Zymomonas mobilis recombinant strain for synthesizing R-(-)-linalool as well as construction method and application of zymomonas mobilis recombinant strain

Abstract

The invention discloses a Zymomonas mobilis recombinant strain for synthesizing R-(-)-linalool as well as a construction method and application of the Zymomonas mobilis recombinant strain. A ZMO1094 site of a genome of the recombinant strain is replaced by Pgap-IspA2S82F, a ZMO1650 site of the recombinant strain is replaced by Pet-tSlNDPS1-ApLS, and a ZMO0959 gene of the genome is knocked out; wherein the nucleotide sequence of the gene tSlNDPS1 is as shown in SEQ ID NO: 1, the nucleotide sequence of the gene ApLS is as shown in SEQ ID NO: 2, the nucleotide sequence of the IspA2S82F is as shown in SEQ ID NO: 3, Pet is an inducible promoter, and Pgap is a constitutive promoter. In the technical scheme provided by the invention, the Zymomonas mobilis recombinant strain constructed through a multi-dimensional metabolic engineering strategy shows significant advantages in the field of green biosynthesis of R-(-)-linalool.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant strain of Zymomonas mobilis for synthesizing R-(-)-linalool, a construction method thereof, and an application thereof. Background Art

[0002] Linalool, as an important monoterpene compound, is widely used in fragrances, daily chemicals, and pharmaceuticals. Its two optical isomers, R-(-)-linalool and S-(+)-linalool, play unique roles in different fields. Currently, the synthesis methods of linalool mainly include plant extraction, chemical synthesis, and biosynthesis. Although plant extraction can obtain pure and natural linalool with good effects, it is difficult to achieve large-scale production due to the limitation of plant raw materials. Although chemical synthesis can meet large-scale demand, it has the problems of non-uniform linalool configuration and high pollution in the synthesis process. Biosynthesis has the advantages of being green and environmentally friendly and having pure products. However, factors such as low linalool yield and high toxicity to chassis cells have seriously restricted its large-scale production process.

[0003] Zymomonas mobilis, a naturally ethanol-producing facultative anaerobic Gram-negative bacterium, possesses a unique Entner-Doudoroff (ED) pathway and a high sugar conversion rate. It not only has high ethanol production, strong alcohol tolerance, and resistance to high osmotic pressure, but also does not require additional oxygen during the fermentation process, making it an ideal cell factory. Currently, the production and fermentation of various products such as PHB, 2,3-butanediol, isobutanol, and lactic acid have been successfully achieved in Zymomonas mobilis. At the same time, the expression of the endogenous MEP pathway in this bacterium is stable and has a high flux, and the synthesis of the sesquiterpene compound farnesene has also been achieved, all of which fully demonstrate its great potential in the field of biosynthesis.

[0004] Given the limitations of existing linalool synthesis methods and the numerous advantages of Z. mobilis in biosynthesis, constructing a recombinant strain of Z. mobilis capable of efficiently synthesizing R-(-)-linalool is of great significance. Modifying Z. mobilis through genetic engineering and other means is expected to address challenges such as low linalool yield and high cytotoxicity in biosynthesis, providing a new pathway for large-scale, green production of R-(-)-linalool and promoting the sustainable development of related industries. Summary of the Invention

[0005] The main purpose of the present invention is to propose a recombinant strain of Zymomonas mobilis that synthesizes R-(-)-linalool, as well as a construction method and application thereof, in order to solve the problems of low yield and high toxicity to chassis cells in the existing biosynthesis of linalool. By improving the catalytic efficiency of NPP synthase and linalool synthase, increasing the supply of precursors, increasing the permeability of the cell wall and optimizing the bottling volume of the culture medium, a recombinant Zymomonas mobilis strain that produces R-(-)-linalool is constructed and optimized, thereby increasing the yield of R-(-)-linalool.

[0006] To achieve the above object, the present invention proposes a recombinant strain of Zymomonas mobilis for synthesizing R-(-)-linalool, wherein the genomic ZMO1094 site of the recombinant strain is replaced with Pgap-IspA2 S82F , the ZMO1650 site is replaced with Ptet-tSlNDPS1-ApLS, and the ZMO0959 gene on the genome is knocked out, and an expression plasmid containing the gene tSlNDPS1 and the gene ApLS is transferred, or the recombinant strain is transferred with an expression plasmid containing the gene tSlNDPS1 and the gene ApLS D101M The expression plasmid, or the recombinant strain is transformed with the gene tSlNDPS1 and gene ApLS R294F or the recombinant strain is transformed into an expression plasmid containing the gene tSlNDPS1, a linker sequence, and the gene ApLS, or the recombinant strain is transformed into an expression plasmid containing the gene tSlNDPS1, ApLS and an expression plasmid containing the gene dxs1 / dxs2, ispG;

[0007] The nucleotide sequence of the gene tSlNDPS1 is shown in SEQ ID NO: 1, the nucleotide sequence of the gene ApLS is shown in SEQ ID NO: 2, and the nucleotide sequence of the gene IspA2 is shown in SEQ ID NO: S82F The nucleotide sequence of the gene ApLS is shown in SEQ ID NO: 3. D101M The nucleotide sequence of the gene ApLS is shown in SEQ ID NO: 4. R294F The nucleotide sequence of gene dxs1 is shown in SEQ ID NO: 5, the nucleotide sequence of gene dxs1 is shown in SEQ ID NO: 6, the nucleotide sequence of gene dxs2 is shown in SEQ ID NO: 7, the nucleotide sequence of gene ispG is shown in SEQ ID NO: 8, Ptet is an inducible promoter, the nucleotide sequence of which is shown in SEQ ID NO: 10, and Pgap is a constitutive promoter, the nucleotide sequence of which is shown in SEQ ID NO: 12.

[0008] Preferably, the genome ZMO1094 site of the recombinant strain is replaced with Pgap-IspA2 S82F, the ZMO1650 site was replaced with Ptet-tSlNDPS1-ApLS, the ZMO0959 gene on the genome was knocked out, and an expression plasmid containing the genes tSlNDPS1 and ApLS was transferred into the cell.

[0009] Preferably, the gene tSlNDPS1 is a truncated gene tSlNDPS1 of nerolidol pyrophosphate synthase from tomato;

[0010] The gene ApLS is the linalool synthase gene ApLS derived from Pleurotus ostreatus.

[0011] Preferably, the gene ApLS D101M A mutant of the linalool synthase gene ApLS from Agaricus truncatus D101M .

[0012] Preferably, the gene ApLS R294F A mutant of the linalool synthase gene ApLS from Agaricus truncatus R294F .

[0013] Preferably, the linker sequence is a nucleotide sequence encoded by a connecting peptide (PT)4P, and tSlNDPS1 is fused with ApLS via the connecting peptide (PT)4P.

[0014] Preferably, the genes dxs1 / dxs2 and ispG are derived from Zymomonas mobilis.

[0015] The present invention also provides a method for constructing a recombinant strain of Zymomonas mobilis for synthesizing R-(-)-linalool as described above, comprising the following steps:

[0016] S1. Construction of expression plasmid or editing plasmid;

[0017] S2. Transforming the expression plasmid or editing plasmid into Zymomonas mobilis to obtain transformants.

[0018] The present invention also provides a use of the above-mentioned recombinant Zymomonas mobilis strain for synthesizing R-(-)-linalool in the production of R-(-)-linalool.

[0019] The present invention also provides a method for producing R-(-)-linalool, which comprises the following steps: culturing the recombinant strain of Zymomonas mobilis, adding isopropyl myristate to carry out two-phase fermentation to obtain a fermentation broth, and extracting the isopropyl myristate phase of the fermentation broth; wherein the culture medium is RMG5 culture medium; during the fermentation process.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention constructs a recombinant strain of Zymomonas mobilis that produces R-(-)-linalool. The strain utilizes the powerful restriction and modification system of Zymomonas mobilis itself, has the advantage of being resistant to phage contamination, and has stronger phage resistance than commonly used engineered strains such as Escherichia coli. The recombinant strain is used to ferment R-(-)-linalool, which reduces production costs. Since Zymomonas mobilis is a facultative anaerobic microorganism, no additional dissolved oxygen control equipment is required during the fermentation process, which can effectively reduce production costs. The Zymomonas mobilis R-(-)-linalool-producing strain obtained by the present invention has an accurate configuration of linalool. Due to the biosafety characteristics of Zymomonas mobilis, the R-(-)-linalool produced by the strain can be widely used in cosmetics, medicine, food additives, etc.

[0022] (2) The present invention improves the yield of R-(-)-linalool and reduces cytotoxicity through multi-dimensional technical means: First, the R-(-)-linalool synthase (ApLS) is rationally designed, and NPPS (tSlNDPS1) and ApLS are expressed by linker fusion to promote product synthesis; secondly, the IspA2 mutant is overexpressed at the genomic level, and the expression of the endogenous MEP pathway rate-limiting enzymes DXS and IspG is upregulated to enhance the supply of the precursor substance GPP; then, the penicillin-binding protein-related gene ZMO0959 is knocked out to increase cell membrane permeability, promote the efflux of R-(-)-linalool, and reduce its toxic side effects on cells; finally, the yield is further improved by optimizing the culture medium. The recombinant strain of Z. mobilis constructed by the present invention through a multi-dimensional metabolic engineering strategy has shown significant advantages in the field of green biosynthesis of R-(-)-linalool. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The diagram (A) shows the construction of the recombinant bacteria NP-PtSA and the control strain NP-Pt of the present invention, as well as the growth status of the recombinant strain fermentation in a culture medium supplemented with 0.5 mg / L tetracycline and the yield of R-(-)-linalool (B).

[0025] Figure 2 The diagram (A) shows the construction of the recombinant bacteria NP-101 and NP-294 of the present invention and the yield of R-(-)-linalool during fermentation in a culture medium supplemented with 0.5 mg / L tetracycline (B).

[0026] Figure 3 The diagram (A) shows the construction of the recombinant bacterium NP-(PT)4P of the present invention, its growth status during fermentation in a culture medium supplemented with 0.5 mg / L tetracycline, and its yield of R-(-)-linalool (B).

[0027] Figure 4 IspA of Escherichia coli and its mutant IspA of the present invention S80F and mutant IspA2 of Zymomonas mobilis S82F Protein structure comparison diagram, red is E. coli IspA, cyan is E. coli IspA S80F , purple is Zymomonas mobilis IspA2 S82F .

[0028] Figure 5 This is a graph showing the yield of R-(-)-linalool detected by fermentation of the recombinant strain NP-A-PtSA of the present invention in a culture medium supplemented with 0.5 mg / L tetracycline.

[0029] Figure 6 The diagram (A) shows the construction of the recombinant strains NP-PtSA-P1 and NP-PtSA-P2 of the present invention and the yield of R-(-)-linalool detected by fermentation in a culture medium supplemented with 0.5 mg / L tetracycline (B).

[0030] Figure 7 Figure 1 shows the growth status of the recombinant strains NP-PtSA, NP-101 and NP-(PT)4P in culture medium supplemented with 0.5 mg / L tetracycline (A) and 0 mg / L tetracycline (B) and the changes in the yield of R-(-)-linalool.

[0031] Figure 8 Figure 2 shows the growth status of the recombinant strain NP-PtSA of the present invention in culture medium supplemented with 0, 0.5, 0.8, 1.0, 1.2 and 1.5 mg / L tetracycline (A) and the yield change of R-(-)-linalool (B).

[0032] Figure 9 This is a graph showing the growth status of the recombinant strain NPΔ0959-PtSA during fermentation and the yield of R-(-)-linalool.

[0033] Figure 10 The diagram (A) shows the construction of the recombinant strain NP-L-PtSA of the present invention, its growth status during fermentation, and its yield of R-(-)-linalool (B).

[0034] Figure 11Figure 2 shows the growth status (A) and the yield of R-(-)-linalool (B) of the recombinant strain NP-L-PtSA of the present invention when fermented at 40%, 60% and 80% of the bottling volume.

[0035] Figure 12 This is a summary diagram of the strain transformation strategy of the present invention (red is the overexpressed endogenous enzyme, blue is the exogenously introduced enzyme, and green is the overexpressed transformed enzyme).

[0036] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0039] The culture medium formula is as follows:

[0040]

[0041] The culture medium formulas for different bottling volumes are as follows:

[0042]

[0043] Example 1 Vector Expression

[0044] (1) PCR was used to amplify the gene sequences (Table 1) using primer sequences shown in Table 2. The reverse amplification was performed using the overexpression vector pEZ15A. The PCR amplification program was set as follows: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 10 s, and 72°C extension (set at 1 kb / 10 s based on the fragment length) for 30 cycles; after the cycle reaction, the reaction was maintained at 72°C for 5 min; the product was purified and stored at -20°C. The PCR amplification conditions are shown in Table 3.

[0045] Table 1 Related genes and sequences

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] Table 2 Primer sequence list

[0055]

[0056]

[0057] Table 3 PCR amplification conditions

[0058]

[0059] Example 2 Construction of recombinant plasmid pEZ15A-Ptet-tS1NDPS1-ApLS

[0060] Specific primers were designed based on the sequences of the truncated neryl diphosphate synthase (NPPS) gene tSlNDPS1 from tomato (Solanum lycopersicum) and the linalool synthase gene ApLS from Agaricus truncatula (Agrocybe pediades) (primer sequences are shown in Table 2). PCR amplification was performed using the designed primers. The tSlNDPS1 and ApLS genes were driven by the inducible promoter Ptet and ligated using RBS15000 to create the final recombinant plasmid pEZ15A-Ptet-tSlNDPS1-ApLS.

[0061] Example 3 Recombinant plasmid pEZ15A-Ptet-tS1NDPS1-ApLS D101M and pEZ15A-Ptet-tSlNDPS1-ApLS R294F Construction

[0062] The stability of the ApLS protein was predicted using the online tool FIREPROT (FireProt: Design stable proteins! (muni.cz)), and the single point mutation sites D101M and R294F were identified. The tSlNDPS1 and ApLS mutants were constructed with the Ptet promoter driving expression. The two genes were connected using RBS15000 to obtain the recombinant plasmid pEZ15A-Ptet-tSlNDPS1-P-ApLS. D101M and pEZ15A-Ptet-tSlNDPS1-ApLS R294F .

[0063] Example 4 Construction of recombinant plasmid pEZ15A-Ptet-tS1NDPS1-(PT)4P-ApLS

[0064] The NPPS gene tSlNDPS1 and the linalool synthase gene ApLS were expressed on the plasmid using the inducible promoter Ptet, and the two genes were connected with a linker to obtain the plasmid pEZ15A-Ptet-tSlNDPS1-(PT)4P-ApLS.

[0065] The gene fragments obtained in Examples 1 to 4 above were mixed with the vector in a ratio of 3:1. After the reaction system was prepared according to Table 4 below, it was allowed to stand on ice for 5 minutes, and then E. coli competent cells were added and transformed using the general method. Screening was performed using spectinomycin-resistant plates, and single colonies were picked. The plasmids of pL2R, pEZ15A, and pEZ39P were verified by colony PCR using pEZ15A-F / R, ptet-check-F / R, and 39p-check-F primers, respectively (PCR amplification program was set as: 98°C pre-denaturation for 3 minutes; 98°C denaturation for 10 seconds, 55°C annealing for 10 seconds, and 72°C extension for 80 seconds, for a total of 30 cycles). Band sizes consistent with expectations were verified by sequencing. The correct strain was maintained, the plasmid was extracted, and then transformed into Zymomonas mobilis by electroporation.

[0066] Table 4 Reaction system

[0067]

[0068] Example 5 Plasmid Transformation

[0069] (1) Preparation of competent cells

[0070] Take out the frozen bacteria from the -80℃ freezer, take 100μL and inoculate it into a cryovial containing 1mL RMG5, and place it in a 30℃ incubator to activate the strain. After the culture becomes turbid, transfer it to a 250mL blue cap bottle containing 200mL RMG5 liquid medium to make the initial OD 600nm In the range of 0.02 to 0.3, culture in a 30°C incubator until OD 600nm When the pH value exceeded 0.3, the cells were collected at room temperature and 100 rpm, then washed once with sterile water and twice with 10% glycerol. Finally, the cells were slowly resuspended with 1-2 mL of 10% glycerol and aliquoted into 55 μL competent cells into 1.5 mL EP tubes.

[0071] (2) Plasmid electroporation method

[0072] Add 0.5 mg of the above-constructed plasmid to a 1.5 mL EP tube containing 55 μL of competent cells, mix gently, and transfer to a 1 mm electroporation cuvette. Set the electroporator to 200 Ω, 25 μF capacitance, and 1.6 kV. Place the cuvette in the electroporator for electroporation. Immediately after electroporation, add 1 mL of RMG5 liquid culture medium, mix thoroughly, and transfer to a sterile EP tube. Seal the tube with parafilm and incubate in a 30°C incubator for 4-6 hours. Evenly spread 100 μL of the bacterial solution onto an RMG5 plate containing 200 μg / mL spectinomycin. Seal the plate with parafilm and incubate it upside down in a 30°C incubator.

[0073] (3) Colony PCR verification method

[0074] After single colonies grew on the plates, PCR verification of the pL2R, pEZ15A, and pEZ39P plasmids was performed using primers pEZ15A-F / R, ptet-check-F / R, and 39p-check-F, respectively. The PCR system and procedure were the same as for colony PCR described above. Correct positive clones were activated in RMG5 liquid medium (containing 200 μg / mL spectinomycin) and maintained in glycerol stock.

[0075] Example 6 Recombinant strain fermentation

[0076] The recombinant strain was first activated in RMG5 medium, and then the seed liquid was cultured in RMG5 (10 g / L yeast extract, 50 g / L glucose, 2 g / L KH2PO4) medium. The seed liquid was then inoculated into a 50 mL shake flask containing 30 mL RMG5 medium and 5 mL isopropyl myristate (IPM) for culture (30°C, 100 rpm), and antibiotics were added if necessary. The final concentration of the antibiotic spectinomycin was 200 μg / mL.

[0077] Example 7 Growth curve test based on shake flask

[0078] For shake flask fermentation, take samples of the bacterial solution at regular intervals to see if it is turbid (if it is very turbid, dilute it) and test its OD using a spectrophotometer. 600nm The values ​​were recorded and the growth curve was drawn using GraphPad Prism software until OD 600nm Stop sampling when the value stabilizes.

[0079] Example 8 Detection of R-(-)-linalool Yield

[0080] (1) HPLC detection of R-(-)-linalool yield

[0081] The fermentation broth supernatant (IPM) was centrifuged at 12,000 rpm for 5 minutes and then filtered through a 0.22 μm filter into a high-performance liquid chromatography (HPLC) vial. The concentration of R-(-)-linalool in the IPM was determined using a WIN-3ER548E UV detector (LC-2030 Plus, Shimadzu) equipped with a Shimadzu Shim-pack GIST-C18 column (5 μm, 4.6 × 250 mm C18 column). The mobile phase was 60% acetonitrile at a flow rate of 1 mL / min, the column temperature was 30°C, the detection wavelength was 205 nm, and the injection volume was 10 μL.

[0082] (2) Gas chromatography detection of R-(-)-linalool yield

[0083] The fermentation broth (IPM) was dehydrated by adding anhydrous sodium sulfate and centrifuged at 12,000 rpm for 5 minutes. The supernatant (IPM) was filtered through a 0.22 μm filter. 100 μL of the filtered sample was transferred to the insert of a gas chromatography (GC) vial. The R-(-)-linalool content was analyzed by GC. The gas chromatograph was a Shimadzu GC-2010 Pro, using a J&W HP-5 column (30 m × 0.320 mm, 0.25 μm, Agilent Technologies), nitrogen as the carrier gas, and a 1 μL injection volume. The GC oven temperature program was as follows: initial temperature 80°C, held for 2 minutes, then increased at 10°C / min to 110°C, then increased at 40°C / min to 250°C, held for 10 minutes. The retention time of R-(-)-linalool was 5.7 minutes.

[0084] The recombinant plasmid pEZ15A-Ptet-tS1NDPS1-ApLS obtained in Example 2 was transformed into the ZMNP strain by electroporation to obtain NP-PtSA ( Figure 1 A). The NP-PtSA strain was fermented in a culture medium supplemented with 0.5 mg / L tetracycline using the method of this example. The growth of the recombinant strain and the yield of R-(-)-linalool were detected. After 48 h of fermentation, 5.95 mg / L of R-(-)-linalool ( Figure 1 B).

[0085] The recombinant plasmid pEZ15A-Ptet-tS1NDPS1-P-ApLS obtained in Example 3 was D101M and pEZ15A-Ptet-tSlNDPS1-ApLS R294F NP-101 and NP-294 were obtained by electroporation into ZMNP strains. Figure 2 A). The method of this example was used to ferment the recombinant strains NP-101 and NP-294 in a culture medium supplemented with 0.5 mg / L tetracycline to detect the production of R-(-)-linalool. 48 h produced 6.70 and 6.28 mg / L of R-(-)-linalool, respectively. Figure 2 B).

[0086] The recombinant plasmid pEZ15A-Ptet-tS1NDPS1-(PT)4P-ApLS obtained in Example 4 was transformed into the ZMNP strain by electroporation to obtain NP-(PT)4P ( Figure 3A). The recombinant strain NP-(PT)4P was fermented in a medium supplemented with 0.5 mg / L tetracycline. The growth of the recombinant strain and the yield of R-(-)-linalool were detected. R-(-)-linalool was produced in 48 hours at 7.24 mg / L ( Figure 3 B).

[0087] Example 9: Increasing the supply of precursors to increase the yield of R-(-)-linalool

[0088] To regulate the metabolic flux of terpenoid precursors, the inventors rationally designed the bifunctional enzyme IspA, with the core goal of inhibiting its activity in catalyzing the conversion of geranyl diphosphate (GPP) to farnesyl diphosphate (FPP). Analysis showed that the IspA variant significantly reduced the intracellular FPP concentration while increasing the accumulation level of GPP, thereby providing sufficient precursor supply for linalool biosynthesis. In microbial metabolic engineering applications, the IspA variant derived from Escherichia coli was introduced into Escherichia coli and Pantoea ananas. S80F After the mutants were identified, the production of R-(-)-linalool was improved. Further focusing on Zymomonas mobilis, its endogenous IspA2 has dual catalytic activity, which can not only condense isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to synthesize GPP, but also catalyze the condensation of GPP and IPP to generate FPP. Through structural comparison, it was found that Escherichia coli IspA S80F The site corresponds to the S82F site of Zymomonas mobilis IspA2 ( Figure 4 Molecular docking was used to investigate the interaction between Zymomonas mobilis IspA2 mutants and their substrates. The results showed that the S82F mutation caused a steric conflict between IspA2 and FPP, significantly enhancing its binding affinity for GPP. This property provides a structural biological basis for the targeted regulation of terpenoid metabolic flux.

[0089] In this example, ZMNP was used as the background strain and the ZMO1094 site on the ZMNP genome was replaced with Pgap-ispA2 on the editing plasmid pL2R-gRNA-1094. S82F The recombinant strain NP-A was obtained. The plasmid pEZ15A-Ptet-tSlNDPS1-ApLS was transferred into the NP-A recombinant strain by electroporation to obtain NP-A-PtSA. The recombinant strain NP-A-PtSA was fermented in a medium supplemented with 0.5 mg / L tetracycline to detect the production of R-(-)-linalool. 6.67 mg / L ( Figure 5).

[0090] Example 10: Enhancing the endogenous MEP pathway to increase R-(-)-linalool production

[0091] In this example, metabolic engineering was performed using the NP-PtSA background strain, focusing on enhancing key precursor synthesis steps. Plasmid-based overexpression of the endogenous Z. mobilis deoxyxylulose 5-phosphate synthase (DXS) genes, dxs1 and dxs2, and the HMBPP synthase gene, ispG, was performed. DXS, the rate-limiting enzyme in the non-mevalonate (MEP) pathway, significantly improved the synthesis efficiency of the precursor deoxyxylulose 5-phosphate (DXP). The ispG gene catalyzes the production of (E)-4-hydroxy-3-methyl-2-(E)-butenyl 4-diphosphate (HMBPP), also a key intermediate in terpenoid synthesis. During the plasmid construction process, the strong promoter Peno was selected to drive gene expression to ensure efficient transcription of the target gene. At the same time, RBS10000 was used to connect dxs1 and ispG, and dxs2 and ispG. This ribosome binding site (RBS) has a high translation initiation efficiency, which can ensure efficient translation of the gene after transcription. Through the above strategy, the recombinant plasmids pEZ39P-Peno-dxs1-ispG and pEZ39P-Peno-dxs2-ispG ( Figure 6 A). And then transformed into NP-PtSA strain by electroporation to obtain NP-PtSA-P1 and NP-PtSA-P2. The recombinant strains NP-PtSA-P1 and NP-PtSA-P2 were fermented in a medium supplemented with 0.5 mg / L tetracycline to detect the production of R-(-)-linalool. After 48 hours, 8.30 and 7.84 mg / L of R-(-)-linalool were produced, respectively. Figure 6 B).

[0092] Example 11 Effect of Tetracycline Concentration on R-(-)-Linalool Yield

[0093] In their research on terpenoid biosynthesis, the inventors discovered a negative correlation between the growth rate of the recombinant strain and the production of R-(-)-linalool. This phenomenon is hypothesized to be related to the dual mechanism of action of tetracycline: on the one hand, tetracycline acts as an inducer, activating the expression of the Ptet promoter-driven NPPS (tSlNDPS1) and R-(-)-linalool synthase (ApLS) genes; on the other hand, its antibiotic activity may inhibit the growth of the host bacteria.

[0094] To verify the above hypothesis, the recombinant strains NP-PtSA, NP-101, and NP-(PT)4P were tested for changes in R-(-)-linalool production in 0 and 0.5 mg / L tetracycline medium. The results showed that after adding 0.5 mg / L tetracycline, the specific growth rates of NP-101 and NP-(PT)4P slowed down, while the specific growth rate of NP-PtSA was not significantly different from that without tetracycline addition ( Figure 7 ). When 0.5 mg / L tetracycline was added, the yield of R-(-)-linalool of NP-101 was the highest (6.62±0.55 mg / L). When no tetracycline was added, the yield of NP-PtSAR-(-)-(-)-linalool was the highest (8.74 mg / L). The recombinant strain with ApLS modification and NPP synthase fusion expression had a higher yield of R-(-)-linalool after adding 0.5 mg / L tetracycline than when no tetracycline was added. The opposite was true for NP-PtSA. After adding 0.5 mg / L tetracycline, the yield of R-(-)-linalool was lower than when no tetracycline was added.

[0095] It is worth noting that the strains with ApLS modification and NPP synthase fusion expression (such as NP-(PT)4P) showed increased production after the addition of tetracycline, while the opposite was true for NP-PtSA.

[0096] Since the R-(-)-linalool yield of NP-PtSA after adding 0.5 mg / L tetracycline was lower than that without tetracycline, in order to confirm whether the yield was highest without tetracycline, the R-(-)-linalool yield of NP-PtSA in the medium with 0, 0.5, 0.8, 1.0, 1.2 and 1.5 mg / L tetracycline was further compared. The maximum biomass OD of the recombinant strain when 1.5 mg / L tetracycline was added 600nm Down to 3.23 ( Figure 8 A). The production of R-(-)-linalool decreased after adding tetracycline. The higher the tetracycline concentration, the lower the production of R-(-)-linalool ( Figure 8 B), when tetracycline was not added, the maximum linalool production of the recombinant strain NP-PtSAR-(-)-linalool reached 9.84 mg / L.

[0097] Example 12 Penicillin Binding Proteins (PBPs) Deficiency Strains Favor R-(-)-Linalool Efflux

[0098] In this embodiment, ZMNP was used as the background strain, and the recombinant strain NPΔ0959 was obtained by editing the plasmid pL2R-gRNA-0959 and knocking out the ZMO0959 site of the ZMNP genome by electroporation. The plasmid pEZ15A-Ptet-tSlNDPS1-ApLS was transferred to the NPΔ0959 recombinant strain by electroporation to obtain NPΔ0959-PtSA. The recombinant strain NPΔ0959-PtSA was fermented, and the growth status of the recombinant strain and the yield of R-(-)-linalool were detected. 10.78 mg / L of LR-(-)-linalool ( Figure 9 ).

[0099] Example 13 Effective Strategy Integration

[0100] In the present invention, the ZMNP background strain was used to knock out ZMO0959, replace ZMO1094 with Pgap-IspA2S82F, and replace ZMO1650 with Ptet-tSlNDPS1-ApLS to obtain the strain NP-L. The plasmid pEZ15A-Ptet-tSlNDPS1-ApLS was then transferred into the NP-L recombinant strain NP-L-PtSA ( Figure 10 A). The recombinant strain NP-L-PtSA was fermented to detect the growth of the recombinant strain and the yield of R-(-)-linalool. 12.01 mg / L R-(-)-linalool was produced in 48 h ( Figure 10 B).

[0101] Example 14 Optimization of culture medium bottling volume

[0102] The recombinant strain NP-L-PtSA, which currently has the highest R-(-)-linalool strategy, was fermented at 40%, 60% and 80% of the bottling volume to test the growth of the recombinant strain and the yield of R-(-)-linalool. The results showed that there was no significant difference in the specific growth rate of NP-L-PtSA at different bottling volumes, but it can be seen that the maximum biomass of 60% (OD 600nm :4.81) than 40% (OD 600nm : 5.26) is lower, but 80% of the bottling volume has the maximum biomass at OD 600nm Only 2.81, a significant decrease ( Figure 11 A). 80% of the bottling volume produced 8.31 mg / L of R-(-)-linalool, 60% of the bottling volume produced 11.17 mg / L of R-(-)-linalool, and 40% of the bottling volume produced the highest R-(-)-linalool yield, reaching 12.24 mg / L ( Figure 11 B).

[0103] The present invention uses multi-dimensional technical means to improve the yield of R-(-)-linalool and reduce cytotoxicity: First, the R-(-)-linalool synthase (ApLS) is rationally designed, and NPPS (tSlNDPS1) and ApLS are expressed by linker fusion to promote product synthesis; secondly, the IspA2 mutant is overexpressed at the genomic level, and the expression of the endogenous MEP pathway rate-limiting enzymes DXS and IspG is upregulated to enhance the supply of the precursor substance GPP; then, the penicillin-binding protein-related gene ZMO0959 is knocked out to increase cell wall permeability, promote the efflux of R-(-)-linalool, and reduce its toxic side effects on cells; finally, the yield is further improved by optimizing the bottling volume of the culture medium. A summary of the above strain transformation strategies can be found in Figure 12 .

[0104] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A recombinant strain of Zymomonas mobilis for synthesizing R-(-)-linalool, characterized in that The genome ZMO1094 site of the recombinant strain is replaced with Pgap-IspA2 S82F , the ZMO1650 site is replaced with Ptet-tSlNDPS1-ApLS, and the ZMO0959 gene on the genome is knocked out, and an expression plasmid containing the gene tSlNDPS1 and the gene ApLS is transferred, or the recombinant strain is transferred with an expression plasmid containing the gene tSlNDPS1 and the gene ApLS D101M The expression plasmid, or the recombinant strain is transformed with the gene tSlNDPS1 and gene ApLS R294F or the recombinant strain is transformed into an expression plasmid containing the gene tSlNDPS1, a linker sequence, and the gene ApLS, or the recombinant strain is transformed into an expression plasmid containing the gene tSlNDPS1, ApLS and an expression plasmid containing the gene dxs1 / dxs2, ispG; The nucleotide sequence of the gene tSlNDPS1 is shown in SEQ ID NO: 1, the nucleotide sequence of the gene ApLS is shown in SEQ ID NO: 2, and the nucleotide sequence of the gene IspA2 is shown in SEQ ID NO: S82F The nucleotide sequence of the gene ApLS is shown in SEQ ID NO:

3. D101M The nucleotide sequence of the gene ApLS is shown in SEQ ID NO:

4. R294F The nucleotide sequence of gene dxs1 is shown in SEQ ID NO: 5, the nucleotide sequence of gene dxs1 is shown in SEQ ID NO: 6, the nucleotide sequence of gene dxs2 is shown in SEQ ID NO: 7, the nucleotide sequence of gene ispG is shown in SEQ ID NO: 8, Ptet is an inducible promoter, the nucleotide sequence of which is shown in SEQ ID NO: 10, and Pgap is a constitutive promoter, the nucleotide sequence of which is shown in SEQ ID NO:

12.

2. The recombinant strain according to claim 1, characterized in that The genome ZMO1094 site of the recombinant strain is replaced with Pgap-IspA2 S82F , the ZMO1650 site was replaced with Ptet-tSlNDPS1-ApLS, the ZMO0959 gene on the genome was knocked out, and an expression plasmid containing the genes tSlNDPS1 and ApLS was transferred into the cell.

3. The recombinant strain according to claim 1, characterized in that The gene tSlNDPS1 is a truncated gene of neryl pyrophosphate synthase tSlNDPS1 from tomato; The gene ApLS is the linalool synthase gene ApLS derived from Pleurotus ostreatus.

4. The recombinant strain according to claim 1, wherein The gene ApLS D101M A mutant of the linalool synthase gene ApLS from Agaricus truncatus D101M .

5. The recombinant strain according to claim 1, characterized in that The gene ApLS R294F A mutant of the linalool synthase gene ApLS from Agaricus truncatus R294F .

6. The recombinant strain according to claim 1, characterized in that The linker sequence is a nucleotide sequence encoded by a connecting peptide (PT)4P, and tSlNDPS1 is fused with ApLS via the connecting peptide (PT)4P.

7. The recombinant strain according to claim 1, characterized in that The genes dxs1 / dxs2 and ispG are derived from Zymomonas mobilis.

8. A method for constructing a recombinant strain of Zymomonas mobilis for synthesizing R-(-)-linalool according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Construction of expression plasmid or editing plasmid; S2. Transforming the expression plasmid or editing plasmid into Zymomonas mobilis to obtain transformants.

9. Use of the recombinant Zymomonas mobilis strain for synthesizing R-(-)-linalool according to any one of claims 1 to 7 in the production of R-(-)-linalool.

10. A method for producing R-(-)-linalool, characterized in that, The method comprises the following steps: culturing the recombinant Zymomonas mobilis strain according to any one of claims 1 to 11, adding isopropyl myristate to carry out two-phase fermentation to obtain a fermentation broth, and extracting the isopropyl myristate phase of the fermentation broth; wherein the culture medium is RMG5 culture medium.

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