Construction method and application of perillyl alcohol production strain

Abstract

The invention discloses a construction method of a perillyl alcohol production strain and application of the perillyl alcohol production strain. The construction method comprises the following steps: transferring a plasmid pESKKDI carrying a mevalonic acid pathway, and a plasmid pNLsP450Fdx carrying a tomato Solanum lypersicum sourced NPP synthetase, a lemon Citrus limon sourced gene Ls and a Mycobacterium sp gene P450 gene into a gene knockout defective bacterium delta YqhD of wild type escherichia coli BL21 (DE3); the obtained perillyl alcohol production strain is used for production of perillyl alcohol, the yield of perillyl alcohol is improved and generation of a by-product perillyl aldehyde is reduced by further over-expressing electron transfer protein (Fdx) and optimizing the concentration of initial glucose in a fermentation culture medium, and a favorable guidance direction is provided for production of perillyl alcohol by escherichia coli.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method for constructing a perillyl alcohol-producing strain and its application. Background Art

[0002] Limonene is a monoterpene compound that is widely used in the food, pharmaceutical, and biofuel industries. Oxygenated derivatives of limonene have medicinal properties, such as perillyl alcohol, which has anti-cancer functions. This compound is produced by the directed oxygenation of limonene through whole-cell biotransformation.

[0003] In recent years, a large number of studies have shown that introducing and optimizing the MVA metabolic pathway in Escherichia coli can increase the supply of IPP and DMAPP, thereby improving the ability to synthesize limonene and its derivatives. By combining the MK gene promoter project to re-adjust the translation of EfMvaE and EfMvaS, the yield of limonene reached 1.29 g / L. At the same time, if an in-situ product removal method and a glycerol-limited fed-batch fermentation process are used, the yield of limonene can be further increased to 3.63 g / L.

[0004] Although the above methods have successfully produced a large amount of precursor compounds, the production of perillyl alcohol from glucose through the introduced heterologous MVA pathway in Escherichia coli is still limited. Although the current yield of perillyl alcohol in shake flasks is 100 mg / L and in a 5-L bioreactor is 453 mg / L. However, during the synthesis of perillyl alcohol, the formation of perillaldehyde as a by-product often affects the purity and yield of the target product. Perillaldehyde has strong toxicity, which may interfere with the normal metabolism of microorganisms and reduce the fermentation efficiency. To improve the production efficiency of perillyl alcohol, reducing the formation of the by-product perillaldehyde is an important optimization direction. Currently, although there are some methods attempting to regulate the expression of related genes through metabolic engineering, there are still few reports on engineering strains that can reduce the formation of perillaldehyde while ensuring high yields of perillyl alcohol. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this part, the abstract, and the title. However, such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for constructing a perillyl alcohol-producing strain.

[0008] To solve the above technical problems, the present invention provides the following technical solutions: including,

[0009] Knock out the ethanol dehydrogenase gene YqhD of Escherichia coli BL21(DE3) to obtain an Escherichia coli defective strain ΔYqhD, and the nucleotide sequence of the ethanol dehydrogenase gene YqhD is as shown in SEQ ID NO: 1;

[0010] The NPP synthase from Solanum lycopersicum with a nucleotide sequence as shown in SEQ ID NO: 2, the gene Ls from Citrus limon with a nucleotide sequence as shown in SEQ ID NO: 3, and the P450 synthase gene from Mycobacterium sp are codon-optimized and synthesized to obtain the plasmid pET-NLsP450;

[0011] The plasmids pESKKDI and pET-NLsP450 are co-transformed into the Escherichia coli defective strain ΔYqhD, and then the Fdx gene is repeatedly expressed to construct a perillyl alcohol-producing strain.

[0012] As a preferred scheme of the method for constructing the perillyl alcohol-producing strain of the present invention, wherein: the gene knockout operation of Escherichia coli is carried out by the CRISPR-Cas9 gene editing technology.

[0013] As a preferred scheme of the method for constructing the perillyl alcohol-producing strain of the present invention, wherein: the repeated expression of the Fdx gene is to express together with the P450 operon containing Cyp450, Fdx, and Fdr based on pET-NLsP450.

[0014] Another object of the present invention is to provide a perillyl alcohol-producing strain.

[0015] Another object of the present invention is to provide an application of the perillyl alcohol-producing strain in the production of perillyl alcohol.

[0016] Another object of the present invention is to provide a method for producing perillyl alcohol.

[0017] To solve the above technical problems, the present invention provides the following technical solutions: including,

[0018] The overnight seed liquid culture of the perillyl alcohol-producing strain is pre-cultured in LB medium until OD600 is 0.6 to obtain overnight seeds;

[0019] The overnight seeds are inoculated into M9 fermentation medium at an inoculation amount of 1% and cultured until OD600 is 0.6, and an inducer is added and cultured continuously, and the obtained fermentation broth contains perillyl alcohol products.

[0020] As a preferred embodiment of the method for producing perillyl alcohol according to the present invention, the culture temperature for the pre-culture is 37 °C and the rotation speed is 220 rpm.

[0021] As a preferred embodiment of the method for producing perillyl alcohol according to the present invention, the glucose concentration in the M9 fermentation medium is 10-15 g / L.

[0022] As a preferred embodiment of the method for producing perillyl alcohol according to the present invention, the inducer is IPTG.

[0023] As a preferred embodiment of the method for producing perillyl alcohol according to the present invention, the perillyl alcohol production strain has a perillyl alcohol yield increased by more than 30% compared to the wild-type strain, and the yield of the by-product perillaldehyde has decreased by more than 50%.

[0024] Advantages of the present invention:

[0025] The present invention constructs a perillyl alcohol production strain, its construction method and its application. The plasmid pESKKDI carrying the mevalonate pathway, the plasmid pNLsP450Fdx carrying the NPP synthase from Solanum lycopersicum, the gene Ls from Citrus limon, and the P450 gene from Mycobacterium sp are transferred into the gene knockout mutant ΔYqhD of the wild-type Escherichia coli BL21(DE3), which improves the yield of perillyl alcohol and reduces the generation of the by-product perillaldehyde, providing a favorable guiding direction for the production of perillyl alcohol by Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts. Among them:

[0027] Figure 1 It is the construction method of the strain for reducing the generation of the by-product perillaldehyde of perillyl alcohol according to the present invention.

[0028] Figure 2 It is the sequence map of the plasmid pET-NLsP450 constructed in Example 1 of the present invention.

[0029] Figure 3 It is the sequence map of the plasmid pET-Cyp450-P450 constructed in Example 1 of the present invention.

[0030] Figure 4Sequence map of plasmid pET-Cyp450-Fdx constructed in Example 1 of the present invention.

[0031] Figure 5 Sequence map of plasmid pET-Cyp450-Fdr constructed in Example 1 of the present invention.

[0032] Figure 6 Sequence map of plasmid pET-YqhD constructed in Example 1 of the present invention.

[0033] Figure 7 PCR verification of ΔYqhD strain with YqhD gene knocked out in Example 2 of the present invention.

[0034] Figure 8 Result diagram of perillaldehyde production detected by GC-MS in different strains in Example 2 of the present invention.

[0035] Figure 9 Result diagram of perilla alcohol and perillaldehyde production detected by GC-MS in different strains in Example 3 of the present invention.

[0036] Figure 10 Result diagram of perilla alcohol and perillaldehyde production detected by GC-MS in different strains in Example 4 of the present invention.

[0037] Figure 11 Result diagram of perilla alcohol and perillaldehyde production detected by GC-MS in different strains in Example 5 of the present invention. Detailed implementation manners

[0038] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be given in combination with the embodiments of the specification.

[0039] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0040] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that can be included in at least one implementation manner of the present invention. The "in one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selectively exclusive embodiment from other embodiments.

[0041] The raw materials used in the specific embodiments of the present invention are all commonly available in the market without special instructions.

[0042] Unless otherwise specified, the methods used in the specific embodiments of the present invention are conventional operating methods in the art.

[0043] For the construction of the pET-Cyp450 plasmid used in the embodiments of the present invention, reference is made to the plasmid pET-Cyp450 carrying perillyl alcohol synthase derived from Mycobacterium sp in the patent "A High Performance Liquid Chromatography Detection Method for Perillyl Alcohol in a Strain Fermentation Broth", with the publication number CN116559307. The nucleotide sequence is as shown in SEQ ID NO: 1 in the application document.

[0044] For the construction of the pESKKDI plasmid used in the embodiments of the present invention, reference is made to the plasmid pACYC-ESMPD-idi in the patent "A Lycopene Cyclase Mutant and Its Application in Constructing High-Yield Retinal Bacteria", with the publication number CN118562778A. The nucleotide sequence is as shown in SEQ ID NO: 5 in the application document.

[0045] Determination of the concentrations of perillyl alcohol and perillaldehyde in the present invention:

[0046] High performance gas chromatography (GC-MS) was used to quantify perillyl alcohol and perillaldehyde. An RTX-5MS chromatographic column (30 m × 0.32 mm × 0.25 μm) was used, and a gas chromatography-mass spectrometry instrument from Shimadzu, Japan was used to analyze limonene and perillyl alcohol. 1 μL of the ethyl acetate extract was injected with a split ratio of 10, and helium was used as the carrier gas. The initial temperature of the column was maintained at 80 °C for 2 minutes, and then gradually increased to 250 °C at a rate of 20 °C / min and held for 5 minutes. The mass spectrometer was operated in the scan mode (m / z 30–500) for compound identification. Standard samples of perillyl alcohol and perillaldehyde were prepared in ethyl acetate solvents at different concentrations to calibrate the standard curve.

[0047] The plasmids used in the specific embodiments of the present invention are shown in Table 1.

[0048] Table 1

[0049] Plasmids Description Reference pETDuet-1 An expression vector, T7 promoter / lac operator, ColE1 ori, Ampr, two MCS Novagene pESKKDI pACYC-PT7-mvaE-mvaS-PT7-mvK-pmK-pmk-mvaD-Idi; Kanr This study pET-Cyp450-P450 pET-PT7-P450-Fdx-Fdr-PT7-P450, Ampr This study pET-Cyp450-Fdr pET-PT7-P450-Fdx-Fdr-PT7-Fdx, Ampr This study pET-Cyp450-Fdx pET-PT7-P450-Fdx-Fdr-PT7-Fdr, Ampr This study pET-YqhD pET-PT7-YqhD, Ampr This study pNLsP450 pET-PT7-NPP-Ls-PT7-P450-Fdx, Ampr This study pNLsP450Fdx pET-PT7-NPP-Ls-PT7-P450-Fdx, Ampr This study pCas RepA101(Ts) ori, Kanr, Pcas-cas9, ParaC-Red, lacIq Novagene pTarget sgRNA plasmid, pMB1 ori, Sper Novagene

[0050] The strains constructed in the specific embodiments of the present invention are shown in Table 2.

[0051] Table 2

[0052]

[0053]

[0054] The primer sequences used in the specific embodiments of the present invention are shown in Table 3.

[0055] Table 3

[0056]

[0057] In the second specific embodiment of the present invention, the operations of PCR, plasmid extraction, DNA purification, ligation, PCR and restriction digestion are all carried out in accordance with the standard operating procedures of molecular biology and the instructions of the kit. The plasmid construction adopts the Gibson assembly method, and the relevant systems are shown in Tables 4 to 7.

[0058] Table 4 PCR reaction system

[0059]

[0060]

[0061] The PCR reaction conditions are as follows:

[0062]

[0063] Table 5 Restriction digestion reaction system

[0064]

[0065] Table 6 Enzyme ligation reaction system

[0066]

[0067] Table 7 Gibson assembly reaction system

[0068]

[0069]

[0070] Plasmid in the construction process of Example 1

[0071] 1) Entrust Jinweizhi Biotechnology Co., Ltd. in Suzhou to synthesize the NPP synthase from Solanum lycopersicum, the gene Ls from Citrus limon, and the gene P450 synthase (pET-Cyp450 gene) from Mycobacterium sp. through codon optimization. The sequences of the NPP synthase from Solanum lycopersicum and the gene Ls from Citrus limon are shown in SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the plasmid pET-NLsP450 is obtained, as Figure 2 shown.

[0072] 2) Design primers Cyp-F / Cyp-R, Fdr-F / Fdr-R, Fdx-F / Fdx-R respectively, and use the plasmid pET-Cyp450 as a template to amplify the P450, Fdr, and Fdx fragments respectively;

[0073] The plasmid pET-Cyp450 and the fragments P450, Fdr, and Fdx were each double-digested with NdeⅠ and EcoRV, and then ligated using DNA ligase (ligation mix). After sequencing verification, plasmids pET-Cyp450-P450, pET-Cyp450-Fdr, and pET-Cyp450-Fdx were obtained respectively.

[0074] The sequence map of pET-Cyp450-P450 is as Figure 3 shown, and the sequence map of pET-Cyp450-Fdx is as Figure 4 shown, and the sequence map of pET-Cyp450-Fdr is as Figure 5 shown.

[0075] 3) Primers YqhD-F / YqhD-R and pET-F / pET-R were designed respectively. Using BL21(DE3) as a template, the YqhD fragment was amplified, and using pET-Duet as a template, the pET fragment was amplified. The YqhD and pET fragments were assembled by Gibson assembly method to obtain plasmid pET-YqhD, and the sequence map is as Figure 6 shown.

[0076] The Fdx fragment was amplified by primers xhoi-fdx-F and avrii-fdx-R, and the pET-NLsP450 vector fragment was amplified by primers NLsp450-F and NLsp450-R. The Fdx and pET-NLsP450 fragments were assembled by Gibson assembly method to obtain plasmid pNLsP450Fdx.

[0077] Example 2 Knockout of gene YqhD based on wild-type Escherichia coli BL21(DE3)

[0078] 1) The CRISPR-Cas9 gene editing technology was used for gene knockout operation in Escherichia coli. First, the DNA sequences of the gene to be knocked out and its 500 bp upstream and downstream were searched on the website NCBI (National Center Of Biotechnology Information);

[0079] Primers were designed based on the genome of Escherichia coli BL21(DE3). The 500 bp DNA fragment L500 upstream of the target gene (YqhD) and the 500 bp DNA fragment R500 downstream were obtained by PCR. The L500 and R500 were amplified by fusion PCR technology to obtain the template (repair fragment). The nucleotide sequence of the gene YqhD is shown in SEQ ID NO: 1.

[0080] 2) Design sgRNA sequences targeting the target gene using the online website ChopChop (http: / / chopchop.cbu.uib.no / ). Enter the target gene sequence and select Escherichia coli strain BL21 (DE3) (NC 012971.2) as the host. Generate several sets of 23-bp target sequences. Design primers based on the selected appropriate N20 sequence. Perform whole-plasmid PCR using the plasmid pTarget (containing the [Spc] resistance gene) as a template. Replace the N20 fragment of the original plasmid to construct the pTarget plasmid targeting the target gene.

[0081] 3) Prepare competent E. coli BL21(DE3) and transform with plasmid pCas (containing the [Kan] resistance gene). Cultivate overnight in a 30°C incubator and verify positive clones using colony PCR.

[0082] Pick the positive clones and inoculate them into a 50 mL centrifuge tube containing 5 mL of kanamycin-resistant LB medium, and incubate at 30°C, 220 r·min. -1 Overnight culture;

[0083] Transfer the culture to 100 mL of kanamycin-resistant medium at a 1% inoculum volume and culture at 30°C for 1 h. Then, add 1% 1M arabinose (final concentration 10 mM) to induce expression of the recombinant enzyme. When the OD600 reaches 0.6, remove the shake flask and place it in an ice bath for 15 min. Collect the bacterial solution in a 50 mL centrifuge tube and centrifuge at 6000 r / min. -1 Centrifuge for 10 minutes and discard the supernatant.

[0084] Add 25 mL of pre-cooled ultrapure water to the centrifuge tube to fully suspend the bacterial sediment at the bottom, and then centrifuge at 6000 r·min. -1 Centrifuge for 10 min and repeat the above steps;

[0085] Then, 25 mL of pre-cooled 10% glycerol was added to slightly suspend the bacterial precipitate and the mixture was stirred at 6000 r·min. -1 Centrifuge for 10 min and repeat the above steps.

[0086] Finally, resuspend the bacteria in 500uL of 10% glycerol and dispense into 1.5mL centrifuge tubes.

[0087] 4) Add the obtained plasmid pTarget (100 ng) and template (400 ng) in a ratio of 1:4 to the prepared competent cells for electrotransformation. After recovery culture, coat them on a plate containing streptomycin and kanamycin resistance, and incubate at 30 °C. Verify whether the target gene has been successfully knocked out by colony PCR. The successfully knocked-out YqhD strain is ΔYqhD. The verification by colony PCR is shown in the figure. The band with a molecular weight of around 1000 is the positive colony, as Figure 7 shown.

[0088] 5) Transform the empty plasmid pET-Duet into the wild-type Escherichia coli strain and the Escherichia coli mutant ΔYqhD to obtain strains BL21 and ΔYqhD, respectively. Transform the pET-YqhD gene into the wild-type Escherichia coli strain and the Escherichia coli mutant ΔYqhD to obtain strains YqhD and ΔYqhD / YqhD.

[0089] 6) Pre-culture the overnight seed liquid cultures of the above 4 strains in LB medium at 37 °C with 220 rpm. Inoculate the overnight seeds in 5 ml of LB fermentation medium and culture at 37 °C with 220 rpm until the OD600 reaches 0.6. Add 0.5 mM IPTG as an inducer and 100 mg / L of perillyl alcohol as a substrate, and culture at 30 °C and 220 rpm for 24 h.

[0090] 7) Take the fermentation broth and centrifuge it at 13000 rpm for 10 min. Aspirate the supernatant and add an equal volume of ethyl acetate for shaking extraction. Aspirate the upper ethyl acetate extract and add Na2SO4 for water removal. Take the supernatant and use GC-MS to detect perillyl alcohol and perillaldehyde. The results are as Figure 8 shown. Gene knockout resulted in a decrease in the perillaldehyde yield from 7.52 mg / L in the control strain BL21 to 3.89 mg / L in the knockout strain ΔYqhD. In contrast, the overexpression strain pET-YqhD of YqhD increased the perillaldehyde yield to 10.19 mg / L. The perillaldehyde yield was restored to a level comparable to that of the control strain BL21 by the complementation strain ΔYqhD / YqhD of the YqhD gene, indicating that the YqhD gene plays a role in the conversion of perillyl alcohol to perillaldehyde.

[0091] Example 3 Construction of a recombinant bacterium for reducing the by-product perillaldehyde of perillyl alcohol

[0092] 1) Co-transform pESKKDI and pNLsP450 into the BL21(DE3) and ΔYqhD strains to obtain strains POH-1 and POH-2;

[0093] 2) De novo biosynthesis of perillyl alcohol was carried out in M9 medium, and the composition of M9 medium was as follows: 10 g / L glucose, 33.7 mmol of Na2HPO4 -1 , 22.0 mmol of KH2PO4 -1 , 8.55 mmol of NaCl -1 , 9.35 mmol of NH4Cl -1 , 1 mmol of MgSO4 -1 , 0.3 mmol of CaCl2 -1 , 1 mg / L of biotin -1 , 1 mg / L of thiamin -1 , 0.134 mmol / L of EDTA -1 , 31 mmol of FeCl3·6H2O -1 , 6.2 mmol of ZnCl2 -1 , 0.76 mmol of CuCl2·2H2O -1 , 0.42 mmol of CoCl2·2H2O -1 , 1.62 mmol of H3BO3 -1 , 0.081 mmol of MnCl2·4H2O -1 , 3 g / L of yeast -1 .

[0094] 3) The overnight seed cultures of strains POH-1 and POH-2 were pre-cultured in LB medium at 37 °C with 220 rpm. The overnight seeds were inoculated into 20 ml of M9 fermentation medium and cultured at 37 °C with 220 rpm until the OD600 reached 0.6. 0.5 mM IPTG was added as an inducer, and the culture was continued at 30 °C and 220 rpm for 48 h.

[0095] 4) The fermentation broth was centrifuged at 13,000 rpm for 10 min. The supernatant was taken and an equal volume of ethyl acetate was added for shaking extraction. The upper ethyl acetate extract was taken and Na2SO4 was added for water removal. The supernatant was used for GC-MS detection of perillyl alcohol and perillaldehyde. The results were as Figure 9 shown. The YqhD gene knockout strain (POH-2) produced 52.37 mg / L of perillyl alcohol, which was 31.35% higher than that of the wild-type strain (POH-1) at 39.87 mg / L. The content of perillaldehyde decreased from 3.44 mg / L by 52.91% to 1.62 mg / L, demonstrating that YqhD gene knockout could effectively increase perillyl alcohol and reduce the formation of the by-product perillaldehyde.

[0096] Example 4: Increasing the production of perillyl alcohol by overexpressing the electron transfer protein (Fdx)

[0097] The plasmids pET-Cyp450, pET-Cyp450-P450, pET-Cyp450-Fdr, and pET-Cyp450-Fdx were separately transformed into BL21(DE3) bacteria to obtain the strains Cyp-operon, Cyp, Fdr, and Fdx, and a 5 mM limonene substrate was added.

[0098] The GC-MS analysis results showed that: Based on the expression of the Cyp 450 operon, the expression of P450, Fdx, and Fdr within the operon was independently increased, achieving a perillyl alcohol production of 56.54 mg / L, as Figure 10 shown. It was demonstrated that the further enhancement of Fdx expression led to an increase in perillyl alcohol production.

[0099] The plasmids pESKKDI and pNLsP450 were co-transformed into the ΔYqhD strain to obtain the strain POH-1, and the plasmids pESKKDI and pNLsP450Fdx were co-transformed into the ΔYqhD strain to obtain the strain POH-2. The GC-MS analysis results showed that: Compared with the POH-1 strain, the additional expression of the Fdx electron transfer protein further increased the perillyl alcohol production to 117.58 mg / L, and the production increased by 2.25 times.

[0100] Example 5 Optimization of the initial glucose concentration

[0101] The plasmids pESKKDI and pNLsP450Fdx were co-transformed into the ΔYqhD strain, and a 10% dinonyl phthalate (DINP) extractant was added to M9 medium with different initial concentrations of 10 g / L, 15 g / L, and 20 g / L. Fermentation was carried out at 30 °C for 48 h. The upper layer of the DINP extractant was aspirated and diluted with ethyl acetate. The GC-MS analysis results showed that: A glucose concentration of 10 g / L resulted in a perillyl alcohol production of 186.1 mg / L. Increasing the glucose concentration further increased the perillyl alcohol production, and the highest titer observed at 15 g / L glucose was 309.1 mg / L. However, at 20 g / L glucose, the perillyl alcohol production decreased to 208 mg / L, as Figure 11 shown. These results indicate that the optimal glucose concentration is beneficial for increasing the perillyl alcohol production.

[0102] In summary, an engineered Escherichia coli strain that reduces the by-product perillaldehyde of perillyl alcohol was constructed in this invention. When it was used to construct a perillyl alcohol-producing strain, the gene knockout-deficient strain ΔYqhD of the wild-type Escherichia coli BL21(DE3) carrying the plasmid pESKKDI with the mevalonate pathway, the plasmid pNLsP450Fdx carrying the NPP synthase from Solanum lycopersicum, the gene Ls from Citrus limon, and the P450 gene from Mycobacterium sp improved the yield of perillyl alcohol and reduced the formation of the by-product perillaldehyde, providing a favorable guiding direction for the production of perillyl alcohol by Escherichia coli.

[0103] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing a perillyl alcohol-producing strain, characterized by: include, The alcohol dehydrogenase gene YqhD of Escherichia coli BL21 (DE3) was knocked out to obtain the Escherichia coli deficient strain ΔYqhD, wherein the nucleotide sequence of the alcohol dehydrogenase gene YqhD is shown in SEQ ID NO: 1; The NPP synthase from Solanum lycopersicum, a tomato with a nucleotide sequence as shown in SEQ ID NO: 2, the Ls gene from Citrus limon with a nucleotide sequence as shown in SEQ ID NO: 3, and the P450 synthase gene from Mycobacterium sp were synthesized by codon optimization to obtain the plasmid pET-NLsP450. Plasmids pESKKDI and pET-NLsP450 were co-transformed into the defective ΔYqhD strain of Escherichia coli, and the Fdx gene was repeatedly expressed to construct a perillyl alcohol-producing strain.

2. The method for constructing a perillyl alcohol-producing strain according to claim 1, wherein: Gene knockout in Escherichia coli was performed using CRISPR-Cas9 gene editing technology.

3. The method for constructing a perillyl alcohol-producing strain according to claim 2, wherein: The repeated expression of the Fdx gene is performed on the basis of pET-NLsP450 and is expressed together with the P450 operon containing Cyp450, Fdx and Fdr.

4. The perillyl alcohol-producing strain constructed by the construction method according to any one of claims 1 to 3.

5. Use of the perillyl alcohol-producing strain as claimed in claim 4 in producing perillyl alcohol.

6. A method for producing perillyl alcohol, characterized in that: The perillyl alcohol producing strain according to claim 4 further comprises: Overnight seed liquid culture of the perillyl alcohol-producing strain was pre-cultured in LB medium to an OD600 of 0.6 to obtain overnight seeds; The overnight seeds were inoculated into M9 fermentation medium at an inoculum rate of 1% and cultured until OD600 reached 0.

6. An inducer was added and the culture was continued. The obtained fermentation liquid contained perillyl alcohol product.

7. The method for producing perillyl alcohol according to claim 6, wherein: The culture temperature of the pre-culture is 37° C. and the rotation speed is 220 rpm.

8. The method for producing perillyl alcohol according to claim 7, wherein: The glucose concentration in the M9 fermentation medium is 10-15 g / L.

9. The method for producing perillyl alcohol according to claim 6, wherein: The inducer is IPTG.

10. The method for producing perillyl alcohol according to claim 6, wherein: Compared with the wild-type strain, the perillyl alcohol production strain of claim 4 increases the perillyl alcohol production by more than 30%, and reduces the production of the by-product perillaldehyde by more than 50%.

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