CN111041041A

CN111041041A - Saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone and construction method thereof

Info

Publication number: CN111041041A
Application number: CN201911385357.5A
Authority: CN
Inventors: 卢文玉; 张传波
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-12-28
Filing date: 2019-12-28
Publication date: 2020-04-21

Abstract

The invention discloses a saccharomyces cerevisiae recombinant bacterium for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone and a construction method thereof, wherein the method comprises the following steps of introducing an optimized α -lupinene synthase encoding gene ZSS1, an optimized cytochrome P450 enzyme encoding gene CYP71BA1, an optimized cytochrome P450 reductase encoding gene AtCPR1 and an optimized dehydrogenase encoding gene ZSD1 into saccharomyces cerevisiae by utilizing homologous recombination^S114AThe recombinant saccharomyces cerevisiae strain 1 for producing α -lupinene, 8-hydroxyl- α -lupinene and zingiberone is obtained, and experiments prove that the strain is prepared by the methodThe constructed saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone can produce α -lupinene, 8-hydroxy- α -lupenone and zingerone, and lays a foundation for artificial cell synthesis of α -lupinene, 8-hydroxy- α -lupenone and zingerone.

Description

Saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone and construction method thereof

Technical Field

The invention relates to the technical field of biology, in particular to recombinant saccharomyces cerevisiae for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone and a construction method thereof.

Background

The biosynthesis pathway of zingerone is now clear, and similar to the biosynthesis of other sesquiterpene compounds, farnesyl pyrophosphate (FPP) is derived from mevalonate pathway (MVA). FPP is catalyzed by α -lupinene synthase ZSS1 to synthesize α -lupinene, α -lupinene, also known as α -caryophyllene or humulene, and is a natural monocyclic sesquiterpene compound, which is found in essential oils of hops of the Humulaceae family, so it is also known as humulene, α -lupinene in beer has anti-cold effect, α -lupinene has anti-inflammatory effect, and is likely to be an effective drug for treating inflammatory diseases.

The accumulation of terpenoids in plants is less, the extraction and purification by using plant raw materials are low in yield and purity, and a large amount of plant raw materials are needed; the synthesis of terpenes by chemical synthesis is widely used, but for terpenoids with complex structures, it is still difficult and costly. At present, the zingerone is only found in the Zingiberaceae plants, although the biosynthesis pathway is already explained, the synthesis regulation mechanism of the zingerone in plant hosts is not reported, and the improvement of the production of the zingerone by modifying the original hosts by a metabolic engineering means is still difficult. Therefore, the development of a method for efficiently synthesizing natural products is imperative, but no report is found for synthesizing the zerumbone by using a synthetic biology technology at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone.

The second purpose of the invention is to provide a construction method of the saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone.

The third purpose of the invention is to provide the application of the recombinant saccharomyces cerevisiae strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone in the fermentation production of α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

The fourth purpose of the invention is to provide a second saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

The fifth purpose of the invention is to provide a construction method of a second saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone.

The sixth purpose of the invention is to provide the application of the second saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone in the fermentation production of α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

The seventh purpose of the invention is to provide a third saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

The eighth purpose of the invention is to provide a construction method of the third saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone.

The ninth purpose of the invention is to provide the application of the third saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone in the fermentation production of α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

The technical scheme of the invention is summarized as follows:

a construction method of a saccharomyces cerevisiae recombinant bacterium for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone comprises the following steps of introducing an optimized α -lupinene synthase encoding gene ZSS1, an optimized cytochrome P450 enzyme encoding gene CYP71BA1, an optimized cytochrome P450 reductase encoding gene AtCPR1 and an optimized dehydrogenase encoding gene ZSD1 into saccharomyces cerevisiae (ATCC:208352) by utilizing homologous recombination^S114AObtaining a recombinant saccharomyces cerevisiae strain 1 which is called recombinant bacterium 1 for short and produces α -lupinene, 8-hydroxyl- α -lupinene and zingiberone;

the nucleotide sequence of the optimized α -luplin synthase enzyme coding gene ZSS1 is shown as SEQ ID NO. 1;

the nucleotide sequence of the optimized cytochrome P450 enzyme coding gene CYP71BA1 is shown in SEQ ID NO. 2;

the nucleotide sequence of the optimized cytochrome P450 reductase coding gene AtCPR1 is shown in SEQ ID NO. 3;

the optimized dehydrogenase encoding gene ZSD1^S114AThe nucleotide sequence of (A) is shown in SEQ ID NO. 4.

The saccharomyces cerevisiae recombinant strain 1 which is constructed by the method and can produce α -lupinene, 8-hydroxy- α -lupinene and zingerone.

Application of recombinant saccharomyces cerevisiae 1 for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone in fermentation production of α -lupinene, 8-hydroxy- α -lupinene and zingerone.

The second construction method of the saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone comprises the following steps:

introducing a 3-hydroxy-3-methylglutaryl coenzyme A reductase coding gene tHMG1 and a farnesyl pyrophosphate synthase coding gene ERG20 into the recombinant bacterium 1 to obtain recombinant saccharomyces cerevisiae 2 which is used for short and produces α -lupinene, 8-hydroxy- α -lupinene and zingerone of the recombinant bacterium 2;

the nucleotide sequence of the 3-hydroxy-3-methylglutaryl coenzyme A reductase coding gene tHMG1 is shown as SEQ ID NO. 5; the nucleotide sequence of the farnesyl pyrophosphate synthase coding gene ERG20 is shown in SEQ ID NO. 6.

The saccharomyces cerevisiae recombinant strain 2 which is constructed by the method and can produce α -lupinene, 8-hydroxy- α -lupinene and zingerone.

Application of recombinant saccharomyces cerevisiae strain 2 for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone in fermentation production of α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

The third construction method of recombinant Saccharomyces cerevisiae strain producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone includes the following steps_HXT1Replacement of promoter P of squalene synthase-encoding gene ERG9 in recombinant bacterium 2_ERG9Obtaining recombinant bacteria 3;

the promoter P_HXT1The nucleotide sequence of (A) is shown by SEQ ID NO. 7.

The saccharomyces cerevisiae recombinant strain 3 which is constructed by the method and can produce α -lupinene, 8-hydroxy- α -lupinene and zingerone.

Application of recombinant saccharomyces cerevisiae 3 for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone in fermentation production of α -lupinene, 8-hydroxy- α -lupenone and zingerone.

The invention has the advantages that:

experiments prove that, taking saccharomyces cerevisiae ATCC:208352 as an example, α -lupinene synthase ZSS1 encoding gene ZSS1, cytochrome P450 enzyme encoding gene CYP71BA1, cytochrome P450 reductase encoding gene AtCPR1 and dehydrogenase encoding gene are introduced into saccharomyces cerevisiae ATCC:208352^ZSD1S114AThe obtained recombinant bacterium 1 can produce α -lupinene, 8-hydroxy- α -lupinene and zingiberone, and can raise the activity of tHMG1 and ERG20 of recombinant bacterium 1 and can raise the yield of α -lupinene, 8-hydroxy- α -lupinene and zingiberone, and its promoter P can be used_HXT1The original promoter of the squalene synthase coding gene ERG9 is replaced to further improve the yield of α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

The invention successfully constructs the saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone, and lays a foundation for artificial cell synthesis of α -lupinene, 8-hydroxy- α -lupenone and zingerone.

Drawings

FIG. 1 shows the result of GC-MS analysis of α -luptene, (wherein A is a GC-MS detection spectrum of recombinant bacteria 1, W303-1a and α -luptene standard substance, B is a α -luptene standard quality spectrum, and C is a mass spectrum of sample Peak 2)

FIG. 2 shows the GC-MS analysis results of 8-hydroxy- α -lupulone and zerumbone (wherein A is a GC-MS detection spectrum of recombinant bacteria 1, W303-1a and zerumbone standard substance, B is a GC-MS detection spectrum of recombinant bacteria 1, chromatogram peak 3, GC, 8-hydroxy- α -lupulone GC-MS detection spectrum, D is a GC peak 1 spectrum of recombinant bacteria 1, and E is a zerumbone standard quality spectrum)

FIG. 3 shows the comparison of the yields of α -lupinene, 8-hydroxy- α -lupinene and zingerone in the respective Saccharomyces cerevisiae recombinant strains.

Detailed Description

The present invention will be further illustrated by the following specific examples.

The experimental procedures used in the following examples are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Saccharomyces cerevisiae W303-1a, American ATCC208352)

The yeast referred to in the examples is Saccharomyces cerevisiae W303-1a (ATCC 208352) for better understanding of the present invention.

Time of purchase, 2016.6 was purchased from ATCC. Website address: https:// www.atcc.org

E.coli plasmid pUC57, commercially available.

ADE2 was derived from saccharomyces cerevisiae BY4741 (usa,

201388) genome (obtained by PCR)

URA3 and HIS3 were derived from pXP218 and pXP320 (both from adddge)

EXAMPLE 1 preparation of the respective fragment sources

(1) The sequence information of the gene element ZSS1 used by the invention is derived from Zingiber zerumbet (GenBank: AB247331.1) and is synthesized by the company through saccharomyces cerevisiae codon optimization, and the nucleotide sequence of the optimized α -lupinene synthase coding gene ZSS1 is shown as SEQ ID NO. 1;

the sequence information of CYP71BA1 is derived from Zingiber zerumbet (GeneBank: AB331234.1), is synthesized by Saccharomyces cerevisiae codon optimization by a company, and the nucleotide sequence of an optimized cytochrome P450 enzyme encoding gene CYP71BA1 is shown in SEQ ID NO. 2.

The sequence information of AtCPR1 was derived from Arabidopsis thaliana (Arabidopsis thaliana, GeneBank: BT008426), which was synthesized by Saccharomyces cerevisiae codon optimization, and the nucleotide sequence of the optimized cytochrome P450 reductase-encoding gene AtCPR1 is shown in SEQ ID NO. 3.

ZSD1^S114ANucleotide of (A)The sequence is derived from Zingiber zerumbet (GeneBank: AB480831.1), the 114 th serine is replaced by alanine, the serine is optimized by saccharomyces cerevisiae codon, and the optimized dehydrogenase coding gene ZSD1^S114AThe nucleotide sequence of (A) is shown in SEQ ID NO. 4.

The above gene was ligated to E.coli plasmid pUC57 and stored in E.coli,

the nucleotide sequence of ADE2 was derived from saccharomyces cerevisiae BY4741 (usa,

201388) the genome of the plant,

the nucleotide sequence of URA3, and the nucleotide sequence of HIS3 were from plasmids pXP218 (from adddge) and pXP320 (from adddge), respectively.

(2) The saccharomyces cerevisiae endogenous fragment is obtained by extracting a saccharomyces cerevisiae ATCC:208352 genome, taking the genome as a template and carrying out PCR amplification.

The extraction method of the saccharomyces cerevisiae genome comprises the following steps: inoculating Saccharomyces cerevisiae into liquid YPD culture medium, and shake culturing at 30 deg.C overnight; sucking the overnight cultured saccharomyces cerevisiae into a 2mL centrifuge tube, centrifuging for 1min at the rotation speed of 10000rpm, removing supernatant, and collecting bottom thallus precipitate; adding quartz sand with the same volume as the thallus into a centrifuge tube, then adding 400 mu L of STES solution and 400 mu L of phenol/chloroform/isoamylol; placing the mixture in an oscillator, and oscillating for 10 min; adding 400 mu L of TE solution into the centrifuge tube after the oscillation is finished, uniformly mixing, centrifuging to obtain supernatant, and transferring the supernatant into a new 2mL centrifuge tube; adding 1/10 volume of 3M NaAc and 2 times volume of absolute ethyl alcohol into the collected supernatant, uniformly mixing, placing at-20 ℃ for 1h, centrifuging at 10000rpm for 10min, observing that a small amount of white precipitate, namely genome DNA, exists at the bottom of a centrifugal tube, removing the supernatant, and rinsing with 75% ethyl alcohol once; in the ventilation place, make the residual alcohol in the centrifuge tube fully volatilize, then add 100 μ L water, namely finish the extraction of yeast genome, measure the DNA concentration with the microspectrophotometer, preserve in the refrigerator of-20 ℃ for subsequent use.

The formulation of the solution used was as follows:

Tris-HCl (50mmol/L) solution: 50mL of 0.1mol/L LTris solution was mixed with 29.2mL of 0.1mol/L hydrochloric acid, and then made up to 100mL with water.

EDTA (0.5mol/L) solution: 18.16g Na₂EDTA·2H₂O was dissolved in water and made to volume of 100mL, and the pH was adjusted to 8.0 with solid NaOH.

Formulation of TE solution: 200mL of Tris-HCl (50mmol/L) solution and 2mL of EDTA (0.5mol/L) solution were mixed, and water was added thereto to adjust the volume to 1L.

The formula of STES lysate: 20mL of Tris-HCl (50mmol/L) solution, 2mL of Triton X-100, 0.584g of NaCl, and 200. mu.L of EDTA (0.5mol/L) solution were mixed, and then the mixture was made up to 100mL with water.

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

(3) The invention adopts high fidelity polymerase of Vazyme company to amplify DNA fragments, and the reaction system comprises the following components:

the PCR reaction procedure was as follows:

the Tm value is determined according to the annealing temperature of the primer.

(4) The plasmid preserved in the escherichia coli is extracted by using a small Tiangen plasmid extraction kit, and the steps are as follows:

1. inoculating Escherichia coli into LB liquid culture medium containing antibiotics for culture, collecting 1-5mL bacterial liquid in a centrifuge tube, centrifuging at 12000rpm for 1min, discarding supernatant, and collecting thallus at the bottom of the centrifuge tube (the supernatant is removed as much as possible);

2. balancing the adsorption column with 500 μ L BL balancing solution, centrifuging at 12000rpm, and pouring off the waste liquid in the collection tube;

3. adding 250 mu L of P1 solution into a centrifuge tube with the bacterial sediment, and completely suspending the bacterial sediment;

4. adding 250 mu L of P2 lysis solution into the centrifuge tube, and turning the centrifuge tube gently to fully lyse the thallus, wherein the bacteria liquid becomes clear at the moment, and the lysis time cannot be too long to avoid plasmid damage;

5. adding 350 mu L of P3 solution into the centrifuge tube, turning the centrifuge tube up and down to precipitate the protein;

6. centrifuging the centrifuge tube at 12000rpm for 10min, centrifuging the precipitate to the bottom completely, collecting supernatant, placing in adsorption column balanced with BL solution, adsorbing in refrigerator at-20 deg.C for 5min, centrifuging at 12000rpm for 1min, and removing waste liquid in the collection tube;

7. rinsing the adsorption column with the adsorbed plasmid twice with PW solution containing alcohol to remove impurities, centrifuging at 12000rpm for 2min to remove PW as much as possible, and placing the adsorption column in ventilation position to volatilize alcohol.

8. Adding 50-100 μ L deionized water into adsorption column, placing at 37 deg.C for 10min to dissolve plasmid in water, placing adsorption column in centrifuge tube, centrifuging at 12000rpm for 2min, and collecting plasmid.

Example 2 construction of recombinant Saccharomyces cerevisiae 1 producing α -lupinene, 8-hydroxy- α -lupinene and zingerone

(1)δ-up，P_ADH2-ZSS1-T_CYC1Construction of HIS 3-delta-down

PCR was performed using the PCR templates and primers described in Table 1, respectively, to obtain DNA fragments: m1 (delta-up), M2 (P)_ADH2)，M3(ZSS1)，M4(T_CYC1)，M5(HIS3)，M6(δ-down)。

TABLE 1 primer sequences

The fragments M2, M3 and M4 were fused into expression cassette P by fusion PCR_ADH2-ZSS1-T_CYC1；

The fragments M5 and M6 were fused into the HIS 3-delta-down fragment by fusion PCR.

The fragments subjected to fusion PCR have 20-30bp homologous sequences mutually, are mutually overlapped and complemented, and can be mutually used as primers and as templates. To carry outThe fused fragments are mixed according to an equimolar ratio, the total amount is more than 800ng, dNTPs, 2 x Phanta Max Buffer, DNA polymerase and deionized water are added, and a 50uL PCR system is prepared. The PCR program was carried out at an annealing temperature of 60 ℃ and a cycle number of 11cycles, and the extension time was calculated as 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 primers at two ends of the total length of the fragment as primers to perform PCR amplification, and then purifying and recovering to obtain a module delta-up, P_ADH2-ZSS1-T_CYC1，HIS3-δ-down。

(2)rDNA-up、P_PGK1-ZSD1^S114A-T_ADH2、P_TEF1-CYP71BA1-T_ADH1、P_TDH3-AtCPR1-T_TDH2Construction of URA3-rDNA-down

PCR was performed using the PCR templates and primers described in Table 2, respectively, to obtain DNA fragments: m7(rDNA-up), M8 (P)_PGK1)，M9(ZSD1^S114A)，M10(T_ADH2)，M11(P_TEF1)，M12(CYP71BA1)，M13(T_ADH1)，M14(P_TDH3)，M15(AtCPR1)，M16(T_TDH2)，M17(URA3)，M18(rDNA-down)。

TABLE 2 primer sequences

The fragments M8, M9 and M10 were fused into expression cassette P by fusion PCR_PGK1-ZSD1^S114A-T_ADH2；

The fragments M11, M12 and M13 were fused into expression cassette P by fusion PCR_TEF1-CYP71BA1-T_ADH1；

The fragments M14, M15 and M16 were fused into expression cassette P by fusion PCR_TDH3-AtCPR1-T_TDH2；

The fragments M17 and M18 were fused into URA3-rDNA-down fragment by fusion PCR.

The fragments subjected to fusion PCR have 20-30bp homologous sequences mutually, are mutually overlapped and complemented, and can be mutually used as primers and as templates. The fused fragments were mixed in equimolar ratio and the total amount was greater than 800ng, and dNTP was supplementeds, 2 × Phanta Max Buffer, DNA polymerase and deionized water to prepare a 50uL PCR system. The PCR program was carried out at an annealing temperature of 60 ℃ and a cycle number of 11cycles, and the extension time was calculated as 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 primers at two ends of the total length of the fragment as primers to perform PCR amplification, and then purifying and recovering to obtain modules rDNA-up and P_PGK1-ZSD1^S114A-T_ADH2、P_TEF1-CYP71BA1-T_ADH1、P_TDH3-AtCPR1-T_TDH2、URA3-rDNA-down。

(3)δ-up，P_ADH2-ZSS1-T_CYC1HIS 3-delta-down fragment transformation of Saccharomyces cerevisiae W303-1a

1. Saccharomyces cerevisiae W303-1a, American ATCC208352) as a growth-producing strain, inoculating into a test tube YPD, and culturing overnight in a shaking table at 30 ℃;

2. transferring the yeast cultured overnight into a new YPD liquid culture medium in a volume ratio of 1/10, and performing shake culture at 30 deg.C for 4-5h to reach logarithmic phase;

3. taking 1mL of bacterial liquid in a sterile centrifuge tube, centrifuging for 3min at 5000rpm, removing supernatant, collecting bottom bacterial liquid, and washing the thalli once by using 1mL of sterile water;

4. resuspend the washed yeast with 1mL of 100mM LiAc and let stand for 5 min;

5.5000 rpm for 3min, removing LiAc solution, and reserving bottom yeast;

6. preparing a transformation system in a centrifugal tube containing yeast, and specifically adding reagents in the following sequence:

the transformed DNA fragment includes delta-up, P_ADH2-ZSS1-T_CYC1HIS 3-delta-down, each fragment being greater than 300 ng;

7. wherein the salmon sperm DNA is boiled in boiling water for 5min to melt, and then is rapidly transferred to ice bath for yeast transformation;

8. blowing and sucking the prepared conversion system by a pipette or placing the conversion system on a vortex oscillator to vibrate for 1min to fully and uniformly mix the system, placing the system in a 42 ℃ water bath kettle, and thermally exciting for 30 min;

9. centrifuging the yeast after heat shock, removing supernatant with a pipette, adding 1mLYPD culture medium, and recovering in a shaking table at 30 deg.C for 2 hr;

10.5000 rpm for 3min, removing YPD liquid medium, and washing with sterile water for 2 times;

11. 100 μ L of sterile water was added, the yeast cells were resuspended and plated on SC selection medium lacking histidine and cultured in 30 ℃ incubator for 2-3 d.

12. After the single bacterium grows out, colony PCR is carried out, and the genome needs to be crudely extracted. The invention adopts a freeze-thaw method to crudely extract the genome of the saccharomyces cerevisiae, firstly, single colony is picked into 10 mu L NaOH solution with the concentration of 10mM, boiled in boiling water for 10min, then put into a refrigerator with the temperature of minus 20 ℃ for freezing for 10min, and freeze-thaw is repeated for three times, namely, the crude extract of the genome can be directly used as a template for colony verification. The correct strain is verified to be the recombinant strain 1-1.

SC selective medium formulation: 6.7g/L Yeast Nitrogen Base (YNB), 20g/L glucose or galactose, 2g/L corresponding default amino acid mixture. The mixture comprises the following components:

the starting strain Saccharomyces cerevisiae W303-1a of the invention can not grow in the SC culture medium without leucine, tryptophan, uracil, adenine and histidine, so when preparing the SC selective culture medium, corresponding components are supplemented as required, and the final concentration is as follows:

(4) construction of recombinant bacterium 1

Fusing modules rDNA-up, P_PGK1-ZSD1^S114A-T_ADH2、P_TEF1-CYP71BA1-T_ADH1、P_TDH3-AtCPR1-T_TDH2And URA3-rDNA-down transformation (3), wherein the transformation method refers to example 1(3), and the screening culture medium is an SC selection culture medium lacking uracil to obtain the recombinant bacterium 1.

Example 3 construction of recombinant Saccharomyces cerevisiae 2 producing α -lupinene, 8-hydroxy- α -lupinene and zingerone

(1) Construction of tHMG1 expression cassette and ERG20 expression cassette

PCR was performed using the PCR templates and primers described in Table 3, respectively, to obtain DNA fragments: m19 (P)_TDH3)，M20(tHMG1)，M21(T_CYC1)，M22(P_PGK1)，M23(ERG20)，M24(T_CYC1)。

TABLE 3 primer sequences

The fragments M19, M20 and M21 were fused into tHMG1 expression cassette P by fusion PCR_TDH3-tHMG1-T_CYC1(ii) a The fragments M22, M23 and M24 are fused by PCR, and ERG20 expression cassette P_PGK1-ERG20-T_CYC1；

Fragment fusion method reference example 2 (1).

(2) Construction of expression vector P1 containing tHMG1 expression cassette

Double cleavage of P with the restriction enzymes ApaI and PstI_TDH3-tHMG1-T_CYC1And PRS304 plasmid (ATCC in USA), and the digested DNA fragment and plasmid fragment were recovered and purified by agarose gel recovery kit (Tiangen Biotechnology Co., Ltd.). 10 u L of the ligation system including 50ng vector DNA, and the carrier DNA mol ratio of 3:1 insert DNA, 10 xT 4DNA Ligasebuffer 2 u L, T4DNA ligase 1 u L, with ddH2O to total volume of 10 u L, prepared system at 22 degrees C reaction for 30min, then transformed into Escherichia coli TRANS T1 competent cells. The correct clone was obtained by M19-F and M21-RPCR verification, and plasmid P1 was obtained by extracting the plasmid from the large intestine in the manner described in example 1 (4).

(3) Construction of expression vector P2 containing ERG20 expression cassette

With the restriction enzyme PstI and BamHI double enzyme P_PGK1-ERG20-T_CYC1And PRS405 plasmid (ATCC, USA), through agarose gel recovery kit (Tiangen Biotechnology technology limited) recovery purification of cut DNA fragments and plasmid fragments. The 10. mu.L ligation system was used in the construction of ptHMG 1. The ligated plasmid was transformed into E.coli TRANS T1 competent cells. The correct clone was obtained by PCR verification using M12-F and M24-R, and plasmid P2 was obtained by extracting the plasmid from the large intestine in the manner described in example 1 (4).

(4) Construction of recombinant bacterium 2

The constructed plasmids P1 and P2 are digested by restriction enzyme NotI for 1 hour at 37 ℃ for linearization, and the following are obtained respectively: the recombinant strain 2 was obtained by introducing linearized ptHMG1 plasmid and pERG20 plasmid, linearized ptHMG1 plasmid and pERG20 plasmid into recombinant Saccharomyces cerevisiae 1, and the transformation method was the same as in example 2, wherein the fragments were linearized ptHMG1 plasmid and pERG20 plasmid, and the selection medium was SC selection medium lacking tryptophan and leucine, respectively.

Example 4 construction of recombinant Saccharomyces cerevisiae 3 producing α -lupinene, 8-hydroxy- α -lupinene and zingerone

(1)ERG9-ADE2-up，P_HXT1Construction of ERG9-down

PCR was performed using the PCR templates and primers described in Table 3, respectively, to obtain DNA fragments: m25(ERG9-up), M26(ADE2), M27 (P)_HXT1)，M28(ERG9-down)。

TABLE 4 primer sequences

The fragments M25 and M26 were fused into ERG9-ADE2-up fragment by fusion PCR.

The fusion method is described in example 2 (1).

(2) Construction of recombinant Strain 3

Fusion module ERG9-ADE2-up, P_HXT1The ERG9-down transformation method refers to example 2(2), wherein the original strain is recombinant bacteria 2, and the screening culture medium is SC selective culture medium lacking adenine to obtain recombinant bacteria 3.

Example 5 recombinant production of α -lupinene, 8-hydroxy- α -lupinene and zerumbone

(1) Recombinant bacterium culture and product extraction

α -lupinene, 8-hydroxy- α -lupinene and zingerone, fermenting, extracting and detecting the recombinant saccharomyces cerevisiae strain:

fermenting in 30mLYPD liquid culture medium, inoculating seed liquid and 10% n-dodecane to obtain initial OD₆₀₀The yield of α -luplin, 8-hydroxy- α -luplin and zerumbone is measured after fermentation for 5 days at the temperature of 0.05 and 30 ℃ and at the speed of 220rpm, the yield of the n-dodecane phase is directly measured for an extracellular product, the intracellular product is subjected to oscillation extraction by adding quartz sand with equal mass and ethyl acetate with equal volume of fermentation liquor, the ethyl acetate is used for gas phase detection after extraction, and a sample needs to be filtered by a filter membrane with the aperture of 0.22 mu m to remove impurities before gas phase and gas chromatography-mass spectrometry detection.

α -luplin, 8-hydroxy- α -luplin and zingiberone gas chromatography detection conditions comprise chromatographic column DB-SWAX, nitrogen flow rate of 1.8mL/min, sample injection temperature of 250 ℃, sample injection split ratio of 1:24, sample injection amount of 1 muL, furnace temperature of 80 ℃ for 3min, furnace temperature of 5 ℃/min for rising to 180 ℃, then 10 ℃/min for rising to 240 ℃, FID detector of 250 ℃, α -luplin, hydroxy α -luplin and zingerone GC-MS detection conditions, chromatographic conditions and gas chromatography, ion source temperature of 230 ℃, ion scanning range of 50-600m/z, α -luplin, 8-hydroxy- α -luplin and zingerone retention time of 12.77, 26.03 and 25.33 respectively.

(2) The result of the detection

A. α -lupinene, 8-hydroxy- α -lupinene and zerumbone were not detected by Saccharomyces cerevisiae W303-1 a;

B. 1, recombinant bacteria:

extracting a fermentation product of the recombinant bacterium 1, wherein a small amount of α -lupinene, 8-hydroxy- α -lupinene and zingiberone can be detected, and the yield is 12.61mg/L, 2.32mg/L and 1.25mg/L respectively;

C. recombinant strain 2, extracting the fermentation product of recombinant strain 2, wherein the contents of α -lupinene, 8-hydroxy- α -lupinene and zingiberone are 20.45mg/L, 11.52mg/L and 8.83mg/L respectively;

D. recombinant strain 3 extraction of fermentation product of recombinant strain 3 can detect α -lupinene, 8-hydroxy- α -lupinene and zingiberone content of 48.85mg/L, 29.58mg/L and 25.52mg/L respectively

The statistics of the results of the recombinant bacteria are shown in FIG. 3.

Sequence listing

<110> Tianjin university

<120> saccharomyces cerevisiae recombinant bacterium for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone and construction method thereof

<160>63

<170>SIPOSequenceListing 1.0

<210>1

<211>1647

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

atggaaagac aatctatggc tttggttggt gacaaggaag aaatcatcag aaagtctttc 60

gaataccacc caactgtttg gggtgactac ttcatcagaa actactcttg tttgccattg 120

gaaaaggaat gtatgatcaa gagagttgaa gaattgaagg acagagttag aaacttgttc 180

gaagaaactc acgacgtttt gcaaatcatg atcttggttg actctatcca attgttgggt 240

ttggactacc acttcgaaaa ggaaatcact gctgctttga gattgatcta cgaagctgac 300

gttgaaaact acggtttgta cgaagtttct ttgagattca gattgttgag acaacacggt 360

tacaacttgt ctccagacgt tttcaacaag ttcaaggacg acaagggtag attcttgcca 420

actttgaacg gtgacgctaa gggtttgttg aacttgtaca acgctgctta cttgggtact 480

cacgaagaaa ctatcttgga cgaagctatc tctttcacta agtgtcaatt ggaatctttg 540

ttgggtgaat tggaacaacc attggctatc gaagtttctt tgttcttgga aactccattg 600

tacagaagaa ctagaagatt gttggttaga aagtacatcc caatctacca agaaaaggtt 660

atgagaaacg acactatctt ggaattggct aagttggact tcaacttgtt gcaatctttg 720

caccaagaag aagttaagaa gatcactatc tggtggaacg acttggcttt gactaagtct 780

ttgaagttcg ctagagacag agttgttgaa tgttactact ggatcgttgc tgtttacttc 840

gaaccacaat actctagagc tagagttatc acttctaagg ctatctcttt gatgtctatc 900

atggacgaca tctacgacaa ctactctact ttggaagaat ctagattgtt gactgaagct 960

atcgaaagat gggaaccaca agctgttgac tgtgttccag aatacttgaa ggacttctac 1020

ttgaagttgt tgaagactta caaggacttc gaagacgaat tggaaccaaa cgaaaagtac 1080

agaatcccat acttgcaaga agaaatcaag gttttgtcta gagcttactt ccaagaagct 1140

aagtggggtg ttgaaagata cgttccagct ttggaagaac acttgttggt ttctttgatc 1200

actgctggtt acttcgctgt tgcttgtgct tcttacgttg gtttgggtga agacgctact 1260

aaggaaactt tcgaatgggt tgcttcttct ccaaagatct tgaagtcttg ttctatccac 1320

tgtagattga tggacgacat cacttctcac caaagagaac aagaaagaga ccacttcgct 1380

tctactgttg aatcttacat gaaggaacac ggtacttctg ctaaggttgc ttgtgaaaag 1440

ttgcaagtta tggttgaaca aaagtggaag gacttgaacg aagaatgttt gagaccaact 1500

caagttgcta gaccattgat cgaaatcatc ttgaacttgt ctagagctat ggaagacatc 1560

tacaagcaca aggacactta cactaactct aacactagaa tgaaggacaa cgtttctttg 1620

atcttcgttg aatctttctt gatctaa 1647

<210>2

<211>1542

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atggaagcca tctctttgtt ctctccattc ttcttcatta ccttgttctt gggtttcttc 60

atcacgctgt tgatcaagag atcttccaga tcttctgttc acaagcaaca agttttgttg 120

gcttctttgc caccatctcc accaagattg ccattgattg gtaacatcca tcaattggtt 180

ggtggtaacc cacatagaat cttgttgcaa ttggctagaa ctcatggtcc attgatttgt 240

ttgagattgg gtcaagttga ccaagttgtt gcttcttcag ttgaagctgt tgaagaaatc 300

atcaagagac acgatttgaa gttcgctgat agaccaagag atttgacttt ctccagaatc 360

ttcttctacg atggtaacgc tgttgttatg actccatatg gtggtgaatg gaagcaaatg 420

agaaaaatct acgccatgga actgttgaac tccagaagag ttaagtcttt cgctgccatt 480

agagaagatg ttgctagaaa attgaccggt gaaattgctc ataaggcttt tgctcaaacc 540

ccagttatta acttgtccga aatggtcatg tccatgatta acgccatcgt tattagagtt 600

gctttcggtg ataagtgcaa acaacaagct tacttcctgc acttggtaaa agaagctatg 660

tcctacgttt cctctttctc tgttgctgatatgtacccat ccttgaagtt cttggatact 720

ttgactggtc tgaagtctaa gttggaaggt gttcatggta aactggataa ggttttcgac 780

gaaattatcg ctcaaagaca agctgctttg gctgctgaac aagctgaaga ggatttgatt 840

atcgatgtcc tgttgaagtt gaaggacgaa ggtaatcaag agttcccaat tacttacacc 900

tctgttaagg ctatcgtgat ggaaattttc ttggctggta ctgaaacctc ctcctctgtt 960

attgattggg ttatgtccga gttgattaag aacccaaaag ccatggaaaa ggtccagaaa 1020

gaaatgagag aagccatgca aggtaagacc aaattggaag aatctgacat cccaaagttc 1080

agctacttga acttggttat caaagaaacc ttgagattgc atccaccagg tcctttgttg 1140

tttccaagag aatgtagaga aacctgcgaa gttatgggtt atagagttcc agctggtgct 1200

agattattga tcaacgcttt tgctttgtcc agggacgaaa agtattgggg ttctgatgct 1260

gaatctttca agccagaaag attcgaaggt atctccgttg attttaaggg ttccaacttt 1320

gagtttatgc catttggtgc tggtagaaga atttgtcctg gtatgacttt cggtatctct 1380

tctgtagaag ttgctttggc acatttgttg ttccatttcg attggcaatt accacagggt 1440

atgaagatcg aagatttgga tatgatggaa gtctctggta tgtctgctac tagaagatct 1500

ccattattgg ttttggccaa gttgattatc ccactgccat aa 1542

<210>3

<211>2079

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atgacttctg cattatacgc atcagactta tttaagcagt tgaaatctat aatgggaaca 60

gactcattgt cagacgacgt cgttttagtt attgctacta cttcattggc tttggttgct 120

ggatttgttg ttttattgtg gaaaaagaca acagctgata ggtctggtga attaaagcca 180

ttaatgatac ctaaatcttt aatggctaag gacgaggacg acgacttgga tttaggatca 240

ggaaagacta gagtctctat atttttcgga actcagacag gaacagctga gggattcgca 300

aaggctttat cagaagagat taaagcaagg tacgagaagg ctgctgtcaa agttatagat 360

ttggatgact acgcagctga tgacgaccag tacgaggaaa agttgaaaaa ggaaactttg 420

gcatttttct gtgttgcaac atacggtgac ggtgagccaa ctgacaacgc tgctaggttc 480

tacaaatggt tcacagagga aaatgagaga gacattaaat tgcagcagtt ggcttacggt 540

gtcttcgcat tgggaaacag gcaatatgaa catttcaata agattggaat tgtcttggac 600

gaagaattat gcaaaaaagg agctaagagg ttgatagagg tcggtttggg tgacgatgac 660

cagtcaatag aggacgactt caatgcatgg aaagagtcat tgtggtcaga gttagataag 720

ttattaaaag acgaagacga caagtcagtc gcaacacctt acacagcagt catacctgag 780

tatagggtcg tcactcacga cccaagattc actactcaaa agtcaatgga gtcaaatgtc 840

gcaaacggaa atactactat tgacattcat cacccatgca gggttgacgt cgctgtccag 900

aaagagttac acactcacga gtctgacagg tcatgcattc acttggagtt cgatatttca 960

agaactggta ttacttacga aacaggtgac cacgttggtg tctacgctga gaaccacgtc 1020

gagattgtcg aggaagctgg aaagttgttg ggacattctt tagatttggt cttctcaatt 1080

catgctgaca aagaggacgg ttcaccattg gagtctgctg ttccaccacc attccctgga 1140

ccatgcactttaggtactgg tttggcaagg tacgcagact tattgaaccc acctaggaag 1200

tcagctttag ttgcattggc tgcatatgca acagaaccat ctgaggcaga gaaattaaag 1260

cacttgactt ctcctgacgg taaggacgag tactcacagt ggatagtcgc atctcagagg 1320

tcattgttgg aggtcatggc agcatttcca tcagcaaagc cacctttagg tgttttcttc 1380

gcagctatag cacctagatt gcagcctagg tattattcaa tatcttcttc acctaggttg 1440

gctccatcta gggtccacgt cacatcagct ttggtttacg gacctactcc tacaggaagg 1500

atacataaag gagtctgctc tacttggatg aagaacgctg tcccagcaga gaagtctcat 1560

gagtgctcag gagctcctat ttttattagg gcatcaaatt tcaaattgcc ttcaaaccca 1620

tctactccaa tagtcatggt cggaccagga acaggtttgg ctcctttcag gggatttttg 1680

caggagagga tggctttgaa ggaggatggt gaggaattgg gatcatcttt gttgttcttt 1740

ggttgtagga ataggcaaat ggacttcatt tatgaggacg aattgaacaa ctttgttgat 1800

caaggagtca tatcagagtt aattatggct ttctcaaggg agggtgcaca aaaggaatac 1860

gtccaacaca agatgatgga aaaggctgca caggtctggg acttgattaa ggaggaggga 1920

tacttatatg tctgcggtga cgcaaagggt atggcaagag acgtccacag gactttgcac 1980

acaattgtcc aggaacagga gggtgtttct tcatctgaag cagaggctat tgttaaaaag 2040

ttgcaaactg aaggtaggta cttgagggac gtctggtaa 2079

<210>4

<211>804

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

atgaggttgg aaggtaaagt tgctttggtt actggtggtg cttctggtat tggtgaatct 60

attgctaggt tgttcattga acatggtgct aagatctgca tcgttgatgt tcaagatgaa 120

ttgggtcaac aagtctccca aagattaggt ggtgatccac atgcttgtta cttccattgt 180

gatgttaccg ttgaagatga tgttagaagg gctgttgatt tcactgctga aaagtacggt 240

actatcgata tcatggttaa caacgctggt attaccggtg ataaggttat tgatattaga 300

gatgccgact tcaacgagtt caaaaaggtt ttcgatatca acgtcaacgg tgtgtttttg 360

ggtatgaagc acgctgctag aattatgatt ccaaagatga agggctcgat cgtttctttg 420

gcttctgttg cttctgttat tgctggtgct ggtccacatg gttatactgg tgctaaacat 480

gctgttgttg gtttgacaaa atctgttgct gctgaattag gtagacacgg tattagagtt 540

aactgcgttt ctccatatgc tgttccaact agattgtcta tgccatactt gccagaatcc 600

gaaatgcaag aagatgcttt gagaggtttc ttgacctttg tcagatctaa cgctaatttg 660

aagggtgttg acttgatgcc aaatgatgtt gctgaagctg tcttgtattt ggctactgaa 720

gaatccaaat acgtgtccgg tttgaacttg gttatagatg gtggtttttc cattgccaac 780

cataccttgc aagttttcga atga 804

<210>5

<211>1509

<212>DNA

<213> Saccharomyces cerevisiae

<400>5

atggttttaa ccaataaaac agtcatttct ggatcgaaag tcaaaagttt atcatctgcg 60

caatcgagct catcaggacc ttcatcatct agtgaggaagatgattcccg cgatattgaa 120

agcttggata agaaaatacg tcctttagaa gaattagaag cattattaag tagtggaaat 180

acaaaacaat tgaagaacaa agaggtcgct gccttggtta ttcacggtaa gttacctttg 240

tacgctttgg agaaaaaatt aggtgatact acgagagcgg ttgcggtacg taggaaggct 300

ctttcaattt tggcagaagc tcctgtatta gcatctgatc gtttaccata taaaaattat 360

gactacgacc gcgtatttgg cgcttgttgt gaaaatgtta taggttacat gcctttgccc 420

gttggtgtta taggcccctt ggttatcgat ggtacatctt atcatatacc aatggcaact 480

acagagggtt gtttggtagc ttctgccatg cgtggctgta aggcaatcaa tgctggcggt 540

ggtgcaacaa ctgttttaac taaggatggt atgacaagag gcccagtagt ccgtttccca 600

actttgaaaa gatctggtgc ctgtaagata tggttagact cagaagaggg acaaaacgca 660

attaaaaaag cttttaactc tacatcaaga tttgcacgtc tgcaacatat tcaaacttgt 720

ctagcaggag atttactctt catgagattt agaacaacta ctggtgacgc aatgggtatg 780

aatatgattt ctaaaggtgt cgaatactca ttaaagcaaa tggtagaaga gtatggctgg 840

gaagatatgg aggttgtctc cgtttctggt aactactgta ccgacaaaaa accagctgcc 900

atcaactgga tcgaaggtcg tggtaagagt gtcgtcgcag aagctactat tcctggtgat 960

gttgtcagaa aagtgttaaa aagtgatgtt tccgcattgg ttgagttgaa cattgctaag 1020

aatttggttg gatctgcaat ggctgggtct gttggtggat ttaacgcaca tgcagctaat 1080

ttagtgacag ctgttttctt ggcattagga caagatcctg cacaaaatgt tgaaagttcc 1140

aactgtataa cattgatgaa agaagtggac ggtgatttga gaatttccgt atccatgcca 1200

tccatcgaag taggtaccat cggtggtggt actgttctag aaccacaagg tgccatgttg 1260

gacttattag gtgtaagagg cccgcatgct accgctcctg gtaccaacgc acgtcaatta 1320

gcaagaatag ttgcctgtgc cgtcttggca ggtgaattat ccttatgtgc tgccctagca 1380

gccggccatt tggttcaaag tcatatgacc cacaacagga aacctgctga accaacaaaa 1440

cctaacaatt tggacgccac tgatataaat cgtttgaaag atgggtccgt cacctgcatt 1500

aaatcctaa 1509

<210>6

<211>1059

<212>DNA

<213> Saccharomyces cerevisiae

<400>6

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 cttacttctt ggtcgccgat 300

gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360

gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420

aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480

accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540

gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaagactgcttac 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>7

<211>1121

<212>DNA

<213> Saccharomyces cerevisiae

<400>7

caggtctcat ctggaatata attcccccct cctgaagcaa atttttcctt tgagccggaa 60

tttttgatat tccgagttct ttttttccat tcgcggaggt tattccattc ctaaacgagt 120

ggccacaatg aaacttcaat tcatatcgac cgactatttt tctccgaacc aaaaaaatag 180

cagggcgaga ttggagctgc ggaaaaaaga ggaaaaaatt ttttcgtagt tttcttgtgc 240

aaattagggt gtaaggtttc tagggcttat tggttcaagc agaagagaca acaattgtag 300

gtcctaaatt caaggcggat gtaaggagta ttggtttcga aagtttttcc gaagcggcat 360

ggcagggact acttgcgcat gcgctcggat tatcttcatt tttgcttgca aaaacgtaga 420

atcatggtaaattacatgaa gaattctctt tttttttttt tttttttttt ttttacctct 480

aaagagtgtt gaccaactga aaaaaccctt cttcaagaga gttaaactaa gactaaccat 540

cataacttcc aaggaattaa tcgatatctt gcactcctga tttttcttca aagagacagc 600

gcaaaggatt atgacactgt tgcattgagt caaaagtttt tccgaagtga cccagtgctc 660

tttttttttt tccgtgaagg actgacaaat atgcgcacaa gatccaatac gtaatggaaa 720

ttcggaaaaa ctaggaagaa atgctgcagg gcattgccgt gccgatcttt tgtctttcag 780

atatatgaga aaaagaatat tcatcaagtg ctgatagaag aataccactc atatgacgtg 840

ggcagaagac agcaaacgta aacatgagct gctgcgacat ttgatggctt ttatccgaca 900

agccaggaaa ctccaccatt atctaatgta gcaaaatatt tcttaacacc cgaagttgcg 960

tgtccccctc acgtttttaa tcatttgaat tagtatattg aaattatata taaaggcaac 1020

aatgtcccca taatcaattc catctggggt ctcatgttct ttccccacct taaaatctat 1080

aaagatatca taatcgtcaa ctagttgata tacgtaaaat c 1121

<210>8

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gcttcggtta cttctaagga agtccacac 29

<210>9

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gtctcttcaa acaaacattg gaaagtcatt ag 32

<210>10

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ctaatgactt tccaatgttt gtttgaagag ac 32

<210>11

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

catagattgt ctttccattg tgtattacga tatagttaat ag 42

<210>12

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ctattaacta tatcgtaata cacaatggaa agacaatcta tg 42

<210>13

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

agcgtgacat aactaattta gatcaagaaa gattcaac 38

<210>14

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

gaatctttct tgatctaaat tagttatgtc acgcttacat tc 42

<210>15

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

agctcttaaa acgcaaatta aagccttcga gcg 33

<210>16

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

cgaaggcttt aatttgcgtt ttaagagctt ggtg 34

<210>17

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

tctcttgaac tcgaatgttg gaatag 26

<210>18

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

tcttgaactc gaatgttgga atagaaatc 29

<210>19

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

cacaggcgct accatgagaa ttg 23

<210>20

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

tattttagat tcctgacttc aactc 25

<210>21

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

tataatatct gtgcgttgtt tttatatttg ttgtaaaaag 40

<210>22

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

caacaaatat aaaaacaacg cacagatatt ataacatctg 40

<210>23

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

cttccaacct cattgtttta tatttgttgt aaaaag 36

<210>24

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

caacaaatat aaaacaatga ggttggaagg taaagt 36

<210>25

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

acataagaga tccgctcatt cgaaaacttg caaggt 36

<210>26

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

agttttcgaa tgagcggatc tcttatgtct ttac 34

<210>27

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>27

tgtgtggggg atcacttaga attatataac ttgatg 36

<210>28

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

tatataattc taagtgatcc cccacacacc at 32

<210>29

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

gagatggctt ccattttgta attaaaactt agat 34

<210>30

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>30

agttttaatt acaaaatgga agccatctct ttgttc 36

<210>31

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

cataagaaat tcgcttatgg cagtgggata atc 33

<210>32

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>32

atcccactgc cataagcgaa tttcttatga tttatg 36

<210>33

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>33

ctaacattca acgctagtat agatcatgat acataaaagc 40

<210>34

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>34

gcttttatgt atcatgatct atactagcgt tgaatgttag 40

<210>35

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>35

atgcagaagt cattttgttt gtttatgtgt gtttattc 38

<210>36

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>36

acataaacaa acaaaatgac ttctgcatta tacg 34

<210>37

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>37

ttaaggagtt aaatttacca gacgtccctc aag 33

<210>38

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>38

ggacgtctgg taaatttaac tccttaagtt actttaatg 39

<210>39

<211>36

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

ctgcaggcat gcaagcgcga aaagccaatt agtgtg 36

<210>40

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

ctaattggct tttcgcgctt gcatgcctgc aggtc 35

<210>41

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

ataagaaatt cgcgaattcg agctcggtac ccg 33

<210>42

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

taccgagctc gaattcgcga atttcttatg atttatg 37

<210>43

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

agatcatgat acataaaagc gatataac 28

<210>44

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

cggggcccat actagcgttg aatgttagc 29

<210>45

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

tgttttattg gttaaaacca ttttgtttgt ttatgtgtgt tt 42

<210>46

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

aaacacacat aaacaaacaa aatggtttta accaataaaa ca 42

<210>47

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>47

tgttttattg gttaaaacca ttttgtttgt ttatgtgtgt tt 42

<210>48

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

gtcacctgca ttaaatccta aacaggcccc ttttcctttg tc 42

<210>49

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

gctgcagaag cagacgctac taaggaaa 28

<210>50

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

taactgcagt attttagatt cctgacttca actc 34

<210>51

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

aatttctttt tctgaagcca ttgttttata tttgttgtaa aaag 44

<210>52

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

ctttttacaa caaatataaa acaatggctt cagaaaaaga aatt 44

<210>53

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

gacaaaggaa aaggggcctg tctatttgct tctcttgtaa ac 42

<210>54

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

gtttacaaga gaagcaaata gacaggcccc ttttcctttg tc 42

<210>55

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

atggatccgg ccgcaaatta aagccttc 28

<210>56

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>56

agtgcagctc agagccccca gcac 24

<210>57

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>57

cacgatacgg cgttatgtgt gtgtgtgata tgtg 34

<210>58

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>58

catatcacac acacacataa cgccgtatcg tgattaacg 39

<210>59

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>59

gaattatatt ccagatgaga cctgcgctat cctcggttct gcattg 46

<210>60

<211>46

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>60

caatgcagaa ccgaggatag cgcaggtctc atctggaata taattc 46

<210>61

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>61

gccaattgta atagctttcc catgatttta cgtatatcaa ctagttgacg 50

<210>62

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>62

cgtcaactag ttgatatacg taaaatcatg ggaaagctat tacaattggc 50

<210>63

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>63

acacgtcgta gtcgtggacg gtttgcaac 29

Claims

1. The construction method of the saccharomyces cerevisiae recombinant bacteria for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone is characterized by comprising the following steps of introducing an optimized α -lupinene synthase encoding gene ZSS1, an optimized cytochrome P450 enzyme encoding gene CYP71BA1, an optimized cytochrome P450 reductase encoding gene AtCPR1 and an optimized dehydrogenase encoding gene ZSD1 into saccharomyces cerevisiae by utilizing homologous recombination^S114AObtaining a recombinant saccharomyces cerevisiae strain 1 which is called recombinant bacterium 1 for short and produces α -lupinene, 8-hydroxyl- α -lupinene and zingiberone;

2. The recombinant saccharomyces cerevisiae strain 1 which is constructed by the method of claim 1 and can produce α -lupinene, 8-hydroxy- α -lupinene and zingiberone.

3. The application of the α -lupinene, 8-hydroxy- α -lupinene and zingiberone-producing saccharomyces cerevisiae recombinant bacterium 1 in the claim 2 in producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone through fermentation.

4. The construction method of the saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone is characterized by comprising the following steps:

the nucleotide sequence of the 3-hydroxy-3-methylglutaryl coenzyme A reductase coding gene tHMG1 is shown in SEQ ID NO. 5; the nucleotide sequence of the farnesyl pyrophosphate synthase coding gene ERG20 is shown in SEQ ID NO. 6.

5. The recombinant Saccharomyces cerevisiae strain 2 capable of producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone, which is constructed by the method of claim 4.

6. The application of the α -lupinene, 8-hydroxy- α -lupinene and zingiberone-producing saccharomyces cerevisiae recombinant bacterium 2 in the production of α -lupinene, 8-hydroxy- α -lupinene and zingiberone by fermentation of the saccharomyces cerevisiae recombinant bacterium in claim 5.

7. The construction method of the saccharomyces cerevisiae recombinant strain for producing α -lupinene, 8-hydroxy- α -lupinene and zingerone is characterized by comprising the following steps of utilizing a promoter P for homologous recombination_HXT1Replacement of promoter P of squalene synthase-encoding gene ERG9 in recombinant bacterium 2_ERG9Obtaining recombinant bacteria 3;

the promoter P_HXT1The nucleotide sequence of (A) is shown by SEQ ID NO. 7.

8. The recombinant saccharomyces cerevisiae 3 capable of producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone, which is constructed by the method of claim 7.

9. The application of the α -lupinene, 8-hydroxy- α -lupinene and zingiberone-producing recombinant saccharomyces cerevisiae 3 of claim 6 in producing α -lupinene, 8-hydroxy- α -lupinene and zingiberone through fermentation.