Escherichia coli to geraniol.

[0028] In a preferred embodiment, the method comprises the following steps: (1) treating wild-type Escherichia coli with ARTP mutagenesis and screening to obtain a geraniol-tolerant mutant strain; (2) overexpressing the transcriptional regulators marA and rob in the mutant strain obtained in step (1) to construct a geraniol-tolerant engineering strain that regulates the efflux system.

[0029] There is no particular limitation on the type of Escherichia coli host in the present invention as long as it can overexpress the transcriptional regulators marA and rob. In a preferred embodiment, the Escherichia coli host is E. coli Trans1T1.

[0030] In a preferred embodiment, 1) the regulatory factor marA is the DNA-binding transcriptional dual regulator MarA, with GeneID 947613, whose amino acid sequence is as shown in SEQ ID NO. 1 and whose nucleotide sequence is as shown in SEQ ID NO. 2; and / or 2) the regulatory factor rob is the DNA-binding transcriptional dual regulator Rob, with GeneID 948916, whose amino acid sequence is as shown in SEQ ID NO. 3 and whose nucleotide sequence is as shown in SEQ ID NO. 4.

[0031] In a preferred embodiment, the method of the present invention comprises the following steps: Step 1, ARTP mutagenesis and strain screening: (1) Select E.coli Trans1T1 (purchased from TransGen Biotech, Beijing, China) as the chassis cell. After transferring and culturing the cells to the logarithmic growth phase, collect the bacterial cells by low-temperature centrifugation, resuspend, dilute with sterile deionized water, and prepare a bacterial suspension, controlling the OD 600 to be 0.6 - 0.8; (2) In a laminar flow hood, pipette the bacterial suspension onto a special slide for mutagenesis, and transfer the slide to the sterilized breeding instrument operation room for mutagenesis treatment. Using 99.99% helium as the working gas, at a power of 120 W and a working gas flow rate of 10 SLM (Standard Liter per Minute), the mutagenesis time is 0.5 - 1 min; (3) Put the mutagenized slide into physiological saline and shake well to obtain a mixed bacterial solution; (4) After diluting the mixed bacterial solution, take an appropriate amount and spread it on an LB solid plate containing geraniol, and culture it overnight at 37 °C. Select the colonies growing well in the high-concentration area and inoculate them into the solid medium; (5) Pick the strain cells from the solid medium and culture and screen them with a 96-well plate. The geraniol concentration in a single well is 3 g / L; (6) After the culture is completed, evaluate the cell growth performance by measuring the OD 600 , and screen the best-growing strain from the forward-growing strains as the evolved strain tolerant to geraniol, named E.coli TS. This strain E.coli TS was preserved in the China General Microbiological Culture Collection Center (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Zip Code: 100101) on April 2, 2025, and its corresponding preservation number is CGMCC No. 34069, and the taxonomic name is Escherichia coli.

[0032] Step 2, efflux engineering to further improve the tolerance of the evolved strain: (1) Connect the transcriptional dual regulators marA and rob to the expression plasmid pSB1C3 to obtain the recombinant plasmid pMR; (2) Introduce the recombinant plasmid pMR obtained in step (1) into the Escherichia coli host E.coli TS by electroporation or chemical transformation, and (3) Screen for positive clones (for example, on an LB plate containing chloramphenicol) to obtain the engineered Escherichia coli TS-pMR.

[0033] In a specific embodiment, the expression vector pSB1C3 contains the J23100 constitutive promoter, lambdat0 terminator, and CmR resistance marker.

[0034] Further, the mutagenesis treatment in step (2) of step one is plasma irradiation. The mutagenesis time for 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.

[0035] Further, in step (4) of step one, the diluted Trans1T1 mixed bacterial solution is spread on an LB solid plate containing 3 g / L geraniol.

[0036] Further, the culture conditions for the 96 - well plate in step (5) of step one are 37 °C and 1000 rpm / min.

[0037] Further, the highest geraniol concentration that the evolved strain TS can tolerate in step (6) of step one is 10 g / L.

[0038] Further, in step (1) of step two, the regulatory factor marA, with GeneID 947613, has 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, with GeneID 948916, has an amino acid sequence as shown in SEQ ID NO. 3 and a nucleotide sequence as shown in SEQ ID NO. 4.

[0039] Further, in step (3) of step two E.coli The highest geraniol tolerance concentration of TS - pMR is 11 g / L.

[0040] All patents and publications mentioned in this application are incorporated into the present invention by reference in their entirety. Those skilled in the art should recognize that certain changes can be made to the present invention without departing from the concept or scope of the present invention. The following examples further illustrate the present invention in detail and should not be considered as limiting the present invention or the scope of the specific methods described in the present invention.

[0041] Example Example 1 Investigation of the tolerance of different Escherichia coli original strains to geraniol

[0042] Escherichia coli DH5α, Trans1T1 (purchased from TransGen Biotech, Beijing), MG1655, TOP10, and BL21(DE3) were inoculated into LB medium and cultured overnight in a shaker at 37 °C and 200 rpm. The next day, they were transferred at 1% (v / v) and cultured until the logarithmic growth phase (OD600 =0.8-1). 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 the plate titration method to take an appropriate amount of 10 diluted -1 Up to 10 -4 The bacterial solution was dripped onto plates containing different concentrations of geraniol (CAS 106-24-1, purchased from Aladdin, catalog number G107517), and the colony growth was observed after overnight culture. The results showed that Trans1T1 had the best tolerance to geraniol, and could tolerate up to 1000 mg / L, that is, it had weak growth at this concentration. Therefore, Trans1T1 was selected as the starting base strain for ARTP mutagenesis breeding. Example 2 ARTP mutagenesis improves the tolerance of Escherichia coli to geraniol (1) ARTP-induced mutagenesis of Escherichia coli Trans1T1

[0043] After the starting bacteria Trans1T1 was activated on the plate, a single colony was picked and inoculated into a 50 mL shaking tube for overnight culture. Then, the inoculation volume was 1% (v / v) and transferred to a 100 mL shaking flask. The cells were cultured at 37 °C and 200 rpm until the logarithmic growth phase. The cells were collected by low-temperature centrifugation and the Trans1T1 bacterial suspension was prepared with sterile deionized water. The OD 600 0.6-0.8. In the clean bench, take a proper amount of bacterial suspension and drop it on the slide for mutagenesis. Move the slide to the sterilized breeding instrument operation room for mutagenesis. In order to find the best mutagenesis time, the strain was irradiated with plasma for 15-120 s. Then the induced sample was diluted and plated, and the lethality curve of the bottom plate bacteria was drawn. Figure 1 As shown in the figure, the optimal mutagenesis time for Trans1T1 is 0.5-1 min. The power of the mutagenesis device was set to 120 W and the gas flow rate was set to 10 SLM.

[0044] The mutagenized slide was placed in an EP tube containing 1 mL of physiological saline, shaken to make the strain fall off the slide, and then diluted 10, 100, and 1000 times. The gradient concentrations of Trans1T1 mutagenized bacteria were spread on LB solid plates containing 3 g / L geraniol, cultured overnight at 37 °C, and the colonies with good growth in the high concentration area were selected and inoculated into the solid culture medium.

[0045] (2) Screening of Escherichia coli Evolved Strains Pick up the cells of each strain mutagenized by ARTP from the solid medium and inoculate them into a 96-well plate containing LB medium. The geraniol concentration in a single well is 3 g / L; culture in a shaker at 37 °C and 1000 rpm / min. After 16 h, take an appropriate amount of the bacterial liquid to measure OD 600 , and evaluate the growth performance of the cells after mutagenesis through OD 600 . A total of 48 Trans1T1 evolved strains that grew well at a geraniol concentration of 3 g / L were obtained; gradually increase the geraniol concentration and repeat the fermentation steps. Finally, select the evolved strain with the strongest tolerance and stable tolerance phenotype from the above 48 evolved bacteria, and name it E.coli TS. This strain E.coli TS was preserved in the China General Microbiological Culture Collection Center (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Postcode: 100101) on April 2, 2025, and its corresponding preservation number is CGMCC No. 34069, and the taxonomic name is Escherichia coli.

[0046] E.coli TS grew well at a geraniol concentration of 3 g / L and also had positive growth at a geraniol concentration of 10 g / L. Example 3 Tolerance and passage stability of Escherichia coli evolved strains

[0047] Evolved strain E.coli The tolerance verification process and results of TS are as follows: Pick up the evolved strain E.coli TS and the starting strain Trans1T1 screened through solid plate rejuvenation in Example 2. Pick a single colony and inoculate it into a 50 mL test tube, and culture it overnight at 37 °C and 200 rpm to obtain a seed solution. The seed solution was transferred to a 100 mL conical flask containing 20 mL of LB medium at an inoculation amount of 1% (V / V), and gradient concentrations of geraniol were added respectively. Place it in a shaker at 37 °C and 200 rpm for culture, and draw a tolerance curve ( Figure 2 ). The experimental results show that the starting strain trans1T1 did not have positive growth in the liquid medium containing 1 g / L geraniol, E.coli TS can grow at a geraniol concentration of up to 10 g / L. Compared with the starting strain, E.coli TS has significant geraniol tolerance.

[0048] Evolved strain E.coli The genetic stability verification process and results of TS are as follows: Pick up the evolved strain screened through solid plate rejuvenation in Example 2E.coli For TS and the starting strain Trans1T1, single colonies were picked and inoculated into 50 mL shaking flasks, and cultured overnight at 37 °C with 200 rpm to obtain seed cultures. The seed cultures were transferred to 100 mL conical flasks containing 20 mL of LB medium at an inoculation amount of 1% (V / V), and cultured on a shaker at 37 °C with 200 rpm, and the growth curves were plotted. As Figure 3 shown, E.coli the logarithmic growth phase of TS was 2 - 12 h, similar to that of the control strain, and the growth condition was good. The bacterial liquid at the 12th h was transferred to a shaking flask containing fresh LB medium at an inoculation amount of 1% (V / V), and cultured on a shaker at 37 °C with 200 rpm for 12 h. Part of the bacterial liquid was taken for strain preservation, and part was continuously transferred for culture, and so on, until the 20th generation. Table 1 shows the OD 600 comparison results of the first-generation strain and the 20th-generation strain, indicating that the Escherichia coli evolved strain obtained by ARTP mutagenesis combined with subculture domestication has good genetic stability.

[0049]

[0050] Example 4 Construction of engineered strains of efflux systems

[0051] The gene sequences of marA (GeneID: 947613) and rob (GeneID: 948916) were retrieved from NCBI, and corresponding primers were designed for the amplification of the target genes and the vector pSB1C3 (commercial source: iGEM) (the specific nucleotide sequences are shown in Table 2). The amplified target fragments and vectors were recovered using a gel extraction kit. The recovered and purified target fragments and vectors with homologous ends were added to the same tube of Master Mix, and pipetted 5 - 10 times, and then the reaction solution was placed in a 50 °C metal bath for reaction for 5 - 15 min to obtain the recombinant plasmid pMR (i.e., pSB1C3 - marA - rob, Figure 4 ).

[0052] The recombinant plasmid was introduced into E.coli TS competent cells by electroporation to obtain engineered Escherichia coli TS - pMR with further improved geraniol tolerance.

[0053] The specific nucleotide sequences of the primers used in the construction process of the engineered strains are shown in Table 2.

[0054]

[0055] Example 5 Growth of engineered Escherichia coli with improved geraniol tolerance under stress

[0056] The engineered Escherichia coli obtained in Example 4 E.coli TS-pMR was inoculated into a 50 mL shaking flask and cultured overnight at 37 °C and 200 rpm to obtain a seed solution. The seed solution was transferred to a 100 mL conical flask containing 20 mL of LB medium at an inoculation amount of 1% (V / V) and cultured at 37 °C and 200 rpm until the logarithmic growth phase (OD 600 = 0.8 - 1.0). 100 μL of the bacterial solution was taken aseptically into a 2 mL EP tube, and 900 μL of sterile normal saline was added and mixed well by shaking to prepare a 1:10 dilution. The bacterial solution was diluted according to the same operation steps until the concentration was 10 -4 times. Using the plate titration method, the diluted 10 -1 , 10 -2 , 10 -3 , 10 -4 times of the bacterial solution was dropped onto plates containing different geraniol concentrations. After 12 h of culture, it was found that the engineered Escherichia coli overexpressing the marA+roB gene had larger, more complete and rounder colonies compared with the control strain, indicating that the genes marA and roB can effectively enhance the geraniol tolerance of Escherichia coli by regulating the efflux system. The results showed that through the regulation of the efflux system engineering, the transcription factors marA and rob were overexpressed in TS, and the constructed engineered strain TS-pMR had a geraniol tolerance concentration of 11 g / L, which was 11 times higher than that of the original strain, breaking through the product toxicity tolerance threshold in microbial synthesis.

[0057] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0058] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable way. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0059] In addition, any combination can be made between different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for improving the geraniol tolerance of Escherichia coli, comprising the following steps: Step A. Mutagenize wild-type Escherichia coli with atmospheric and room temperature plasma, and screen to obtain geraniol-tolerant mutants; and Step B. Overexpress the transcriptional regulator marA and the transcriptional regulator rob in the mutants obtained in Step A to construct a geraniol-tolerant engineering strain with a regulated efflux system.

2. The method according to claim 1, wherein the wild-type Escherichia coli is Escherichia coli Trans1T1.

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

4.

4. The method according to claim 1 or 2, wherein Step A includes: (1) Centrifuge and collect the wild-type Escherichia coli cultured to the logarithmic growth phase, resuspend and prepare a bacterial suspension, and control the OD 600 to be 0.6 to 0.8; (2) Drop the bacterial suspension on a slide, and move the slide to the operation room of an atmospheric and room temperature plasma breeding instrument for mutagenesis treatment; (3) Use helium as the working gas for mutagenesis treatment; (4) Put the mutagenized slide into physiological saline and shake well to obtain a mixed bacterial solution; (5) Dilute the mixed bacterial solution and spread it on a solid plate containing geraniol, incubate overnight, and pick well-growing colonies and inoculate them into a solid medium; (6) Pick strain cells from the solid medium for culture and screening; and (7) After the cultivation is completed, measure OD 600 to evaluate the cell growth performance and screen out the geraniol-tolerant mutant strain.

5. The method according to claim 1 or 2, wherein Step B includes: (1) Construct a recombinant plasmid overexpressing the transcriptional regulator marA and the transcriptional regulator rob; and (2) Introduce the recombinant plasmid obtained in (1) into the mutants obtained in Step A to obtain an engineering strain with further improved geraniol tolerance.

6. The method according to claim 1 or 2, wherein: The geraniol-tolerant mutant obtained in Step A has a deposit number of CGMCC No. 34069, and / or the highest geraniol concentration it can tolerate is 10 g / L.

7. The method according to claim 1 or 2, wherein: The highest geraniol concentration that the geraniol-tolerant engineering strain obtained in Step B can tolerate is 11 g / L.

8. A geraniol-tolerant engineering strain obtained by the method according to any one of claims 1 to 7.

9. The geraniol-tolerant engineering strain according to claim 8, wherein the geraniol-tolerant mutant obtained in Step A in the method according to any one of claims 1 to 7 has a deposit number of CGMCC No. 34069.

10. Use of the geraniol-tolerant engineering strain according to claim 8 or 9 in the production of geraniol.

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