CN111440733A - Recombinant saccharomyces cerevisiae for producing terpineol, construction method and application - Google Patents

Abstract

The invention discloses a recombinant saccharomyces cerevisiae for producing terpineol, a construction method and application thereof, wherein the construction method of the recombinant saccharomyces cerevisiae for producing the terpineol comprises the following steps: using a method of homologous recombination, the strain of saccharomyces cerevisiae ATCC: 208352, introducing a truncated terpineol synthase coding gene delta Ts to obtain a recombinant bacterium 1; introducing a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding gene tHMG1 and an isopentenyl pyrophosphate isomerase encoding gene IDI1 into the recombinant bacterium 1 to obtain a recombinant bacterium 2; experiments prove that the recombinant saccharomyces cerevisiae for producing the terpineol is fermented, so that the yield of the terpineol is high. The invention successfully constructs the recombinant saccharomyces cerevisiae for producing the terpineol. Lays a foundation for the synthesis of terpineol by artificial cells.

Description

Recombinant Saccharomyces cerevisiae producing terpineol and construction method and application

technical field

The invention relates to the field of biotechnology, in particular to a terpineol-producing recombinant Saccharomyces cerevisiae and a construction method and application.

Background technique

Terpineol is a monoterpene alcohol compound with a skeleton of 10 carbon atoms. It is the main component of many plant essential oils. It has a lilac fragrance. Solvents and deodorants are also used in medicine, pesticides, plastics, soaps, and ink industries, and are also solvents for color on glassware. At present, its preparation is mainly based on plant extraction or turpentine oil as raw material, adding a small amount of peregalin in sulfuric acid as an emulsifier, and performing a hydration reaction at room temperature, so that the main component of the turpentine oil, pinene, generates hydrated terpene glycol, and then dehydrates to obtain crude terpineol. , obtained by fractional distillation.

Synthetic biology technology has attracted much attention since its inception. Compared with genetic engineering, it involves large-scale genetic modification and focuses on changes in metabolic flux. It is an interdisciplinary subject involving biology, genetics, chemistry, computer science and engineering. . Many terpenoids have high application value, and some of them have little content in plants and cannot be well developed and utilized. The preparation of chemical synthesis methods is cumbersome and complicated. With synthetic biology technology, the microbial platform is used to synthesize plant-derived high Value-added terpenoids have become the research hotspot of scientists in this field, such as using Saccharomyces cerevisiae to synthesize artemisinic acid, the precursor of antimalarial drug artemisinin; Saccharomyces cerevisiae synthesized tanshinone diene, the precursor of antibacterial and anti-inflammatory drug tanshinone, etc.

However, the synthesis of monoterpene terpineol using Saccharomyces cerevisiae as a chassis has not been reported.

SUMMARY OF THE INVENTION

One object of the present invention is to overcome the deficiencies of the prior art, and to provide a recombinant Saccharomyces cerevisiae producing terpineol.

The second object of the present invention is to provide a second terpineol-producing recombinant Saccharomyces cerevisiae.

The third object of the present invention is to provide a third terpineol-producing recombinant Saccharomyces cerevisiae.

The fourth object of the present invention is to provide a fourth terpineol-producing recombinant Saccharomyces cerevisiae.

The fifth object of the present invention is to provide a fifth terpineol-producing recombinant Saccharomyces cerevisiae.

The sixth object of the present invention is to provide a method for constructing the above-mentioned terpineol-producing recombinant Saccharomyces cerevisiae.

The seventh object of the present invention is to provide the application of the above-mentioned terpineol-producing recombinant Saccharomyces cerevisiae for preparing terpineol by fermentation.

The technical scheme of the present invention is summarized as follows:

A recombinant Saccharomyces cerevisiae construction method for producing terpineol, comprising the steps:

Using the method of homologous recombination, the truncated terpineol synthase encoding gene ΔTs was introduced into Saccharomyces cerevisiae ATCC: 208352 to obtain recombinant strain 1;

The nucleotide sequence of the truncated terpineol synthase encoding gene ΔTs is shown in SEQ ID NO.1.

The second method for constructing a recombinant Saccharomyces cerevisiae producing terpineol, comprising the steps:

Using the method of homologous recombination, the truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 and isopentenyl pyrophosphate isomerase encoding gene IDI1 were introduced into recombinant bacteria 1 to obtain recombinant bacteria 2;

The nucleotide sequence of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 is shown in SEQ ID NO.5;

The nucleotide sequence of the isopentenyl pyrophosphate isomerase encoding gene IDI1 is shown in SEQ ID NO.6.

The third terpineol-producing recombinant Saccharomyces cerevisiae construction method comprises the following steps:

Using the method of homologous recombination, the mutated farnesyl pyrophosphate synthase-encoding gene MErg20 was introduced into the ERG20 site in recombinant bacteria 2 to obtain recombinant bacteria 3;

The nucleotide sequence of the mutated farnesyl pyrophosphate synthase encoding gene MErg20 is shown in SEQ ID NO.11.

The fourth terpineol-producing recombinant Saccharomyces cerevisiae construction method comprises the following steps:

Using the method of homologous recombination, the fusion protein encoding gene ERG20FTs of mutated farnesyl pyrophosphate synthase and truncated terpineol synthase was introduced into recombinant bacteria 3 to obtain recombinant bacteria 4;

The nucleotide sequence of the encoding gene ERG20FTs is shown in SEQ ID NO.12.

The fifth terpineol-producing recombinant Saccharomyces cerevisiae construction method, comprising the following steps:

Using the method of homologous recombination, the squalene synthase encoding gene ERG9 was introduced into the recombinant bacteria 4 to obtain the recombinant bacteria 5;

The nucleotide sequence of the squalene synthase encoding gene ERG9 is shown in SEQ ID NO.13.

The terpineol-producing recombinant Saccharomyces cerevisiae constructed by the above methods.

Application of the above-mentioned terpineol-producing recombinant Saccharomyces cerevisiae in the production of terpineol.

The experiment of the present invention proves that the terpineol-producing recombinant Saccharomyces cerevisiae of the present invention is fermented, so that the yield of terpineol is high. The present invention successfully constructs the recombinant Saccharomyces cerevisiae producing terpineol. It laid the foundation for the synthesis of terpineol by artificial cells.

Description of drawings

Figure 1 shows the results of GC-MS analysis of terpineol. a in Figure 1 is the gas chromatogram of the detection. A in a is the GC-MS detection spectrum of W303-1a; B is the GC-MS detection spectrum of recombinant bacteria 1; C is the GC-MS detection spectrum of terpineol standard product; 1 is the chromatographic peak of terpineol standard product ; 2 is the chromatographic peak of the product produced by recombinant bacteria 1. b in Figure 1 is the mass spectrum of the product produced by recombinant bacteria 1, that is, the mass spectrum corresponding to peak 2 in a. c in Figure 1 is the mass spectrum of the terpineol standard, that is, the mass spectrum corresponding to peak 1 in a.

Figure 2 is a comparison of the terpineol production of each recombinant Saccharomyces cerevisiae strain.

Detailed ways

The present invention will be further described below through specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Saccharomyces cerevisiae (Saccharomyces cerevisiaeW303-1a, US ATCC: 208352) Purchase time, 2016.6 Website: https://www.atcc.org/

Embodiment 1, the preparation method of each fragment source

(1) ΔTS sequence of the original gene used in the present invention (truncated ΔTS, the original TS is from Vitisvinifera); MErg20 sequence (mutated ERG20, the original ERG20 is from Saccharomyces cerevisiae), ERG20FTs Sequence (mutated ERG20 and ΔTS fusion protein encoding gene).

URA3 sequence, ADE2 sequence, TRP1 sequence, LEU2 sequence, HIS3 sequence are all derived from Saccharomyces cerevisiae), fully synthesized by the company, and connected to E. coli plasmid pUC57, and stored in E. coli.

The nucleotide sequence of the ΔTS is shown in SEQ ID NO.1, the nucleotide sequence of MErg20 is shown in SEQ ID NO.11, and the nucleotide sequence of ERG20FTs (the artificial sequence of the gene encoding the fusion protein) is shown in SEQ ID NO.11. ID NO.12, the nucleotide sequence of URA3 is shown in SEQ ID NO.16, the nucleotide sequence of ADE2 is shown in SEQ ID NO.4, and the nucleotide sequence of TRP1 is shown in SEQ ID NO.17 The nucleotide sequence of LEU2 is shown in SEQ ID NO.15, and the nucleotide sequence of HIS3 is shown in SEQ ID NO.14.

(2) The endogenous fragment of Saccharomyces cerevisiae used in the present invention is obtained by extracting the genome of Saccharomyces cerevisiae, using it as a template, and amplifying by PCR.

The extraction method of Saccharomyces cerevisiae genome is as follows: inoculate Saccharomyces cerevisiae (W303-1a) in liquid YPD medium, and cultivate overnight on a shaker at 30°C; transfer the overnight cultured Saccharomyces cerevisiae into a 2 mL centrifuge tube, centrifuge at 10,000 rpm for 1 min, and discard. Supernatant, collect the bottom cell pellet; add quartz sand equal to the cell volume into the centrifuge tube, then add 400 μL of STES lysis solution, 400 μL phenol/chloroform/isoamyl alcohol; put the above mixture on a shaker and shake 10min; add 400 μL of TE solution to the centrifuge tube after shaking, mix well, centrifuge to take the supernatant, transfer the supernatant to a new 2mL centrifuge tube; then add 1/10 volume to the collected supernatant 3M NaAc, 2 times the volume of absolute ethanol, mixed evenly, placed at -20 °C for 1 h, centrifuged at 10,000 rpm for 10 min, you can see a small amount of white precipitate at the bottom of the centrifuge tube, that is, genomic DNA, remove the supernatant, and use 75% ethanol. Rinse once; in a ventilated place, fully volatilize the residual alcohol in the centrifuge tube, then add 100 μL of water to complete the yeast genome extraction, measure the DNA concentration with a microspectrophotometer, and store it in a -20°C refrigerator for later use.

The solution formulations used are as follows:

1. Tris·HCl (50 mmol/L) solution: Mix 50 mL of 0.1 mol/L Tris solution with 29.2 mL of 0.1 mol/L hydrochloric acid, and then make up to 100 mL with water.

2. EDTA (0.5mol/L) solution: 18.16g Na ₂ EDTA·2H ₂ O was dissolved in water, and the volume was adjusted to 100 mL, and the pH was adjusted to 8.0 with solid NaOH.

3. The formula of TE solution: 200ml of Tris·HCl (50mmol/L) solution, 2mL of EDTA (0.5mol/L) solution, add water after mixing, and make up to 1L.

4. The formula of STES lysis solution: take 20 mL of Tris·HCl (50 mmol/L) solution, 2 mL of Triton X-100, 0.584 g NaCl, 200 μL of EDTA (0.5 mol/L) solution, mix them evenly, and rinse with water. Make up to 100mL.

5. Phenol:chloroform:isoamyl alcohol=25:24:1 in the phenol/chloroform/isoamyl alcohol mixture.

(3) the present invention adopts the high-fidelity polymerase of Vazyme company to amplify the DNA fragment, and the reaction system is as follows:

The PCR reaction program is as follows:

The Tm value is determined according to the annealing temperature of the primers.

(4) The plasmid stored in Escherichia coli is extracted with Tiangen Plasmid Mini Kit, and the steps are as follows:

1. Inoculate Escherichia coli into LB liquid medium containing antibiotics for culture, collect 1-5mL bacterial liquid in a centrifuge tube, centrifuge at 12000rpm for 1min, discard the supernatant, and collect the bacteria at the bottom of the centrifuge tube (the supernatant should be as much as possible) remove);

2. Equilibrate the adsorption column with 500 μL of BL equilibration solution, centrifuge at 12,000 rpm, and discard the waste liquid in the collection tube;

3. Add 250 μL of P1 solution to the centrifuge tube with bacterial cell precipitation, and suspend the bacterial cell precipitation completely;

4. Add 250 μL of P2 lysis solution to the centrifuge tube, and turn the centrifuge tube gently to fully lyse the bacteria. At this time, the bacteria solution will become clear, and the lysis time should not be too long to avoid plasmid damage;

5. Add 350 μL of P3 solution to the centrifuge tube, and turn the centrifuge tube upside down to precipitate the protein;

6. Centrifuge the above centrifuge tube at 12,000 rpm for 10 min, and thoroughly centrifuge the precipitate to the bottom. Take the supernatant into an adsorption column equilibrated with BL solution, adsorb in a -20°C refrigerator for 5 min, centrifuge at 12,000 rpm for 1 min, and pour it into the collection tube. waste liquid;

7. Rinse the adsorption column that has adsorbed the plasmid twice with alcohol-containing PW solution to remove impurities, then centrifuge at 12,000 rpm for 2 minutes to remove PW as much as possible, and then place the adsorption column in a ventilated place to allow the alcohol to evaporate.

8. Add 50-100 μL of deionized water to the adsorption column, put it at 37°C for 10 minutes to dissolve the plasmid in water, put the adsorption column in a centrifuge tube, and centrifuge at 12000 rpm for 2 minutes to collect the plasmid.

Embodiment 2. Construction of recombinant Saccharomyces cerevisiae (recombinant bacteria 1) producing terpineol

(1) Construction of URA3-up, P _Tef1 -ΔTs-T _Cyc1 , HIS3-URA3-down

PCR was performed with the PCR templates and primers described in Table 1 to obtain DNA fragments: M1(URA3-up), M2(P _Tef1 ), M3(ΔTs), M4(T _Cyc1 ), M5(HIS3), M6(URA3- down).

Table 1 Primer sequences

Fragments M2, M3 and M4 were fused into expression cassette P _Tef1 -ΔTs-T _Cyc1 by fusion PCR;

Fragments M5 and M6 were fused into HIS3-URA3-down fragments by fusion PCR.

The fragments for fusion PCR have 20-30 bp homologous sequences with each other, which overlap and complement each other, and the fragments can serve as primers and templates for each other. The fused fragments were mixed in an equimolar ratio, and the total amount was greater than 800 ng, supplemented with dNTPs, 2 × Phanta Max Buffer, DNA polymerase, and deionized water to prepare a 50 uL PCR system. The PCR program was an annealing temperature of 60 °C, the number of cycles was 11 cycles, and the extension time was calculated based on the total length of the module. The fusion PCR system can be directly used as a PCR template. Using the fused fragment as a template and the primers at both ends of the total length of the fragment as primers, PCR amplification was performed, followed by purification and recovery to obtain modules URA3-up, P _Tef1 -ΔTs-T _Cyc1 , HIS3-URA3-down.

(2) Construction of recombinant bacteria 1

1. The starting bacterium Saccharomyces cerevisiaeW303-1a was inoculated into a test tube YPD, and cultured overnight on a shaker at 30°C; 2. The yeast cultured overnight was transferred to a new test tube YPD liquid medium at a volume ratio of 1/10 3. Take 1 mL of bacterial liquid into a sterile centrifuge tube, centrifuge at 5000 rpm for 3 min, remove the supernatant, collect the bottom bacterial liquid, and use 1 mL of sterile Wash the cells once with water; 4. Resuspend the washed yeast with 1 mL of 100 mM LiAc, and let stand for 5 min; centrifuge at 5.5000 rpm for 3 min, remove the LiAc solution, and keep the yeast at the bottom; 6. In a centrifuge tube containing yeast The transformation system was prepared in , and the specific reagents were added and the sequence was as follows:

The transformed DNA fragments include URA3-up, P _Tef1 -ΔTs-T _Cyc1 , HIS3-URA3-down, and each fragment is greater than 300ng;

7. The salmon sperm DNA needs to be boiled in boiling water for 5 minutes to melt it, and then quickly transferred to an ice bath on ice for yeast transformation;

8. Blow up the prepared transformation system with a pipette, or shake it on a vortex shaker for 1 min to fully mix the system, put it in a 42°C water bath, and heat for 30 min;

9. Centrifuge the heat-shocked yeast, remove the supernatant with a pipette, add 1 mL of LYPD medium, and recover in a shaker at 30°C for 2 hours;

Centrifuge at 10.5000rpm for 3min, remove YPD liquid medium, and wash twice with sterile water;

11. Add 100 μL of sterile water, resuspend the yeast cells, spread them on the SC selection medium lacking histidine, and culture in a 30°C incubator for 2-3 days.

12. After a single colony is grown, colony PCR is performed, and the genome needs to be roughly extracted. The invention adopts the freeze-thaw method to crudely extract the Saccharomyces cerevisiae genome. First, a single colony is picked into 10 μL of a NaOH solution with a concentration of 10 mM, boiled in boiling water for 10 minutes, then placed in a -20° C. refrigerator for freezing for 10 minutes, and the freeze-thaw is repeated three times. The genome is roughly extracted, which can be directly used as a template for colony verification. The correct strain was verified as recombinant strain 1 (T1).

SC selection medium formula: 6.7g/L Yeast Nitrogen Base (YNB), 20g/L glucose or galactose, 2g/L corresponding default amino acid mixture. Mixture ingredients include:

The starting bacterium Saccharomyces cerevisiae W303-1a of the present invention lacks the genes Leu2, Trp1, Ura3, Ade2, His3, and is used for screening markers, so when preparing the SC selection medium, fill up the corresponding components as needed, and the final concentration is: Leucine Leu 0.1g/L; Tryptophan Trp 0.02g/L; Histidine His 0.02g/L; Uracil Ura 0.02g/L; Adenine Ade 0.02g/L.

Embodiment 3, the construction of the recombinant Saccharomyces cerevisiae (recombinant bacteria 2) producing terpineol

(1) Construction of δ-up, P _Pgk1 -tHMG1-T _Pgk1 , P _Tdh3 -IDI1-T _Adh1 , LEU2-δ-down

PCR was performed with the PCR templates and primers described in Table 2 to obtain DNA fragments: M7 (δ-up), M8 (P _Pgk1 ), M9 (tHMG1 ), M10 (T _Pgk1 ), M11 (P _Tdh3 ), M12 (IDI1 ), M13 (T _Adh1 ), M14 (LEU2), M15 (δ-down). The nucleotide sequence of tHMG1 is shown in SEQ ID NO.5; the nucleotide sequence of IDI1 is shown in SEQ ID NO.6;

Table 2 Primer sequences

Fragments M8, M9 and M10 were fused into expression _cassette Pgk1-tHMG1-T _Pgk1 by fusion PCR;

Fragments M11, M12, and M13 were fused into expression cassettes P _Tdh3 -IDI1-T _Adh1 by fusion PCR;

Fragments M14 and M15 were fused into LEU2-δ-down fragment by fusion PCR.

For the fusion method, refer to Example 2(1).

(2) Construction of recombinant bacteria 2 (T2)

The fusion module δ-up, P _Pgk1 -tHMG1-T _Pgk1 , P _Tdh3 -IDI1-T _Adh1 , LEU2-δ-down transformation method refer to Example 2 (2), wherein the starting strain is recombinant bacteria 1, and the screening medium used is Leucine-depleted SC selection medium. Recombinant strain 2 (T2) was obtained.

Embodiment 4, the construction of the recombinant Saccharomyces cerevisiae (recombinant bacteria 3) producing terpineol

(1) Construction of ERG20-up, P _Pgk1 -MErg20-T _Cyc1 , URA3-ERG20-down

PCR was performed with the PCR templates and primers described in Table 3 to obtain DNA fragments: M16 (ERG20-up), M17 (P _Pgk1 ), M18 (MErg20), M19 (T _Cyc1 ), M20 (URA3), M21 (ERG20- down).

Table 3 Primer sequences

Fragments M17, M18, M19 were fused into expression _cassette Pgk1-MErg20-T _Cyc1 by fusion PCR;

Fragments M20 and M21 were fused into URA3-ERG20-down fragment by fusion PCR.

For the fusion method, refer to Example 2(1).

(2) Construction of recombinant strain 3 (T3)

Fusion modules ERG20-up, P _Pgk1 -MErg20-T _Cyc1 , URA3-ERG20-down transformation method refer to Example 2 (2), wherein the starting strain is recombinant bacteria 2, and the screening medium used is SC selection medium lacking uracil , the recombinant strain 3 (T3) was obtained.

Embodiment 5. Construction of the recombinant Saccharomyces cerevisiae (recombinant bacteria 4) producing terpineol

(1) Construction of rDNA-up, P _Pgk1 -ERG20FTs-T _Adh1 , TRP1-rDNA-down

PCR was performed with the PCR templates and primers described in Table 4 to obtain DNA fragments: M22 (rDNA-up), M23 (P _Pgk1 ), M24 (ERG20FTs), M25 (T _Adh1 ), M26 (TRP1), M27 (rDNA- down).

Table 4 Primer sequences

Fragments M23, M24, M25 were fused into expression _cassette Pgk1-ERG20FTs-T _Adh1 by fusion PCR;

Fragments M26 and M27 were fused into TRP1-rDNA-down by fusion PCR.

For the fusion method, refer to Example 2(1).

(2) Construction of recombinant bacteria 4 (T4)

Fusion module rDNA-up, P _Pgk1 -ERG20FTs-T _Adh1 , TRP1-rDNA-down transformation method refer to Example 2 (2), wherein the starting strain is recombinant bacteria 3, and the screening medium used is the SC selection culture lacking tryptophan base to obtain recombinant bacteria 4 (T4).

Embodiment 6. Construction of the recombinant Saccharomyces cerevisiae (recombinant bacteria 5) producing terpineol

(1) Construction of HO-up, P _Tdh3 -ERG9-T _Adh1 , ADE2-HO-down

PCR was performed with the PCR templates and primers described in Table 4 to obtain DNA fragments: M28 (HO-up), M29 (P _Tdh3 ), M30 (ERG9), M31 (T _Adh1 ), M32 (ADE2), M33 (HO- down).

The nucleotide sequence of the squalene synthase encoding gene ERG9 is shown in SEQ ID NO.13.

Table 5 Primer sequences

Fragments M29, M30 and M31 were fused into expression cassette P _Tdh3 -ERG9-T _Adh1 by fusion PCR;

Fragments M32 and M33 were fused into ADE2-HO-down by fusion PCR.

For the fusion method, refer to Example 2(1).

(2) Construction of recombinant strain 5 (T5)

Fusion module HO-up, P _Tdh3 -ERG9-T _Adh1 , ADE2-HO-down transformation method refer to Example 2 (2), wherein the starting strain is recombinant bacteria 4, and the screening medium used is SC selection medium lacking adenine , the recombinant strain 5 (T5) was obtained.

Embodiment 7, recombinant bacteria generate terpineol

(1) Recombinant bacteria culture and product extraction

Fermentation, extraction and detection of terpineol:

Fermentation was carried out in 30 mL of LYPD liquid medium, and the seed liquid was inserted to make the initial OD ₆₀₀ 0.05. After 12 h, 3 mL of n-dodecane was added for biphasic fermentation, and the fermentation was carried out at 30 °C and 220 rpm for 5 d, and the yield of terpineol was measured. Terpineol belongs to monoterpenoids and is easy to volatilize. In order to reduce the volatilization loss in the fermentation process, the present invention adds n-dodecane in the fermentation process for biphasic fermentation. For the determination of terpineol, the n-dodecane phase can be directly taken for detection. Before the gas phase, GC/MS detection, the sample needs to be filtered with a 0.22μm pore size filter to remove impurities.

Gas chromatography detection conditions for terpineol: chromatographic column DB-SWAX, nitrogen flow rate 1mL/min, injection temperature 250℃, injection split ratio 1:20, injection volume 1μL; oven temperature: 80℃ for 1min, 10℃ /min to 180°C, then 30°C/min to 250°C; FID detector: 250°C. GC-MS detection conditions of terpineol: chromatographic conditions are the same as those of gas chromatography; ion source temperature is 230 °C, and ion scanning range is 50-600 m/z. The retention time of terpineol was 11.01 min.

(2) Test results

A. Saccharomyces cerevisiae W303-1a did not detect terpineol;

B. Recombinant bacteria 1 (T1): extracting the T1 fermentation product, a trace amount of terpineol can be detected, and its output is 100ug/L;

C. Recombinant bacteria 2 (T1): extract the T2 fermentation product, and the terpineol content can be detected to be 550ug/L;

D. Recombinant bacteria 3 (T3): extract the T3 fermentation product, and the terpineol content can be detected to be 832ug/L;

E. Recombinant bacteria 4 (T4): extract the T4 fermentation product, and the terpineol content can be detected to be 2.39 mg/L;

F. Recombinant bacteria 5 (T5): The T5 fermentation product was extracted, and the content of terpineol could be detected to be 3.32 mg/L.

The statistical results of the above recombinant bacteria are shown in Figure 2.

Embodiment 8, the 5L tank fermentation of recombinant bacteria T5:

First, a single colony of T5 was inoculated into 200 mL of LYPD medium, and cultured at 30 °C on a shaker at 220 rpm for 24 h to prepare a seed solution. The fermenter was initially loaded with YPD medium with a volume of 2L, and 1L of feed solution was prepared. After the seed liquid was inoculated into the medium for 24 hours, the feed solution was supplemented. The dissolved oxygen content was kept at 40%, and the fermentation was controlled by the feed solution and ammonia water. At pH 5.5, after 120h fermentation, the yield of terpineol was 21.88mg/L.

The feed solution formula is: 500g/L glucose, KH ₂ PO ₄ 9g/L, K ₂ SO ₄ 3.5g/L, Na ₂ SO ₄ 0.28g/L, MgSO ₄ ·7H ₂ O 0.5g/L, 10ml trace Elemental mother liquor, 12ml vitamin mother liquor.

The formula of trace element mother liquor is: ZnSO ₄ ·7H ₂ O 10.2g/L, EDTANa ₂ ·2H ₂ O 15g/L, FeSO ₄ ·7H ₂ O 5.12g/L, anhydrous CuSO ₄ 0.5g/L, MnCl ₂ · 4H ₂ O 0.5 g/L, CoCl ₂ ·6H ₂ O 0.86 g/L, CaCl ₂ ·2H ₂ O 3.84 g/L, Na ₂ MoO ₄ ·2H ₂ O 0.56 g/L.

The formula of vitamin mother liquor is: inositol 25g/L, biotin 0.05g/L, niacin 1g/L, calcium pantothenate 1g/L, thiamine HCl 1g/L, pyridoxol HCl 1g/L, para-aminobenzoic acid 0.2g /L.

sequence listing

<110> Tianjin University

<120> Recombinant Saccharomyces cerevisiae producing terpineol and its construction method and application

<160> 77

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1692

<212> DNA

<213> Artificial Sequence

<400> 1

atggcttcac caagaggtat taaagttaag atcggtaatt ctaactgtga agaaatcatc 60

gttagaagaa ctgcaaacta ccatccaaca atctgggatt acgattacgt tcaatcattg 120

agatctgatt acgttggtga aacttacaca agaagattag ataaattgaa gagagatgtt 180

aagccaatgt tgggtaaagt taagaaacca ttggatcaat tggaattaat cgatgttttg 240

caaagattgg gtatctatta ccatttcaag gatgaaatta aaagaatttt aaattctatc 300

tataatcaat acaatagaca tgaagaatgg caaaaagatg atttgtatgc tactgctttg 360

gagtttagat tgttaagaca acatggttac gatgttccac aagatgtttt tagtagattc 420

aaagatgata caggttcttt taaagcttgt ttgtgtgaag atatgaaggg catgttgtgt 480

ttgtacgaag catcatactt gtgtgttcag ggtgaatcta ctatggaaca agctagagat 540

tttgcacata gacatttggg taaaggtttg gaacaaaaca tcgatcaaaa tttggctatc 600

gaagttaagc atgcattgga attaccattg cattggagaa tgccaagatt ggaagctaga 660

tggttcatcg atgtttacga aaagagacaa gatatgaacc caatcttgtt agaatttgct 720

aagttggatt ttaatatggt tcaagcaact catcaagaag atttgagaca tatgtcatct 780

tggtggtcat ctacaagatt gggtgaaaag ttgaacttcg ctagagatag attgatggaa 840

aatttcttgt ggactgttgg tgttattttc gaaccacaat acggttactg tagaagaatg 900

tctacaaagg ttaacacttt gatcacaatc attgatgatg tttatgatgt ttacggtact 960

atggatgaat tagaattgtt tacagatgtt gttgatagat gggatattaa tgctatggac 1020

ccattgccag aatacatgaa gttgtgtttc ttggcattgt acaactcaac aaacgaaatg 1080

gcttacgatg cattgaagga acatggtttg catatcgttt cttatttgag aaaggcttgg 1140

tcagatttgt gtaagtctta cttgttagaa gcaaagtggt actactcaag atacactcca 1200

tctttgcaag aatacatctc aaattcttgg atctcaatct ctggtccagt tattttggtt 1260

catgcttact ttttggttgc aaacccaatc acaaaggaag ctttgcaatc attggaaaga 1320

taccataaca tcatcagatg gtcatctatg atcttgagat tgtcagatga tttgggtact 1380

tctttagatg aattgaagag aggtgacgtt ccaaagtcta tccaatgtta catgtacgaa 1440

acaggtgctt cagaagaaga tgcaagaaag catacttctt atttgatcgg tgaaacatgg 1500

aagaaattga atgaagatgg tgctgttgaa tcaccattcc cagaaacttt tattggtatc 1560

gcaatgaatt tggctagaat ggcacaatgt atgtatcaac atggtgacgg tcatggtatt 1620

gaatacggtg aaacagaaga tagagttttg tctttgttgg ttgaaccaat cccatcattg 1680

tcttttgaat aa 1692

<210> 2

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 2

cacataaaca aacaaaatgg gaaagctatt acaattggc 39

<210> 3

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 3

cataaatcat aagaaattcg ctcacgctct gtgtaaagtg tat 43

<210> 4

<211> 2668

<212> DNA

<213> Saccharomyces cerevisiae

<400> 4

attaacgtat tacataagtt acaggattca tgcttatggg ttagctattt cgcccaatgt 60

gtccatctga cattactatt ttgcatttta atttaattag aacttgacta gcgcactacc 120

agtatatcat ctcatttccg taaataccaa atgtattata tattgaaagc ttttgaccag 180

gttattataa aagaaacttc atgctcgaaa aagatcattt cgaaaagttg cctagtttca 240

tgaaatttta aagcagttta tataaatttt accttttgat gcggaattga ctttttcttg 300

aataatacat aacttttctt aaaagaatca aagacagata aaatttaaga gatattaaat 360

attagtgaga agccgagaat tttgtaacac caacataaca ctgacatctt taacaacttt 420

taattatgat acatttctta cgtcatgatt gattattaca gctatgctga caaatgactc 480

ttgttgcatg gctacgaacc gggtaatact aagtgattga ctcttgctga ccttttatta 540

agaactaaat ggacaatatt atggagcatt tcatgtataa attggtgcgt aaaatcgttg 600

gatctctctt ctaagtacat cctactataa caatcaagaa aaacaagaaa atcggacaaa 660

acaatcaagt atggattcta gaacagttgg tatattagga gggggacaat tgggacgtat 720

gattgttgag gcagcaaaca ggctcaacat taagacggta atactagatg ctgaaaattc 780

tcctgccaaa caaataagca actccaatga ccacgttaat ggctcctttt ccaatcctct 840

tgatatcgaa aaactagctg aaaaatgtga tgtgctaacg attgagattg agcatgttga 900

tgttcctaca ctaaagaatc ttcaagtaaa acatcccaaa ttaaaaattt acccttctcc 960

agaaacaatc agatgatac aagacaaata tattcaaaaa gagcatttaa tcaaaaatgg 1020

tatagcagtt acccaaagtg ttcctgtgga acaagccagt gagacgtccc tattgaatgt 1080

tggaagagat ttgggttttc cattcgtctt gaagtcgagg actttggcat acgatggaag 1140

aggtaacttc gttgtaaaga ataaggaaat gattccggaa gctttggaag tactgaagga 1200

tcgtcctttg tacgccgaaa aatgggcacc atttactaaa gaattagcag tcatgattgt 1260

gagatctgtt aacggtttag tgttttctta cccaattgta gagactatcc acaaggacaa 1320

tatttgtgac ttatgttatg cgcctgctag agttccggac tccgttcaac ttaaggcgaa 1380

gttgttggca gaaaatgcaa tcaaatcttt tcccggttgt ggtatatttg gtgtggaaat 1440

gttctattta gaaacagggg aattgcttat taacgaaatt gccccaaggc ctcacaactc 1500

tggacattat accattgatg cttgcgtcac ttctcaattt gaagctcatt tgagatcaat 1560

attggatttg ccaatgccaa agaatttcac atctttctcc accattacaa cgaacgccat 1620

tatgctaaat gttcttggag acaaacatac aaaagataaa gagctagaaa cttgcgaaag 1680

agcattggcg actccaggtt cctcagtgta cttatatgga aaagagtcta gacctaacag 1740

aaaagtaggt cacataaata ttattgcctc cagtatggcg gaatgtgaac aaaggctgaa 1800

ctacattaca ggtagaactg atattccaat caaaatctct gtcgctcaaa agttggactt 1860

ggaagcaatg gtcaaaccat tggttggaat catcatggga tcagactctg acttgccggt 1920

aatgtctgcc gcatgtgcgg ttttaaaaga ttttggcgtt ccatttgaag tgacaatagt 1980

ctctgctcat agaactccac ataggatgtc agcatatgct atttccgcaa gcaagcgtgg 2040

aattaaaaca attatcgctg gagctggtgg ggctgctcac ttgccaggta tggtggctgc 2100

aatgacacca cttcctgtca tcggtgtgcc cgtaaaaggt tcttgtctag atggagtaga 2160

ttctttacat tcaattgtgc aaatgcctag aggtgttcca gtagctaccg tcgctattaa 2220

taatagtacg aacgctgcgc tgttggctgt cagactgctt ggcgcttatg attcaagtta 2280

tacaacgaaa atggaacagt ttttattaaa gcaagaagaa gaagttcttg tcaaagcaca 2340

aaagttagaa actgtcggtt acgaagctta tctagaaaac aagtaatata taagtttatt 2400

gatatacttg tacagcaaat aattataaaa tgatatacct attttttagg ctttgttatg 2460

attacatcaa atgtggactt catacataga aatcaacgct tacaggtgtc cttttttaag 2520

aatttcatac ataagatcac ttattataca tacatacata tccagtaaca agaagcaagg 2580

aataattacc tgcttaagtc tgcgattaaa aaaataacgt ttcgatacag ttcatataag 2640

gcggctcaat gcagaaccga ggatagcg 2668

<210> 5

<211> 1512

<212> DNA

<213> Saccharomyces cerevisiae

<400> 5

atgccagttt taaccaataa aacagtcatt tctggatcga aagtcaaaag tttatcatct 60

gcgcaatcga gctcatcagg accttcatca tctagtgagg aagatgattc ccgcgatatt 120

gaaagcttgg ataagaaaat acgtccttta gaagaattag aagcattatt aagtagtgga 180

aatacaaaac aattgaagaa caaagaggtc gctgccttgg ttattcacgg taagttacct 240

ttgtacgctt tggagaaaaa attaggtgat actacgagag cggttgcggt acgtaggaag 300

gctctttcaa ttttggcaga agctcctgta ttagcatctg atcgtttacc atataaaaat 360

tatgactacg accgcgtatt tggcgcttgt tgtgaaaatg ttataggtta catgcctttg 420

cccgttggtg ttataggccc cttggttatc gatggtacat cttatcatat accaatggca 480

actacagagg gttgtttggt agcttctgcc atgcgtggct gtaaggcaat caatgctggc 540

ggtggtgcaa caactgtttt aactaaggat ggtatgacaa gaggcccagt agtccgtttc 600

ccaactttga aaagatctgg tgcctgtaag atatggttag actcagaaga gggacaaaac 660

gcaattaaaa aagcttttaa ctctacatca agatttgcac gtctgcaaca tattcaaact 720

tgtctagcag gagatttact cttcatgaga tttagaacaa ctactggtga cgcaatgggt 780

atgaatatga tttctaaagg tgtcgaatac tcattaaagc aaatggtaga agagtatggc 840

tgggaagata tggaggttgt ctccgtttct ggtaactact gtaccgacaa aaaaccagct 900

gccatcaact ggatcgaagg tcgtggtaag agtgtcgtcg cagaagctac tattcctggt 960

gatgttgtca gaaaagtgtt aaaaagtgat gtttccgcat tggttgagtt gaacattgct 1020

aagaatttgg ttggatctgc aatggctggg tctgttggtg gatttaacgc acatgcagct 1080

aatttagtga cagctgtttt cttggcatta ggacaagatc ctgcacaaaa tgttgaaagt 1140

tccaactgta taacattgat gaaagaagtg gacggtgatt tgagaatttc cgtatccatg 1200

ccatccatcg aagtaggtac catcggtggt ggtactgttc tagaaccaca aggtgccatg 1260

ttggacttat taggtgtaag aggcccgcat gctaccgctc ctggtaccaa cgcacgtcaa 1320

ttagcaagaa tagttgcctg tgccgtcttg gcaggtgaat tatccttatg tgctgcccta 1380

gcagccggcc atttggttca aagtcatatg acccacaaca ggaaacctgc tgaaccaaca 1440

aaacctaaca atttggacgc cactgatata aatcgtttga aagatgggtc cgtcacctgc 1500

attaaatcct aa 1512

<210> 6

<211> 867

<212> DNA

<213> Saccharomyces cerevisiae

<400> 6

atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60

caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120

agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180

ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240

gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300

ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360

caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420

catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480

ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540

actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600

gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660

aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720

gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780

tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840

gacaggcaaa ttcatagaat gctataa 867

<210> 7

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 7

acgatacggc gttaagatca tgatacataa aagc 34

<210> 8

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 8

gtatcatgat cttaacgccg tatcgtgatt aac 33

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 9

ctgacatacc aagacgctat cctcggttct gcattg 36

<210> 10

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 10

accgaggata gcgtcttggt atgtcagcta ctg 33

<210> 11

<211> 1059

<212> DNA

<213> Artificial Sequence

<400> 11

atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60

gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120

gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180

gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240

aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttactggtt ggtcgccgat 300

gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360

gttgggggaaa ttgccatctg ggacgcattc atgttagagg ctgctatcta caagcttttg 420

aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480

accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540

gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600

tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660

gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720

gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780

gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840

aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900

attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960

gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020

actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059

<210> 12

<211> 2778

<212> DNA

<213> Artificial Sequence

<400> 12

atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60

gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120

gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180

gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240

aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttactggtt ggtcgccgat 300

gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360

gttgggggaaa ttgccatctg ggacgcattc atgttagagg ctgctatcta caagcttttg 420

aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480

accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540

gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600

tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660

gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720

gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780

gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840

aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900

attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960

gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020

actgcgttct tgaacaaagt ttacaagaga agcaaaggtt ctggttctgg ttctggttct 1080

ggttctatgg cttcaccaag aggtattaaa gttaagatcg gtaattctaa ctgtgaagaa 1140

atcatcgtta gaagaactgc aaactaccat ccaacaatct gggattacga ttacgttcaa 1200

tcattgagat ctgattacgt tggtgaaact tacacaagaa gattagataa attgaagaga 1260

gatgttaagc caatgttggg taaagttaag aaaccattgg atcaattgga attaatcgat 1320

gttttgcaaa gattgggtat ctattaccat ttcaaggatg aaattaaaag aattttaaat 1380

tctatctata atcaatacaa tagacatgaa gaatggcaaa aagatgattt gtatgctact 1440

gctttggagt ttagattgtt aagacaacat ggttacgatg ttccacaaga tgtttttagt 1500

agattcaaag atgatacagg ttcttttaaa gcttgtttgt gtgaagatat gaagggcatg 1560

ttgtgtttgt acgaagcatc atacttgtgt gttcagggtg aatctactat ggaacaagct 1620

agagattttg cacatagaca tttgggtaaa ggtttggaac aaaacatcga tcaaaatttg 1680

gctatcgaag ttaagcatgc attggaatta ccattgcatt ggagaatgcc aagattggaa 1740

gctagatggt tcatcgatgt ttacgaaaag agacaagata tgaacccaat cttgttagaa 1800

tttgctaagt tggattttaa tatggttcaa gcaactcatc aagaagattt gagacatatg 1860

tcatcttggt ggtcatctac aagattgggt gaaaagttga acttcgctag agatagattg 1920

atggaaaatt tcttgtggac tgttggtgtt attttcgaac cacaatacgg ttactgtaga 1980

agaatgtcta caaaggttaa cactttgatc acaatcattg atgatgttta tgatgtttac 2040

ggtactatgg atgaattaga attgtttaca gatgttgttg atagatggga tattaatgct 2100

atggacccat tgccagaata catgaagttg tgtttcttgg cattgtacaa ctcaacaaac 2160

gaaatggctt acgatgcatt gaaggaacat ggtttgcata tcgtttctta tttgagaaag 2220

gcttggtcag atttgtgtaa gtcttacttg ttagaagcaa agtggtacta ctcaagatac 2280

actccatctt tgcaagaata catctcaaat tcttggatct caatctctgg tccagttatt 2340

ttggttcatg cttacttttt ggttgcaaac ccaatcacaa aggaagcttt gcaatcattg 2400

gaaagatacc ataacatcat cagatggtca tctatgatct tgagattgtc agatgatttg 2460

ggtacttctt tagatgaatt gaagagaggt gacgttccaa agtctatcca atgttacatg 2520

tacgaaacag gtgcttcaga agaagatgca agaaagcata cttcttattt gatcggtgaa 2580

acatggaaga aattgaatga agatggtgct gttgaatcac cattcccaga aacttttatt 2640

ggtatcgcaa tgaatttggc tagaatggca caatgtatgt atcaacatgg tgacggtcat 2700

ggtattgaat acggtgaaac agaagataga gttttgtctt tgttggttga accaatccca 2760

tcattgtctt ttgaataa 2778

<210> 13

<211> 1335

<212> DNA

<213> Saccharomyces cerevisiae

<400> 13

atgggaaagc tattacaatt ggcattgcat ccggtcgaga tgaaggcagc tttgaagctg 60

aagttttgca gaacaccgct attctccatc tatgatcagt ccacgtctcc atatctcttg 120

cactgtttcg aactgttgaa cttgacctcc agatcgtttg ctgctgtgat cagagagctg 180

catccagaat tgagaaactg tgttactctc ttttatttga ttttaagggc tttggatacc 240

atcgaagacg atatgtccat cgaacacgat ttgaaaattg acttgttgcg tcacttccac 300

gagaaattgt tgttaactaa atggagtttc gacggaaatg cccccgatgt gaaggacaga 360

gccgttttga cagatttcga atcgattctt attgaattcc acaaattgaa accagaatat 420

caagaagtca tcaaggagat caccgagaaa atgggtaatg gtatggccga ctacatctta 480

gatgaaaatt acaacttgaa tgggttgcaa accgtccacg actacgacgt gtactgtcac 540

tacgtagctg gtttggtcgg tgatggtttg acccgtttga ttgtcattgc caagtttgcc 600

aacgaatctt tgtattctaa tgagcaattg tatgaaagca tgggtctttt cctacaaaaa 660

accaacatca tcagagatta caatgaagat ttggtcgatg gtagatcctt ctggcccaag 720

gaaatctggt cacaatacgc tcctcagttg aaggacttca tgaaacctga aaacgaacaa 780

ctggggttgg actgtataaa ccacctcgtc ttaaacgcat tgagtcatgt tatcgatgtg 840

ttgacttatt tggccggtat ccacgagcaa tccactttcc aattttgtgc cattccccaa 900

gttatggcca ttgcaacctt ggctttggta ttcaacaacc gtgaagtgct acatggcaat 960

gtaaagattc gtaagggtac tacctgctat ttaattttga aatcaaggac tttgcgtggc 1020

tgtgtcgaga ttttttgacta ttacttacgt gatatcaaat ctaaattggc tgtgcaagat 1080

ccaaatttct taaaattgaa cattcaaatc tccaagatcg aacagtttat ggaagaaatg 1140

taccaggata aattacctcc taacgtgaag ccaaatgaaa ctccaatttt cttgaaagtt 1200

aaagaaagat ccagatacga tgatgaattg gttccaaccc aacaagaaga agagtacaag 1260

ttcaatatgg ttttatctat catcttgtcc gttcttcttg ggttttatta tatatacact 1320

ttacacagag cgtga 1335

<210> 14

<211> 1277

<212> DNA

<213> Saccharomyces cerevisiae

<400> 14

ctctagagga tccccgggat aacttcgtat agcatacatt atacgaagtt atcgttttaa 60

gagcttggtg agcgctagga gtcactgcca ggtatcgttt gaacacggca ttagtcaggg 120

aagtcataac acagtccttt cccgcaattt tctttttcta ttactcttgg cctcctctgt 180

acactctata ttttttttatg cctcggtaat gattttcatt ttttttttttc cacctagcgg 240

atgactcttt ttttttctta gcgattggca ttatcacata atgaattata cattatataa 300

agtaatgtga tttcttcgaa gaatatacta aaaaatgagc aggcaagata aacgaaggca 360

aagatgacag agcagaaagc cctagtaaag cgtattacaa atgaaaccaa gattcagatt 420

gcgatctctt taaagggtgg tcccctagcg atagagcact cgatcttccc agaaaaagag 480

gcagaagcag tagcagaaca ggccacacaa tcgcaagtga ttaacgtcca cacaggtata 540

gggtttctgg accatatgat acatgctctg gccaagcatt ccggctggtc gctaatcgtt 600

gagtgcattg gtgacttaca catagacgac catcacacca ctgaagactg cgggattgct 660

ctcggtcaag cttttaaaga ggccctaggg gccgtgcgtg gagtaaaaag gtttggatca 720

ggatttgcgc ctttggatga ggcactttcc agagcggtgg tagatctttc gaacaggccg 780

tacgcagttg tcgaacttgg tttgcaaagg gagaaagtag gagatctctc ttgcgagatg 840

atcccgcatt ttcttgaaag ctttgcagag gctagcagaa ttaccctcca cgttgattgt 900

ctgcgaggca agaatgatca tcaccgtagt gagagtgcgt tcaaggctct tgcggttgcc 960

ataagagaag ccacctcgcc caatggtacc aacgatgttc cctccaccaa aggtgttctt 1020

atgtagtgac accgattatt taaagctgca gcatacgata tatatacatg tgtatatatg 1080

tatacctatg aatgtcagta agtatgtata cgaacagtat gatactgaag atgacaaggt 1140

aatgcatcat tctatacgtg tcattctgaa cgaggcgcgc tttccttttt tctttttgct 1200

ttttcttttt ttttctcttg aactcgaata acttcgtata gcatacatta tacgaagtta 1260

tcccgggtac cgagctc 1277

<210> 15

<211> 2053

<212> DNA

<213> Saccharomyces cerevisiae

<400> 15

gtcctgtact tccttgttca tgtgtgttca aaaacgttat atttatagga taattatact 60

ctatttctca acaagtaatt ggttgtttgg ccgagcggtc taaggcgcct gattcaagaa 120

atatcttgac cgcagttaac tgtgggaata ctcaggtatc gtaagatgca agagttcgaa 180

tctcttagca accattattt ttttcctcaa cataacgaga acacacaggg gcgctatcgc 240

acagaatcaa attcgatgac tggaaatttt ttgttaattt cagaggtcgc ctgacgcata 300

tacctttttc aactgaaaaa ttgggagaaa aaggaaaggt gagagcgccg gaaccggctt 360

ttcatataga atagagaagc gttcatgact aaatgcttgc atcacaatac ttgaagttga 420

caatattatt taaggaccta ttgttttttc caataggtgg ttagcaatcg tcttactttc 480

taacttttct taccttttac atttcagcaa tatatatata tatatttcaa ggatatacca 540

ttctaatgtc tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa 600

tcacagccga agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt 660

tcgatttcga aaatcattta attggtggtg ctgctatcga tgctacaggt gttccacttc 720

cagatgaggc gctggaagcc tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg 780

gtcctaaatg gggtaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag 840

aacttcaatt gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact 900

tatctccaat caagccacaa tttgctaaag gtactgactt cgttgttgtc agagaattag 960

tgggaggtat ttactttggt aagagaaagg aagacgatgg tgatggtgtc gcttgggata 1020

gtgaacaata caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc 1080

tacaacatga gccaccattg cctatttggt ccttggataa agctaatgtt ttggcctctt 1140

caagattatg gagaaaaact gtggaggaaa ccatcaagaa cgaattccct acattgaagg 1200

ttcaacatca attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa 1260

atggtattat aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta 1320

tcccaggttc cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca 1380

ccgcatttgg tttgtacgaa ccatgccacg gttctgctcc agatttgcca aagaataagg 1440

tcaaccctat cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc 1500

ctgaagaagg taaggccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa 1560

ctggtgattt aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag 1620

ttaagaaaat ccttgcttaa aaagattctc ttttttttatg atatttgtac ataaacttta 1680

taaatgaaat tcataataga aacgacacga aattacaaaa tggaatatgt tcatagggta 1740

gacgaaacta tatacgcaat ctacatacat ttatcaagaa ggagaaaaag gaggatgtaa 1800

aggaatacag gtaagcaaat tgatactaat ggctcaacgt gataaggaaa aagaattgca 1860

ctttaacatt aatattgaca aggaggaggg caccacacaa aaagttaggt gtaacagaaa 1920

atcatgaaac tatgattcct aatttatata ttggaggatt ttctctaaaa aaaaaaaaat 1980

acaacaaata aaaaacactc aatgacctga ccatttgatg gagtttaagt caataccttc 2040

ttgaaccatt tcc 2053

<210> 16

<211> 1245

<212> DNA

<213> Saccharomyces cerevisiae

<400> 16

gcttgcatgc ctgcaggtcg actctagagg atccccggga taacttcgta taatgtatgc 60

tatacgaagt tatgggtaat aactgatata attaaattga agctctaatt tgtgagttta 120

gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat 180

atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg 240

caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg 300

ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa 360

tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa 420

caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagcta 480

gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt 540

cttccgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg 600

taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat 660

taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg gcggataatg 720

cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc acatgtgttt 780

ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc ttggtggtac 840

gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta aatagcttgg 900

cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt 960

atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact gggcaatttc 1020

atgtttcttc aacaccacat atgcgtatat ataccaatct aagtctgtgc tccttccttc 1080

gttcttcctt ctgctcggag attaccgaat caaaaaaatt tcaaagaaac cggaatcaaa 1140

aaaaagaaca aaaaaaaaaa agatgaattg aaaagcttta tggaccctga taacttcgta 1200

taatgtatgc tatacgaagt tatcccgggt accgagctcg aattc 1245

<210> 17

<211> 1007

<212> DNA

<213> Saccharomyces cerevisiae

<400> 17

aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc gtaatatatg 60

tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta ttgaaaaata 120

gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg attaagaatt 180

cggtcgaaaa aagaaaagga gagggccaag agggagggca ttggtgacta ttgagcacgt 240

gagtatacgt gattaagcac acaaaggcag cttggagtat gtctgttatt aatttcacag 300

gtagttctgg tccattggtg aaagtttgcg gcttgcagag cacagaggcc gcagaatgtg 360

ctctagattc cgatgctgac ttgctgggta ttatatgtgt gcccaataga aagagaacaa 420

ttgacccggt tattgcaagg aaaatttcaa gtcttgtaaa agcatataaa aatagttcag 480

gcactccgaa atacttggtt ggcgtgtttc gtaatcaacc taaggaggat gttttggctc 540

tggtcaatga ttacggcatt gatatcgtcc aactgcatgg agatgagtcg tggcaagaat 600

accaagagtt cctcggtttg ccagttatta aaagactcgt atttccaaaa gactgcaaca 660

tactactcag tgcagcttca cagaaacctc attcgtttat tcccttgttt gattcagaag 720

caggtgggac aggtgaactt ttggattgga actcgatttc tgactgggtt ggaaggcaag 780

agagccccga aagcttacat tttatgttag ctggtggact gacgccagaa aatgttggtg 840

atgcgcttag attaaatggc gttattggtg ttgatgtaag cggaggtgtg gagacaaatg 900

gtgtaaaaga ctctaacaaa atagcaaatt tcgtcaaaaa tgctaagaaa taggttatta 960

ctgagtagta tttatttaag tattgtttgt gcacttgcct gcaggcc 1007

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 18

gacgttgaaa ttgaggctac tgcg 24

<210> 19

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 19

tgggggatca ctgacctaat gcttcaacta ac 32

<210> 20

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 20

tgaagcatta ggtcagtgat cccccacaca ccat 34

<210> 21

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 21

cttggtgaag ccattttgta attaaaactt agat 34

<210> 22

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 22

aagttttaat tacaaaatgg cttcaccaag aggtat 36

<210> 23

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 23

cgtgacataa ctaatttatt caaaagacaa tgatg 35

<210> 24

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 24

tgtcttttga ataaattagt tatgtcacgc ttac 34

<210> 25

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 25

ggatcctcta gagcaaatta aagccttcga gcg 33

<210> 26

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 26

aaggctttaa tttgctctag aggatccccg ggat 34

<210> 27

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 27

gtagagacca catcgagctc ggtacccggg at 32

<210> 28

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 28

gtaccgagct cgatgtggtc tctacaggat c 31

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 29

caagccttgt cccaaggcag cg 22

<210> 30

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 30

gcttcggtta cttctaagga agtccacaca aatcaagatc cgttag 46

<210> 31

<211> 69

<212> DNA

<213> Artificial Sequence

<400> 31

gttataatat ctgtgcgtct tgagttgaag tcaggaatct aaaatattgg aaagtcatta 60

ggtgaggtt 69

<210> 32

<211> 69

<212> DNA

<213> Artificial Sequence

<400> 32

tatgtcagac caccaccaat gttaacctca cctaatgact ttccaatatt ttagattcct 60

gacttcaac 69

<210> 33

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 33

attggttaaa actggcattg ttttatattt gttgtaaaaa gtag 44

<210> 34

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 34

caacaaatat aaaacaatgc cagttttaac caataaaaca gtc 43

<210> 35

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 35

ctatcgattt caattcaatt caatttagga tttaatgcag gtgac 45

<210> 36

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 36

gtcacctgca ttaaatccta aattgaattg aattgaaatc gatag 45

<210> 37

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 37

taacattcaa cgctagtatc actatactgg atctaaagag tacaatag 48

<210> 38

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 38

ctctttagat ccagtatagt gatactagcg ttgaatgtta gcg 43

<210> 39

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 39

ctattgttgt cggcagtcat tttgtttgtt tatgtgtg 38

<210> 40

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 40

cacacataaa caaacaaaat gactgccgac aacaatag 38

<210> 41

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 41

atcataaatc ataagaaatt cgcttatagc attctatgaa tttgcc 46

<210> 42

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 42

ggcaaattca tagaatgcta taagcgaatt tcttatgatt tatgat 46

<210> 43

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 43

catgaacaag gaagtacagg acagatcatg atacataaaa gcgatataac 50

<210> 44

<211> 52

<212> DNA

<213> Artificial Sequence

<400> 44

gttatatcgc ttttatgtat catgatctgt cctgtacttc cttgttcatg tg 52

<210> 45

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 45

gatagttgat ttctattcca acaggaaatg gttcaagaag gtattgac 48

<210> 46

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 46

gtcaatacct tcttgaacca tttcctgttg gaatagaaat caactatc 48

<210> 47

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 47

cacaggcgct accatgagaa ttg 23

<210> 48

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 48

ttggttcttc gacatgatca c 21

<210> 49

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 49

aggaatctaa aatacgtttc gtttagaacc gacg 34

<210> 50

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 50

gttctaaacg aaacgtattt tagattcctg acttc 35

<210> 51

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 51

ctttttctga agccattgtt ttttatatttg ttgt 34

<210> 52

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 52

caaatataaa aacaatggct tcagaaaaag aaat 34

<210> 53

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 53

cgtgacataa ctaatctatt tgcttctctt gtaaac 36

<210> 54

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 54

agagaagcaa atagattagt tatgtcacgc ttac 34

<210> 55

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 55

caggcatgca agccaaatta aagccttcga gcg 33

<210> 56

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 56

gaaggcttta atttggcttg catgcctgca ggtc 34

<210> 57

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 57

catggtcctt atcgaattcg agctcggtac 30

<210> 58

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 58

gagctcgaat tcgataagga ccatgtataa g 31

<210> 59

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 59

atcgatgtaa tgtagag 17

<210> 60

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 60

ccggggcacc tgtcactttg g 21

<210> 61

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 61

caggaatcta aaatatttcc tctaatcagg ttcc 34

<210> 62

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 62

acctgattag aggaaatatt ttagattcct gacttc 36

<210> 63

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 63

ctttttctga agccattgtt ttttatatttg ttgt 34

<210> 64

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 64

aatataaaaa caatggcttc agaaaaagaa attag 35

<210> 65

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 65

tcataagaaa ttcgcttatt caaaagacaa tgatg 35

<210> 66

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 66

ttgtcttttg aataagcgaa ttttcttatga tttatg 36

<210> 67

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 67

tatatagtaa tgtcgttaga tcatgataca taaaagc 37

<210> 68

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 68

tatgtatcat gatctaacga cattactata tatataatat ag 42

<210> 69

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 69

tcataagaaa ttcgcggcct gcaggcaagt gcac 34

<210> 70

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 70

ttgcctgcag gccgcgaatt tcttatgatt tat 33

<210> 71

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 71

agatcatgat acataaaagc gat 23

<210> 72

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 72

tcccaggcgt agaacagttt atcag 25

<210> 73

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 73

tcaacgctag tatgatccaa gctatctact gag 33

<210> 74

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 74

agatagcttg gatcatacta gcgttgaatg ttag 34

<210> 75

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 75

atagctttcc cattttgttt gtttatgtgt gtttattcg 39

<210> 76

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 76

atacacttta cacagagcgt gagcgaattt cttatgattt atg 43

<210> 77

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 77

cacttcacgt gcttctggta catacttg 28

Claims (7)

1. A construction method of recombinant saccharomyces cerevisiae for producing terpineol is characterized by comprising the following steps:

using a method of homologous recombination, the strain of saccharomyces cerevisiae ATCC: 208352, introducing a truncated terpineol synthase coding gene delta Ts to obtain a recombinant bacterium 1;

the nucleotide sequence of the truncated terpineol synthase coding gene delta Ts is shown in SEQ ID NO. 1.

2. A construction method of recombinant saccharomyces cerevisiae for producing terpineol is characterized by comprising the following steps:

introducing a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase encoding gene tHMG1 and an isopentenyl pyrophosphate isomerase encoding gene IDI1 into the recombinant bacterium 1 by utilizing a homologous recombination method to obtain a recombinant bacterium 2;

the nucleotide sequence of the truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase coding gene tHMG1 is shown as SEQ ID NO. 5;

the nucleotide sequence of the isopentenyl pyrophosphate isomerase coding gene IDI1 is shown in SEQ ID NO. 6.

3. A construction method of recombinant saccharomyces cerevisiae for producing terpineol is characterized by comprising the following steps:

introducing a mutated farnesyl pyrophosphate synthase encoding gene MERg20 into an ERG20 site in the recombinant bacterium 2 by using a homologous recombination method to obtain a recombinant bacterium 3;

the nucleotide sequence of the mutated farnesyl pyrophosphate synthase encoding gene MErg20 is shown in SEQ ID No. 11.

4. A construction method of recombinant saccharomyces cerevisiae for producing terpineol is characterized by comprising the following steps:

introducing a fusion protein coding gene ERG20FTs of mutated farnesyl pyrophosphate synthase and truncated terpineol synthase into the recombinant bacterium 3 by using a homologous recombination method to obtain a recombinant bacterium 4;

the nucleotide sequence of the coding gene ERG20FTs is shown in SEQ ID NO. 12.

5. A construction method of recombinant saccharomyces cerevisiae for producing terpineol is characterized by comprising the following steps:

by utilizing a homologous recombination method, introducing a squalene synthase coding gene ERG9 into the recombinant strain 4 to obtain a recombinant strain 5;

the nucleotide sequence of the squalene synthase coding gene ERG9 is shown in SEQ ID NO. 13.

6. A recombinant s.cerevisiae producing terpineol constructed by the method of any one of claims 1-5.

7. Use of the terpineol-producing recombinant saccharomyces cerevisiae of claim 6 in the production of terpineol.

Images