A recombinant bacterium for synthesizing key components of plant essential oil, and its construction method and application

Abstract

The present invention discloses a recombinant bacterium for synthesizing key components of plant essential oil, a construction method thereof and applications. The recombinant bacterium contains exogenous genes; the exogenous genes include genes of terpene synthase, cytochrome P450 and short-chain dehydrogenase; the key components of the plant essential oil include thymol and carvacrol. By introducing the genes of terpene synthase, cytochrome P450 and short-chain dehydrogenase, the present invention successfully constructs a recombinant bacterium capable of synthesizing thymol and carvacrol, realizing the heterologous synthesis of thymol derivatives. Moreover, by using the recombinant bacterium of the present invention to transform and produce thymol and carvacrol, the production efficiency is high, it is green and environmentally friendly, and the cost is low, having good industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology. Specifically, it relates to a recombinant bacterium for synthesizing key components of plant essential oils, and a construction method and application thereof. Background Art

[0002] Thymol, also known as thymol, is mainly derived from plants of the genus Thymus and is the main component of thyme volatile oil. In the fragrance field, thymol has a special aroma and is often used in the preparation of various flavors, such as citrus and mushroom flavors. Thymol is not only widely used in the fragrance field, but also because of its antiseptic property, it can also be used as a medical drug and often appears in some hygienic products with medicinal value, such as toothpaste and soap. At the same time, thymol can also be used to treat wounds and store anatomical specimens. In addition, thymol can also be used as an antioxidant, a special reagent for detecting titanium nitride, a comparison standard for thymol blue indicator, and a primary raw material for synthesizing many chemicals. The genus Thymus contains 300 - 400 species of aromatic herbs, and most of the Thymus genus is often used to treat dry cough, bronchitis, laryngitis, and indigestion. Research shows that the biological activity of the Thymus genus mainly comes from the structure of its phenolic hydroxyl group, especially thymol and carvacrol. The essential oil content in thyme is much higher than that in carvacrol, and these compounds have 30 times higher antibacterial activity than phenol and 4 times lower toxicity than phenol.

[0003] In recent years, thymol and carvacrol have been mostly used in the production of fragrances, preservatives, medical supplies, and insect repellents. Research shows that thymol has various biological activities such as acaricidal and bacteriostatic effects. The viricidal effect of thymol on Rhizoglyphus echinopus was studied, and it was found that thymol has an obvious inhibitory effect on the growth of Bacillus. In recent years, thymol derivatives have received extensive attention from domestic and foreign researchers, and more and more thymol derivatives have been synthesized.

[0004] Currently, the main synthesis method of thymol and its derivatives on the market is chemical synthesis, and there is still a large room for development in biological synthesis. Using microorganisms to synthesize thymol and its derivatives can effectively reduce the production cost of enterprises. Therefore, it is urgent to create a more efficient preparation method for thymol and carvacrol.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant bacterium for synthesizing key components of plant essential oils, and a construction method and application thereof, through which a higher yield of thymol and carvacrol can be produced.

[0007] The present invention is implemented as follows:

[0008] In a first aspect, the present invention provides a recombinant bacterium for synthesizing key components of plant essential oils, which contains exogenous genes; the exogenous genes include genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase;

[0009] The above key components of plant essential oils include thymol and carvacrol.

[0010] In a second aspect, the present invention provides a method for constructing the above recombinant bacterium for synthesizing key components of plant essential oils, which includes the following steps: connecting the genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase after codon optimization to an expression vector, and then introducing the obtained recombinant expression vector into a starting bacterium to obtain the recombinant bacterium.

[0011] In a third aspect, the present invention provides an application of the above recombinant bacterium, which includes using the above recombinant bacterium to ferment and produce thymol and carvacrol.

[0012] The present invention has the following beneficial effects:

[0013] By introducing the genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase, the present invention successfully constructs a recombinant bacterium that can synthesize thymol and carvacrol, realizing the heterologous synthesis of thymol derivatives. Moreover, using the recombinant bacterium of the present invention to transform and produce thymol and carvacrol has high production efficiency, is green and environmentally friendly, and has low cost, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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 use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0015] Figure 1 It is the synthesis pathway of thymol and carvacrol in the present invention;

[0016] Figure 2 It is a schematic diagram of the construction of the recombinant plasmid in the present invention, where Figure 2 -A is TvTPS2-PrTEFin-TvSDR1-T-pcfb4586, Figure 2 -B is TvTPS2-PrTEFin-TvCYP71D507-T-pcfb5791;

[0017] Figure 3 It is the comparison of the dry weight data of the recombinant yeast in Experimental Example 1;

[0018] Figure 4Analysis of products of recombinant yeast at different fermentation times in Experimental Example 2;

[0019] Figure 5 Comparison of product contents of recombinant yeast after fermentation in Experimental Example 2;

[0020] Figure 6 Comparison of product contents of recombinant bacteria constructed with different cytochrome p450 genes in Experimental Example 3. Detailed implementation manners

[0021] To make the objectives, 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. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0022] The present invention provides a recombinant bacterium for synthesizing key components of plant essential oils. The recombinant bacterium contains foreign genes; the foreign genes include genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase; the above-mentioned key components of plant essential oils include thymol and carvacrol.

[0023] In the present invention, the inventors first introduced the genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase in thyme into the starting bacterium, so that geranyl diphosphate is cyclized to γ-terpinene under the action of terpene synthase (TvTPS2). Cytochrome P450 (TvCYP71D507-T) hydroxylates γ-terpinene at C-3 or C-6, and the dienol intermediate is converted to the corresponding ketone by short-chain dehydrogenase (SDR). The synthesis pathway is as Figure 1 shown.

[0024] According to the above reaction principle, the inventors introduced the genes encoding terpene synthase, cytochrome P450, and short-chain dehydrogenase into the starting bacterium to obtain a recombinant bacterium, and then used the recombinant bacterium to ferment and produce thymol and carvacrol.

[0025] In some embodiments, the genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase are derived from thyme.

[0026] In some embodiments, the starting bacterium of the recombinant bacterium is yeast; more preferably, the yeast is Yarrowia lipolytica, specifically, the Yarrowia lipolytica is the Yarrowia lipolytica pO1f strain.

[0027] In the present invention, the starting strain Yarrowia lipolytica is selected because it has a more efficient pentose phosphate pathway and acetyl-CoA metabolism ability compared to other yeasts, which can provide a large amount of NADPH and substrates for fatty acid synthesis. Yarrowia lipolytica cells have a high oil content, reaching more than 30% of the dry weight. Moreover, Yarrowia lipolytica is a safe strain that can be industrially produced, has obtained the GRAS certification from the US FDA, and has mature fermentation production technology, making it an ideal chassis organism for producing terpenoids.

[0028] To obtain the above recombinant strain, the present invention also provides a method for constructing the recombinant strain, which includes ligating the genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase after codon optimization to an expression vector, and then introducing the obtained recombinant expression vector into the starting strain to obtain the recombinant strain.

[0029] In some embodiments, the terpene synthase includes TvTPS2, and the nucleotide sequence of codon-optimized TvTPS2 is shown in SEQ ID NO.1.

[0030] In some embodiments, the cytochrome P450 includes TvCYP71D507-T, and the nucleotide sequence of codon-optimized TvCYP71D507-T is shown in SEQ ID NO.2.

[0031] During the research process, the inventors screened among multiple cytochrome P450 genes and obtained the gene TvCYP71D507-T that can form a synthesis pathway with terpene synthase and short-chain dehydrogenase as shown in Figure 1 This synthesis pathway containing this gene can not only synthesize thymol but also simultaneously synthesize carvacrol.

[0032] In some embodiments, the short-chain dehydrogenase includes TvSDR1-T, and the nucleotide sequence of codon-optimized TvSDR1-T is shown in SEQ ID NO.3.

[0033] In the present invention, TvTPS2 、 TvCYP71D507-T and TvSDR1-T all originate from thyme. Since the original sequences may result in the situation that the genes cannot be expressed or the expression efficiency is extremely low in constructing the recombinant strain, after obtaining the amino acid sequences of TvTPS2 、 TvCYP71D507-T and TvSDR1-T, the inventors carried out codon optimization according to the preference of the starting strain and synthesized three optimized nucleotide sequences.

[0034] In some embodiments, the expression vectors include plasmid pCfB4586, plasmid pCfB4778, and plasmid pCfB5791.

[0035] In the present invention, the genes of the above-mentioned terpene synthase, cytochrome P450, and short-chain dehydrogenase are co-expressed in three gene combinations. For the method of introducing the plasmids carrying the coding genes of the above-mentioned enzymes, any two of the coding genes may be present on the same plasmid, or the three genes may be present on different plasmids respectively, or other introduction methods may be used. The present invention does not limit this.

[0036] Due to the special promoter and terminator elements in plasmid pCfB4586, plasmid pCfB4778, and plasmid pCfB5791, the combination of any two genes can achieve the highest efficiency and can insert the constructed expression cassette as a whole into a specific yeast genome. Therefore, more preferably, any two of the above three genes are used as a group to jointly construct an expression plasmid. For example, the genes encoding terpene synthase and cytochrome P450 are combined to construct a recombinant plasmid, or the genes encoding terpene synthase and short-chain dehydrogenase are combined to construct a recombinant plasmid. The construction schematic diagram is as Figure 2 shown. The constructed recombinant plasmids can be respectively represented as TvTPS2-PrTEFin-TvSDR1-T-pcfb4586( Figure 2 -A) and TvTPS2-PrTEFin-TvCYP71D507-T-pcfb5791( Figure 2 -B).

[0037] The present invention also provides the application of the above-mentioned recombinant bacteria, and the recombinant bacteria can produce thymol and carvacrol by fermentation.

[0038] In some embodiments, the steps of the recombinant bacteria producing thymol and carvacrol by fermentation include: activating the recombinant bacteria, then inoculating them into a liquid medium, and after culturing, subjecting the obtained bacterial liquid to vacuum freeze-drying and grinding to obtain a bacterial powder containing thymol and carvacrol.

[0039] Specifically, the steps include: overnight activating and culturing the above-mentioned recombinant strains at 28 °C, then measuring their respective OD600, adjusting their OD600 to be the same, and then taking the same bacterial liquid and inoculating it into 100 mL of YPD (i.e., yeast extract peptone dextrose medium) liquid medium, culturing at 25 - 30 °C and 200 - 220 rpm for 36 - 72 h, then collecting the cell bacterial liquid and storing it in a -80 °C refrigerator for 1 day, and then placing it in a vacuum freeze-dryer to completely dry it, and finally grinding to obtain the bacterial powder of each strain.

[0040] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0041] Example 1

[0042] This example provides a method for constructing a recombinant bacterium for synthesizing key components of plant essential oil, and its steps include:

[0043] 1. Obtaining of target genes

[0044] The exogenous genes required in this example include TvTPS2, TvCYP71D507-T, and TvSDR1-T, all of which are derived from thyme. The nucleotide sequences after codon optimization are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively.

[0045] 2. Construction of recombinant vectors

[0046] The plasmids used in this example are pCfB4586 plasmid and pCfB5791 plasmid.

[0047] A recombinant plasmid is constructed by combining the genes of TvTPS2 and TvCYP71D507-T, and a recombinant plasmid is constructed by combining the genes of TvTPS2 and TvSDR1-T. The constructed recombinant plasmids can be represented as TvTPS2-PrTEFin-TvSDR1-T-pcfb4586 and TvTPS2-PrTEFin-TvCYP71D507-T-pcfb5791 respectively. The construction method of TvTPS2-PrTEFin-TvSDR1-T-pcfb4586 is as follows:

[0048] (1) The target genes and promoter fragments are amplified by PCR using the high-fidelity enzyme Phusion U Hot Start DNA Polymerase. The primers are shown in the following table, and the system is as follows:

[0049]

[0050]

[0051] The reaction procedure is: 98°C, 1 min → (98°C, 10 s → 54°C, 30 s → 72°C, 1 min) × 35 → 72°C, 5 min → 10°C. After the reaction, the PCR liquid is subjected to agarose gel electrophoresis, and each correct target fragment is recovered to obtain the TvTPS2, PrTEFin, PrGPD, and TvSDR1-T gene fragments.

[0052] (2) Digest the pCfB4586 plasmid with AsiSI first. The system is as shown in the following table:

[0053]

[0054] After reacting at 37 °C for 2 h, gel extraction was performed, followed by digestion with Nb.BsmI. The digestion system was as follows:

[0055]

[0056] React at 65 °C for 1 h, and then perform gel extraction to obtain the end-modified linearized plasmid.

[0057] (3) Assemble each gene fragment onto the pCfB4586 vector using the USER cloning method. The system was as follows:

[0058]

[0059]

[0060] The reaction program was: 37 °C, 25 min → 25 °C, 10 min → 4 °C, hold. Transform the above reaction solution into DH5α. The specific steps refer to...

[0061] (4) Screening and detection of DH5α transformants

[0062] Pick monoclonal colonies and perform colony PCR detection using the PR-14617 / PR-14619 primers. The reaction system was as follows:

[0063]

[0064] The reaction program was: 98 °C, 3 min → (98 °C, 15 s → 56 °C, 15 s → 72 °C, 1 min) × 35 → 72 °C, 5 min → 12 °C, hold. Then detect by DNA gel electrophoresis and confirm by sequencing.

[0065] The construction method of TvTPS2-PrTEFin-TvCYP71D507-T-pcfb5791 was the same as that of TvTPS2-PrTEFin-TvSDR1-T-pcfb4586. The primers used in the above construction process are shown in Table 1:

[0066] Table 1 Primer sequence list

[0067]

[0068]

[0069] 3. Yeast transformation and screening

[0070] The yeast strain used in this example was Yarrowia lipolytica pO1f strain.

[0071] (1) Streak the transformed strain on a YPD plate one day in advance and culture it.

[0072] (2) Take 90 μL of PEG6000 and 5 μL of lithium acetate in a 1.5 mL sterilized EP tube, and then add 5 μL of salmon sperm that has been treated at 100 °C for 5 min to it.

[0073] (3) Then pick a cell mass the size of a mung bean into the EP tube, shake it for 10 - 15 s, and then add 500 ng of linearized expression plasmid; after shaking for 10 - 15 s, react at 30 °C for 10 min, shake it for 10 - 15 s again, repeat this process 3 times, then react at 39 °C for 10 min, and finally add 100 μL of sterilized ddH2O to it, mix well and take 100 μL to coat on the SC-ura plate.

[0074] (4) Place it in an incubator at 28 °C for 2 - 4 days to grow single colonies. When picking the transformed yeast single colonies and streaking them on the SC-ura plate, simultaneously inoculate them into the SC-ura medium, extract their genomes after overnight culture, and use them as templates to perform PCR to detect whether the corresponding genes are integrated into the target sites of the yeast genome.

[0075] For the smooth transformation of the next plasmid and the screening of monoclonal colonies, it is necessary to first remove the URA tag in the positive clones screened in step (4). Since there are LoxP sites at both ends of URA on the constructed vector, the cre-loxp system is used to remove the URA tag. The specific method is as follows:

[0076] Transfer the Cre plasmid into the positive clones obtained in step (4), coat it on the SC-leu plate after transformation. Place it in an incubator at 28 °C for 2 - 4 days to grow single colonies. Then streak the grown monoclonal colonies on the YPD solid plate for two rounds of subculture, and then streak the clones in the second round on YPD, SC-leu, and SC-ura plates. After about 5 - 6 rounds of subculture, there will be monoclonal colonies that only grow on YPD, that is, the transformed yeast with the tag removed.

[0077] After multiple rounds of transformation screening, the target recombinant bacteria are finally obtained.

[0078] Example 2

[0079] This example provides a method for the recombinant bacteria obtained in Example 1 to ferment and produce thymol and carvacrol. The specific steps are as follows:

[0080] Activate and culture the recombinant bacteria obtained in Example 1 overnight at 28 °C, then measure their respective OD600. After adjusting their OD600 to be the same, take the same bacterial liquid and inoculate it into 100 mL of YPD liquid medium, culture it at 28 °C and 220 rpm for 36 h, then collect the cell bacterial liquid and store it in a -80 °C refrigerator for 1 day, and then place it in a vacuum freeze dryer to completely dry it, and finally grind it to obtain the bacterial powder of each strain.

[0081] Example 3

[0082] This example provides a method for the recombinant bacteria obtained in Example 1 to ferment and produce thymol and carvacrol. The specific steps are as follows:

[0083] The recombinant bacteria obtained in Example 1 were activated and cultured overnight at 28°C. Then, their OD600 values were measured. After adjusting their OD600 values to be the same, the same bacterial liquid was taken and inoculated into 100 mL of YPD liquid medium, and cultured at 28°C and 220 rpm for 48 h. Then, the cell bacterial liquid was collected and stored in an -80°C refrigerator for 1 day, and then completely dried in a vacuum freeze dryer, and finally ground to obtain the bacterial powder of each strain.

[0084] Example 4

[0085] This example provides a method for the recombinant bacteria obtained in Example 1 to ferment and produce thymol and carvacrol. The specific steps are as follows:

[0086] The recombinant bacteria obtained in Example 1 were activated and cultured overnight at 28°C. Then, their OD600 values were measured. After adjusting their OD600 values to be the same, the same bacterial liquid was taken and inoculated into 100 mL of YPD liquid medium, and cultured at 28°C and 220 rpm for 54 h. Then, the cell bacterial liquid was collected and stored in an -80°C refrigerator for 1 day, and then completely dried in a vacuum freeze dryer, and finally ground to obtain the bacterial powder of each strain.

[0087] Example 5

[0088] This example provides a method for the recombinant bacteria obtained in Example 1 to ferment and produce thymol and carvacrol. The specific steps are as follows:

[0089] The recombinant bacteria obtained in Example 1 were activated and cultured overnight at 28°C. Then, their OD600 values were measured. After adjusting their OD600 values to be the same, the same bacterial liquid was taken and inoculated into 100 mL of YPD liquid medium, and cultured at 28°C and 220 rpm for 60 h. Then, the cell bacterial liquid was collected and stored in an -80°C refrigerator for 1 day, and then completely dried in a vacuum freeze dryer, and finally ground to obtain the bacterial powder of each strain.

[0090] Example 6

[0091] This example provides a method for the recombinant bacteria obtained in Example 1 to ferment and produce thymol and carvacrol. The specific steps are as follows:

[0092] The recombinant bacteria obtained in Example 1 were activated and cultured overnight at 28°C, and then their respective OD600 values were measured. After adjusting their OD600 values to be the same, the same bacterial solution was taken and inoculated into 100 mL of YPD liquid medium, and cultured at 28°C and 220 rpm for 72 h. Then, the cell bacterial solution was collected and stored in an -80°C refrigerator for 1 day, and then completely dried in a vacuum freeze dryer, and finally ground to obtain the bacterial powder of each strain.

[0093] Example 7

[0094] This example provides a method for fermenting and producing thymol and carvacrol by the recombinant bacteria obtained in Example 1. The specific steps are as follows:

[0095] The recombinant bacteria obtained in Example 1 were activated and cultured overnight at 28°C, and then their respective OD600 values were measured. After adjusting their OD600 values to be the same, the same bacterial solution was taken and inoculated into 100 mL of YPD liquid medium, and cultured at 28°C and 220 rpm for 84 h. Then, the cell bacterial solution was collected and stored in an -80°C refrigerator for 1 day, and then completely dried in a vacuum freeze dryer, and finally ground to obtain the bacterial powder of each strain.

[0096] Experimental Example 1

[0097] The dry weight of the yeast obtained by fermentation culture in Examples 2 - 7 was detected, and the results are as Figure 3 shown.

[0098] From Figure 3 it can be seen that the dry weight of the yeast can reach more than 20 g / L after 60 h of fermentation.

[0099] Experimental Example 2

[0100] The bacterial powder obtained in Examples 3 - 6 was detected by gas chromatography for the products of the corresponding strains. The specific method is as follows:

[0101] 1. Weigh 5 mg of the freeze-dried sample into a 5 ml glass bottle;

[0102] 2. Add 2 ml of ethyl acetate to the glass bottle and extract by ultrasound for 90 min (the temperature does not exceed 30°C);

[0103] 3. Centrifuge the sample after ultrasound at 4000 rpm for 10 min and take the supernatant;

[0104] 4. After filtering the supernatant with a 0.22 μm organic phase filter membrane, transfer it to a gas bottle.

[0105] Gas chromatography detection conditions:

[0106] Chromatographic column: capillary chromatographic column (5% diphenyl / 95% dimethyl polysiloxane), 30.0 m X 0.25 mm × 0.25 μm, or those with equivalent performance;

[0107] Temperature rising program: Maintain at 80°C for 1 min, rise to 120°C at a rate of 10°C / min, then rise to 145°C at a rate of 5°C / min, and finally rise to 250°C at a rate of 35°C / min and maintain for 2 min;

[0108] Carrier gas flow rate: 1.0 mL / min (constant flow mode);

[0109] Injection volume: 1 μL (splitless injection);

[0110] Injection port temperature: 250°C;

[0111] Detector temperature: 300°C;

[0112] Hydrogen flow rate: 30 mL / min;

[0113] Air flow rate: 300 mL / min;

[0114] Make-up gas flow rate: 30 mL / min.

[0115] The detection results are as Figure 4 and Figure 5 shown. It can be seen from this that by introducing TvTPS2, TvCYP71D507-T, and TvSDR1-T to construct a terpene synthesis pathway, thymol and carvacrol were successfully produced in Yarrowia lipolytica, and the total yield of thymol and carvacrol could reach 2 mg / kg.

[0116] Experimental Example 3

[0117] In this experimental example, the cytochrome p450 genes CYP71D179 and TvCYP71D180v1T were respectively combined with TvTPS2 and TvSDR1-T to construct recombinant bacteria. The CYP71D179 and TvCYP71D180v1T genes are derived from thyme, and the optimized nucleotide sequences after codon optimization are as shown in SEQ ID NO.4 and SEQ ID NO.5. The selected plasmids are all pCfB5791, and the constructed recombinant plasmids are TvTPS2-PrTEFin-CYP71D179–pcfb5791 and TvTPS2-PrTEFin-TvCYP71D180v1T–pcfb5791, and their construction methods are the same as those in Example 1. The sequences of the primers involved in the construction process are shown in Table 2:

[0118] Table 2 Primer sequences of CYP71D179 and TvCYP71D180v1T

[0119]

[0120] Then, the constructed recombinant bacteria were used to produce thymol and carvacrol according to the fermentation method of Example 2. The results were compared with those of the recombinant bacteria constructed in Example 1, and the results are as Figure 6 shown. Among them, TvCYP71D507-T is abbreviated as 507-T; CYP71D179 is abbreviated as 179; TvCYP71D180v1T is abbreviated as 180v1t.

[0121] It can be seen from Figure 6 that the recombinant bacteria constructed with different cytochrome p450 genes have an important impact on the products. When the introduced gene is CYP71D179, only a small amount of carvacrol can be produced; when the introduced gene is TvCYP71D180v1T, only a small amount of thymol can be produced; while when the introduced gene is TvCYP71D507-T, the contents of thymol and carvacrol produced are the highest, and the two products can be obtained simultaneously.

[0122] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant bacterium for synthesizing key components of plant essential oil, characterized in that, The recombinant bacterium contains exogenous genes; the exogenous genes include genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase; The key components of the plant essential oil include thymol and carvacrol; The genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase are derived from thyme. Among them, the terpene synthase is TvTPS2, the cytochrome P450 is TvCYP71D507-T, and the short-chain dehydrogenase is TvSDR1-T; The starting bacterium of the recombinant bacterium is Yarrowia lipolytica pO1f.

2. The method for constructing the recombinant bacterium according to claim 1, characterized in that, It includes the following steps: Connect the genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase after codon optimization to an expression vector, and then introduce the obtained recombinant expression vector into the starting bacterium to obtain the recombinant bacterium.

3. The construction method of the recombinant bacterium according to claim 2, wherein, The terpene synthase is TvTPS2, and the nucleotide sequence of TvTPS2 after codon optimization is shown in SEQ ID NO.

1.

4. The method for constructing the recombinant bacterium according to claim 3, characterized in that, The cytochrome P450 is TvCYP71D507-T, and the nucleotide sequence of TvCYP71D507-T after codon optimization is shown in SEQ ID NO.

2.

5. The method for constructing the recombinant bacterium according to claim 4, wherein The short-chain dehydrogenase is TvSDR1-T, and the nucleotide sequence of TvSDR1-T after codon optimization is shown in SEQ ID NO.

3.

6. The method for constructing the recombinant bacterium according to claim 5, wherein Any two of the genes of terpene synthase, cytochrome P450, and short-chain dehydrogenase are connected to the same expression vector.

7. Use of the recombinant bacterium according to claim 1 or the recombinant bacterium obtained by the method for constructing the recombinant bacterium according to any one of claims 2-6, characterized in that, The recombinant bacterium produces thymol and carvacrol through fermentation.

8. The application according to claim 7, characterized in that The steps for the recombinant bacterium to produce thymol and carvacrol through fermentation include: activating the recombinant bacterium, then inoculating it into a liquid medium, and after culturing, subjecting the obtained bacterial liquid to vacuum freeze-drying and grinding to obtain a bacterial powder containing thymol and carvacrol.

9. The application according to claim 8, characterized in that, The liquid medium includes yeast extract peptone dextrose medium.

10. The application according to claim 8, wherein The culture conditions of the recombinant bacterium are: culturing at 25 - 30 °C and 180 - 400 rpm for 24 - 96 h.

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