An engineered Escherichia coli tolerant to monoterpenoids, and its construction method and application

Abstract

The present invention discloses an engineered Escherichia coli strain with monoterpene tolerance, and a construction method and application thereof. The present invention for the first time reveals the function of gene elaD in improving the tolerance of Escherichia coli to monoterpenes. On this basis, an engineered strain with overexpressed gene was constructed by combining metabolic engineering strategies. The engineered strain achieves broad-spectrum tolerance to monoterpenes such as geraniol and limonene (the tolerance concentration is increased by 4-6 times), and significantly improves the production of geraniol (the highest increase is 115.3%). The present invention can solve the problem of poor tolerance of current Escherichia coli to monoterpenes, facilitate the construction of robust engineered Escherichia coli strains, provide an innovative solution with both robustness and high productivity for the efficient microbial synthesis of monoterpenes, and has important industrial application value.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering. More specifically, the present invention relates to an engineered Escherichia coli resistant to monoterpenoids, a method for constructing the same, and applications thereof. Background Art

[0002] Monoterpenoids are terpene substances composed of two isoprene units. As the members of the terpene family with the smallest molecular weight, they are widely present in plant secondary metabolites. Such compounds (such as myrcene, α-terpineol, nerol, geraniol, etc.) not only have significant aromatic odor characteristics, but also show important application values in the fields of food additives, spice manufacturing, and daily chemical products. Recent studies have further revealed that monoterpenoids have multiple pharmacological activities, including immunomodulation, antibacterial and anti-inflammatory, analgesic, hypoglycemic and lipid-lowering, and anti-tumor effects, and can be applied to the fields of preventive medicine and clinical treatment.

[0003] The breakthrough of synthetic biology technology provides a new path for the heterologous synthesis of plant natural products by microorganisms. Currently, model microorganisms such as Escherichia coli and Saccharomyces cerevisiae have been successfully transformed into biosynthesis chassis for monoterpenoids. However, the inherent cytotoxicity of such compounds severely restricts their industrial production process: when the intracellular and extracellular monoterpenoid concentrations reach the threshold, cell proliferation can be inhibited. For example, 0.03% limonene or 0.05% geraniol can completely inhibit the growth of Escherichia coli, and 0.6 mM limonene can completely inhibit the growth of Saccharomyces cerevisiae. In addition, the irreversible damage to the membrane system caused by product toxicity will further lead to a sharp decline in product yield and production intensity during the fermentation process.

[0004] To address the problem of product inhibition, currently, mainly two strategies of in-situ extraction and adaptive evolution are adopted, but they have significant limitations: the former relies on exogenous extractants (such as resins, organic solvents) to construct a two-phase fermentation system, which can reduce the extracellular product concentration, but faces problems such as high costs, significant scale-up effects, and low product recovery rates; the latter screens for tolerant strains through long-term stress, but the evolved phenotypes have high decay rates and narrow substrate specificities, and random mutations may lead to the inactivation of key genes in the metabolic pathway. Summary of the Invention

[0005] To solve the problem of the lack of acid-resistant elements in the prior art, the present invention proposes to construct an engineered strain with a stable tolerance phenotype by systematically mining endogenous stress-resistant elements in Escherichia coli. This strategy can not only avoid the metabolic interference caused by the introduction of exogenous genes and fundamentally solve the cytotoxicity problem, but also obtain broad-spectrum resistance gene elements, providing an innovative solution for breaking through the toxicity bottleneck of monoterpenoids and realizing the efficient synthesis of compounds.

[0006] Therefore, on the one hand, the present invention provides an engineered Escherichia coli with tolerance to monoterpenoids, and the engineered Escherichia coli overexpresses a deubiquitinating protease geneelaD 。

[0007] In a specific embodiment in this regard, the deubiquitinating protease gene elaD has the amino acid sequence shown in SEQ ID NO. 1.

[0008] In a specific embodiment in this regard, the deubiquitinating protease gene elaD (GeneID: 946742) has the nucleotide sequence shown in SEQ ID NO. 2.

[0009] In a specific embodiment in this regard, the Escherichia coli is Escherichia coli BL21(DE3), BL21, BL21 Star(DE3), BL21(DE3)pLysS, DH5a, JM109, Top10, Rosetta(DE3) or Origami(DE3).

[0010] In a specific embodiment in this regard, the monoterpenoid compounds are selected from: geraniol, nerol, linalool, myrcene, limonene, α-terpineol, menthol, (-)-carvone and (-)-borneol.

[0011] Another aspect of the present invention also provides a method for constructing the engineered Escherichia coli according to the present invention, and the method includes the following steps:

[0012] 1) Connect the deubiquitinating protease gene elaD to an expression plasmid (such as pET28a-tac with the sequence shown in SEQ ID NO. 3) to obtain a recombinant plasmid (such as pET28a-tac-elaD);

[0013] 2) Introduce the recombinant plasmid obtained in step 1) into an Escherichia coli host by electrotransformation or chemical transformation (such as E. coli BL21(DE3)); and

[0014] 3) Screen for positive clones (such as on an LB plate containing kanamycin) to obtain the engineered Escherichia coli.

[0015] In a specific embodiment, the expression vector pET28a-tac contains a Ptac inducible promoter, a T7 terminator and a KanR resistance marker.

[0016] Another aspect of the present invention also provides the use of the engineered Escherichia coli according to the present invention in the production of monoterpenoid compounds.

[0017] In a specific embodiment, the monoterpenoid compound is selected from the group consisting of: geraniol, nerol, linalool, myrcene, limonene, α-terpineol, menthol, L-carvone and (-)-borneol.

[0018] Another aspect of the present invention provides a method for improving the monoterpenoid tolerance of Escherichia coli, the method comprising overexpressing a deubiquitinating protease gene in the Escherichia coli. elaD (GeneID: 946742).

[0019] In a specific embodiment of this aspect, the deubiquitinating protease gene elaD The amino acid sequence is shown in SEQ ID NO. 1.

[0020] In a specific embodiment of this aspect, the deubiquitinating protease gene elaD The nucleotide sequence of (GeneID:946742) is shown in SEQ ID NO. 2.

[0021] In a specific embodiment of this aspect, the Escherichia coli is Escherichia coli BL21(DE3), BL21, BL21 Star(DE3), BL21(DE3)pLysS, DH5a, JM109, Top10, Rosetta(DE3) or Origami(DE3).

[0022] In a specific embodiment of this aspect, the monoterpenoid compound is selected from the group consisting of: geraniol, nerol, linalool, myrcene, limonene, α-terpineol, menthol, L-carvone and (-)-borneol.

[0023] In one embodiment of this aspect, the overexpression is carried out by expressing the deubiquitinating protease gene on a plasmid. elaD To achieve it. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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:

[0025] Figure 1 The OD values of the engineered strain BL21-elaD and the starting strain BL21-pET28a under different monoterpenoid stresses are shown in Table 1. 600 value;

[0026] Figure 2 It is a process in which Escherichia coli produces geraniol using the mevalonic acid (MVA) pathway or the 2-methyl-D-erythritol-4-phosphate (MEP) pathway; and

[0027] Figure 3It is a comparison chart of geraniol production. SG1 is a control strain for producing geraniol using the MVA+MEP pathway, and SG2 is a strain that overexpresses the gene elaD on the basis of SG1.

[0028] Sequence Listing Description.

[0029] SEQ ID NO. 1 - Amino acid sequence of the deubiquitinating protease gene elaD

[0030] SEQ ID NO. 2 - Nucleotide sequence of the deubiquitinating protease gene elaD

[0031] SEQ ID NO. 3 - Full sequence of pET28a-tac

[0032] SEQ ID NO. 4 - Nucleotide sequence of the pSC101 ori empty plasmid

[0033] SEQ ID NO. 5 - Primer elaD-F

[0034] SEQ ID NO. 6 - Primer elaD-R

[0035] SEQ ID NO. 7 - Primer Zai-elaD-F

[0036] SEQ ID NO. 8 - Primer Zai-elaD-R Detailed Implementation Modes

[0037] Next, in combination with the implementation modes of the present invention, the technical solutions in the implementation modes of the present invention will be clearly and completely described. Obviously, the described implementation modes are only a part of the implementation modes of the present invention, rather than all of the implementation modes. Based on the implementation modes in the present invention, all other implementation modes obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0038] The experimental methods used in the examples are all conventional methods unless otherwise specified.

[0039] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels.

[0040] ​​Unless otherwise indicated, the terms used herein have the ordinary technical meaning understood by those skilled in the art. For definitions and terms in the art, those skilled in the art are particularly recommended to 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).

[0041] As used herein, the term "comprising" or "including" is an open-ended description, including all specified components or steps described, as well as other specified components or steps that do not substantially 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 have the activity described in the present invention.

[0042] As used herein, the term "and / or" includes all combinations of the items connected by this term, and each combination should be considered to be 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".

[0043] 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 transcription occurs at a high rate. High-rate transcription results in the production of a large amount of mRNA. For the overexpression of the "endogenous gene (such as the deubiquitinating protease gene elaD )" in the present invention, it means that the DNA or RNA or protein expression level of the endogenous gene (such as the deubiquitinating protease gene elaD ) in the engineered Escherichia coli described in the present invention is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or 300% higher than that of the control (not overexpressing the endogenous gene (such as the deubiquitinating protease gene elaD )), or even 4, 5, 6, 7, 8, 9, 10 times or more the DNA or RNA or protein expression level of the endogenous gene (such as the deubiquitinating protease gene

[0044] There is no particular restriction on the type of Escherichia coli host in the present invention, as long as it can overexpress the deubiquitinating protease gene elaD That's all. Typical commercially available Escherichia coli strains used for protein expression in the art include Escherichia coli BL21(DE3), BL21, BL21 Star(DE3), BL21(DE3)pLysS, DH5a, JM109, Top10, Rosetta(DE3) or Origami(DE3).

[0045] When expressing foreign proteins, the BL21 strain reduces the degradation of recombinant proteins and increases the yield of recombinant proteins due to mutations in the lon and ompT genes. However, BL21 does not express T7 RNA polymerase, so it is not suitable for use with plasmid vectors based on the T7 promoter.

[0046] The BL21(DE3) strain is a strain created by integrating the genome of phage λ DE3 into the genome of BL21. This strain contains the T7 RNA polymerase gene controlled by the lacUV5 promoter and is suitable for highly expressing genes cloned in expression vectors containing the T7 promoter (such as the pET series). It can be activated by adding inducers such as IPTG and is used for expressing toxic proteins or for high-level expression in Escherichia coli.

[0047] BL21 Star(DE3) is based on BL21(DE3) with the addition of the rne131 mutation (RNase E coding gene), resulting in defective RNase E expression, enhancing mRNA stability, and further increasing the expression level of foreign proteins.

[0048] BL21(DE3)pLysS is a BL21(DE3) strain carrying the pLysS plasmid and having chloramphenicol resistance, which can express both toxic and non-toxic proteins.

[0049] DH5a is a strain commonly used for plasmid cloning. When transforming with pUC series plasmid vectors, it can achieve α-complementation with the amino terminus of the β-galactosidase encoded by the vector, and is used for blue-white screening to identify recombinant strains.

[0050] When using the JM109 strain for DNA transformation with pUC series plasmid vectors or transfection with M13 phage vectors, due to α-complementation between the LacZa polypeptide produced by the vector DNA and the LacZΔM15 encoded by JM109, β-galactosidase activity is shown, and thus it is very easy to identify recombinant strains. It is suitable for plasmid cloning and genetic engineering operations.

[0051] Top10 This strain is suitable for efficient DNA cloning and plasmid amplification, and can ensure the stable inheritance of high-copy plasmids.

[0052] Rosetta(DE3) is a derivative of the BL21(DE3) strain, which is used to express recombinant proteins with rare codons. It provides tRNAs corresponding to the rare codons (such as AGG, AGA, AUA, CUA, CCC, GGA) in Escherichia coli, solving the problem of codon bias.

[0053] Origami(DE3) is a modified strain, which is particularly suitable for the expression of proteins that require correct disulfide bond formation.

[0054] Aiming at the core bottlenecks of low tolerance of Escherichia coli to monoterpenoids and lack of broad-spectrum resistance gene elements in the prior art, the present invention provides the following innovative solutions:

[0055] 1) Key tolerance gene element: Systematically identify and verify the broad-spectrum monoterpene tolerance function of the deubiquitinating enzyme gene elaD (GeneID:946742) for the first time;

[0056] 2) Modular engineering strain: Construct an Escherichia coli engineering strain with significantly improved tolerance through gene overexpression strategy;

[0057] 3) Application in geraniol fermentation: The tolerance gene is used for the production of geraniol, and the yield is increased by 115.3%, breaking through the toxicity threshold of microbial synthesis.

[0058] The purpose of the present invention is to provide an Escherichia coli engineering strain with high tolerance to monoterpenoids. This engineering strain uses Escherichia coli E. coli BL21(DE3) as the expression host, and overexpresses the deubiquitinating protein elaD in Escherichia coli.

[0059] The gene elaD is obtained by exposing Escherichia coli in the logarithmic growth phase to high concentrations of monoterpenoids. After the cells grow to the stationary phase, the cells are collected, total RNA is extracted for transcriptome sequencing (RNA-seq), differentially expressed genes are screened, and candidate genes with significantly increased expression levels are locked elaD , and then its function of improving the tolerance of Escherichia coli to monoterpenoids is verified and identified by means of gene knockout, gene complementation and gene overexpression.

[0060] Preferably, the Escherichia coli is E. coli BL21(DE3).

[0061] The monoterpenoids are geraniol, nerol, linalool, myrcene, limonene, α-terpineol, menthol, (-)-carvone, and (-)-borneol.

[0062] The present invention provides a method for constructing an engineered Escherichia coli strain with the ability to tolerate monoterpenoids. The construction method includes the following steps:

[0063] 1) Using the genome of Escherichia coli BL21(DE3) as a template, the target fragment of the deubiquitinating protease gene elaD is obtained by PCR amplification, and the target fragment is recovered using a gel extraction kit;

[0064] 2) The elaD target fragment obtained in step 1) is ligated to the expression vector pET28a-tac by Gibson assembly to obtain the recombinant plasmid pET28a-tac-elaD;

[0065] 3) The recombinant plasmid pET28a-tac-elaD obtained in step 2) is introduced into E. coli BL21(DE3) to obtain the engineered Escherichia coli BL21-elaD.

[0066] The present invention also provides the application of the above-mentioned monoterpenoid-tolerant gene in constructing an engineered strain for producing geraniol.

[0067] Preferably, the method for transforming the recombinant plasmid into the host cell in the present invention is the commonly used heat shock transformation method, and positive transformants are screened using an antibiotic selection plate.

[0068] Advantages of the present invention:

[0069] The present invention for the first time confirmed the function of the elaD protein in improving the tolerance of Escherichia coli to monoterpenoids. By overexpressing the genes encoding these proteins in Escherichia coli respectively, an engineered Escherichia coli strain tolerant to monoterpenoids was successfully constructed.

[0070] Testing the OD 600 of the engineered strain BL21-elaD under different monoterpenoid stresses, compared with the starting strain BL21-pET28a, the tolerance of the engineered strain to monoterpenoids was increased by up to 6.23 times at most, proving that these engineered strains have good tolerance effects on linear monoterpenoids: geraniol, nerol, linalool, and myrcene; monocyclic monoterpenoids: limonene, α-terpineol, menthol, and (-)-carvone; bicyclic monoterpenoids: (-)-borneol, etc.

[0071] The tolerance gene identified in the present invention elaDIt has the ability to increase the production of monoterpenoids in Escherichia coli. Taking geraniol production as an example, after overexpressing elaD the tolerance gene in Escherichia coli with a constructed geraniol synthesis pathway, the geraniol production was increased, and the highest increase was 115.3% compared to the control, with a shake flask production of 945.3 mg / L.

[0072] All patents and publications mentioned in this application are incorporated into the present invention by reference as a whole. 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. Examples

[0073] Example 1. Mining and identification of tolerance genes elaD

[0074] Use an inoculation loop to streak and activate the control strain E. coli trans1T1 and the evolved strain E. coli TS that is tolerant to monoterpenoids obtained by adaptive evolution on an LB solid plate. Pick single colonies and inoculate them into 20 mL of LB medium, and culture them in a shaker at 37 °C and 200 rpm. After completion, collect the cells by low-speed centrifugation at 5000 rpm, quickly freeze them with liquid nitrogen, and extract total RNA. Purify and fragment the RNA, perform product ligation, and the ligation product is purified and amplified to obtain the final cDNA library. Finally, sequence the constructed sequencing library using the HiSeq / MGI sequencing platform and perform expression level analysis. Among them, the expression level of the gene elaD in the evolved bacteria was significantly up-regulated compared to the control. These genes were functionally verified to confirm their monoterpenoid tolerance function.

[0075] Example 2. Construction of an engineered strain tolerant to monoterpenoids.

[0076] Found from NCBI elaDFor the gene sequence (GeneID: 946742), corresponding primers were designed to amplify the target gene and the vector pET28a-tac (the sequence is shown in SEQ ID NO. 3) (pET28a was purchased from Novagen, and pET28a-tac was obtained by replacing the promoter in the present invention, which contains the Ptac inducible promoter, the T7 terminator and the KanR resistance marker) (the specific primer nucleotide sequences are shown in Table 1). The amplified target fragment and vector were recovered using a gel extraction kit. The recovered and purified target fragment and vector with homologous ends were added to the same tube of Master Mix, pipetted 5 - 10 times, and then the reaction solution was immediately placed in a 50 °C metal bath for 5 - 15 min to obtain the recombinant plasmid pET28a-tac-elaD.

[0077] The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells by heat shock method to obtain the engineered strain BL21-elaD with the function of monoterpenoid tolerance.

[0078] The specific nucleotide sequences of the primers used in the construction process of the engineered strain are shown in Table 1.

[0079] Table 1 Primer sequences.

[0080] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0081] Example 3. Growth of the tolerant engineered strain under monoterpenoid stress.

[0082] Single colonies of BL21(DE3) containing the pET28a-tac empty plasmid and the engineered strain BL21-elaD were picked and activated in LB liquid medium containing kanamycin resistance to obtain the seed solution. The seed solution was transferred to 20 mL of LB medium, and the initial OD ​ value was controlled to be 0.1, and the culture was continued until the OD 600 value of the strain reached 0.8 - 1. Then, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the expression of the tolerance gene, and at the same time, 0.2 g / L of monoterpenoids such as geraniol (the commercial sources and product catalog numbers of the monoterpenoids are shown in Table 2) were added externally for stress. The temperature of the shaker was controlled and the culture was continued until the stationary phase. An appropriate amount of the bacterial solution was taken out, and the optical absorption value (OD 600 ) of each sample at 600 nm was measured using a spectrophotometer to monitor the growth of the bacteria. 600 )

[0083] Table 2 Commercial sources and product catalog numbers of monoterpenoids

[0084] ​ ​ ​ ​ ​ 106-24-1 ​ G107517 ​ 106-25-2 ​ N101447 ​ 123-35-3 ​ M101322 ​ 5989-54-8 ​ L157739 ​ 10482-56-1 ​ T111437 ​ 2216-51-5 ​ M107059 ​ 6485-40-1 ​ C117707 ​ 464-45-9 ​ B141262

[0085] ​ Yes ​ The light absorption values of the overexpressed engineering strain BL21-elaD and the starting strain BL21-pET28a under different monoterpene compound stresses. According to the measurement results, it can be seen that compared with the starting strain, the tolerance of the engineering strain BL21-elaD to geraniol increased by 251.8%; the tolerance to nerol increased by 474.0%; the tolerance to linalool increased by 142.9%; the tolerance to myrcene increased by 142.8%; the tolerance to limonene increased by 76.5%; the tolerance to α-terpineol increased by 91.4%; the tolerance to menthol increased by 310.4%; the tolerance to carvone increased by 53.3%; the tolerance to (-)-borneol increased by 95.1%.

[0086] Example 4. Application of tolerance genes in geraniol production.

[0087] There is a natural MEP pathway in Escherichia coli: using glucose as a carbon source, the key intermediate metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are synthesized. The substrates IPP and DMAPP are catalyzed by farnesyl diphosphate synthase (ispA) to generate geranyl diphosphate (GPP), and then monoterpene compounds are generated by expressing exogenous monoterpene synthase. ​ It is the process of Escherichia coli producing geraniol using the mevalonate (MVA) pathway or the 2-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, geraniol synthase (GES) from Ocimum basilicum was selected to be introduced. To strengthen the precursor supply, the MVA pathway and geranyl diphosphate synthase (GPPS) from Abies were further introduced, and plasmids were constructed by Gibson assembly.

[0088] Geraniol production plasmid: The production plasmid pET28a-tac-GES-tac-GPPS, including the promoter tac and the genes ​ (GenBank Accession No. AY362553.1) ​ (GenBank Accession No. AF513112.1), named pG1.

[0089] MVA pathway: including plasmids pMH1 and pFZ81, plasmid pMH1 (overexpressing atoB-erg13-thmg1), plasmid pFZ81 (overexpressing erg12-erg8-mvd1-idi).

[0090] Tolerant plasmids: plasmid pG2 (empty plasmid, pSC101 ori) (the sequence is shown in SEQ ID NO. 4), plasmid pG3 (overexpressed in plasmid pG2 by promoter tac ​ ).

[0091] Plasmids pG1, pMH1, pFZ81 and pG2 were transformed into ​ DH5α to obtain strain SG1; plasmids pG1, pMH1, pFZ81 and pG3 were transformed into ​ DH5α to obtain strain SG2. All plasmids, strains and their characteristics are listed in Table 3.

[0092] Table 3

[0093] ​ ​ ​ ​ ​ <![CDATA[Kan, pBR322 ori, P T7 > ​ ​ <![CDATA[Kan, pBR322 ori, P tac > ​ ​ <![CDATA[Kan, pBR322 ori, P tac , ​ > ​ ​ <![CDATA[Cm, p15A ori, P lac , ​ > ​ ​ <![CDATA[Amp, pBBR1 Rep, P lac , ​ > ​ ​ <![CDATA[Kan, pBR322 ori, P tac , ​ > ​ ​ <![CDATA[Spec, pSC101 ori, P tac > ​ ​ <![CDATA[Spec, pSC101 ori, P tac , ​ > ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0094] In Table 3 (Zhu et al., 2014) is In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli. Biotechnology and Bioengineering. 111, 1396 - 1405.

[0095] Colonies of engineered strain SG2 and control strain SG1 were picked from fresh plates and inoculated into LB medium containing the corresponding resistance, and the seed solution was obtained by overnight culture. The above seed solution was transferred to a 100 mL conical flask containing 20 mL of TB medium, and the initial OD 600 value was controlled and cultured at 37 °C and 200 rpm. When it grew to the appropriate OD 600When reaching the value, IPTG was added for induction, and isopropyl myristate was added as the organic phase for two-phase fermentation. The temperature and rotation speed were controlled for culturing for 48 h. After the fermentation ended, 2 mL of the upper organic phase was taken into an EP tube, and centrifuged at 12000 rpm for 10 min to separate the organic phase. Carefully suck the upper clarified organic phase into a clean EP tube, add an appropriate amount of anhydrous sodium sulfate, and let it stand for 10 min to remove water. Centrifuge at 12000 rpm for 5 min, filter the sample through a 0.22 μm organic filter membrane and transfer it to a liquid phase vial sleeve, and the concentration was detected by gas chromatography. The model of the gas chromatograph was Shimadzu GC-2010, and an RTX-5 chromatographic column (30 m×0.25 mm×0.25 μm) was selected to separate geraniol. The detection program was set as follows: the column oven temperature was 70 °C, the detector temperature was 280 °C, and the split ratio was 4:1. The initial temperature of the oven was 70 °C, held for 1 min, heated to 180 °C at a rate of 30 °C / min, held for 1 min, heated to 270 °C at a rate of 30 °C / min, and held for 2 min. By detecting geraniol standard solutions with different concentration gradients, a standard curve of yield and peak area was plotted.

[0096] ​ The fermentation results showed that: the tolerance gene ​ Overexpression in the production chassis could further improve the geraniol yield. The yield was 945.3 mg / L, which was 115.3% higher than that of the strain SG1 before overexpressing the tolerance gene, that is, the yield was increased to 2.15 times. It can be thus proved that the tolerance gene ​ dug out by the present invention

[0097] 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 solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0098] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any appropriate way without contradiction. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0099] Furthermore, any combination can be made between various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should equally be regarded as the content disclosed by the present invention.

Claims

1. Use of engineered Escherichia coli in the production of monoterpenoids, wherein the engineered Escherichia coli overexpresses a deubiquitinating protease gene elaD , and the deubiquitinating protease gene elaD has an amino acid sequence as shown in SEQ ID NO. 1, and wherein the monoterpenoid is selected from: geraniol, neral, linalool, myrcene, limonene, α-terpineol, menthol, carvone and (-)-borneol.

2. The application according to claim 1, wherein the deubiquitinating protease gene elaD has a nucleotide sequence as shown in SEQ ID NO.

2.

3. The application according to claim 1, wherein the Escherichia coli is Escherichia coli BL21(DE3), BL21, BL21 Star(DE3), BL21(DE3)pLysS, DH5a, JM109, Top10, Rosetta(DE3) or Origami(DE3).

4. A method for improving the tolerance of Escherichia coli to monoterpenoids, the method comprising overexpressing a deubiquitinating protease gene in the Escherichia coli elaD , wherein the deubiquitinating protease gene elaD has an amino acid sequence as shown in SEQ ID NO. 1, and wherein the monoterpenoid is selected from: geraniol, nerol, linalool, myrcene, limonene, α-terpineol, menthol, (+)-carvone and (-)-borneol.

5. The method according to claim 4, wherein 1) the nucleotide sequence of the deubiquitinating protease gene elaD is as shown in SEQ ID NO. 2; and / or 2) the Escherichia coli is Escherichia coli BL21(DE3), BL21, BL21Star(DE3), BL21(DE3)pLysS, DH5a, JM109, Top10, Rosetta(DE3) or Origami(DE3).

6. The method according to claim 4 or 5, wherein the overexpression is achieved by expressing the deubiquitinating protease gene on a plasmid elaD to achieve.

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