CN111334522B - Recombinant saccharomyces cerevisiae for producing ambergris alcohol and construction method - Google Patents

Abstract

The invention discloses a recombinant saccharomyces cerevisiae for producing ambergris alcohol and a construction method thereof, wherein the construction method comprises the following steps: introducing a site-directed mutagenesis coding gene delta SHC of a hopene cyclase and a coding gene BmeTC of a tetraphenyl-beta-curcumene synthase into Saccharomyces cerevisiae (Saccharomyces cerevisiaeW-1 a) to obtain recombinant bacteria 1; introducing truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 into recombinant bacterium 1 to obtain recombinant bacterium 2; experiments prove that the recombinant saccharomyces cerevisiae for producing ambergris can ferment and produce ambergris. Lays a foundation for artificially synthesizing ambroxol.

Description

Recombinant saccharomyces cerevisiae for producing ambergris alcohol and construction method

Technical Field

The invention relates to the technical field of biology, in particular to recombinant saccharomyces cerevisiae for producing ambergris and a construction method thereof.

Background

Ambroxol (ambryin) is a tricyclic triterpene compound, is a main component of ambroxol, is a precious animal spice, and is an intestinal metabolite of sperm whales. Ambroxol, which is used as an additive to perfumes due to its long-lasting taste, has been reported to have anti-inflammatory, analgesic, and aphrodisiac activities. Ambroxol has no fragrance, and is degraded and oxidized after being changed in the air to generate ambroxol with special fragrance. Under the constraint of international treaty of rejection of whale, production of ambroxol appears to be in short supply, the chemical synthesis process of ambroxol is complicated in steps, and pollution waste liquid is generated.

The appearance of synthetic biology depends on the grasping and accumulating of structural and functional information of a large number of life basic elements such as genes, proteins and the like, and depends on means such as molecular biology, genetic engineering and the like as a supporting platform. The method utilizes synthetic biology to convert cheap raw materials into available substances such as biofuel, solves the energy problem, or utilizes pollutants through biological decomposition to solve the environmental pollution problem, and can also utilize active complex compounds which are difficult to obtain by chemical and biological synthesis methods produced by synthetic biological students. At present, the design and modification of microbial strains to produce natural products using synthetic biology methods has been considered as a potential method, but the construction and optimization of related biosynthetic pathways in Saccharomyces cerevisiae to produce ambergris has not been reported using synthetic biology methods.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a recombinant saccharomyces cerevisiae for producing ambergris.

The second object of the invention is to provide a construction method of recombinant saccharomyces cerevisiae for producing ambergris alcohol.

The third object of the invention is to provide an application of the recombinant saccharomyces cerevisiae for producing ambergris alcohol in producing ambergris alcohol by fermentation.

A fourth object of the present invention is to provide a second recombinant Saccharomyces cerevisiae for the production of ambergris.

The fifth object of the invention is to provide a second construction method of recombinant saccharomyces cerevisiae for producing ambergris alcohol.

The sixth object of the invention is to provide a second use of the recombinant saccharomyces cerevisiae for producing ambergris alcohol for fermentation production of ambergris alcohol.

A seventh object of the present invention is to provide a third recombinant Saccharomyces cerevisiae for the production of ambergris.

The eighth object of the invention is to provide a third construction method of recombinant saccharomyces cerevisiae for producing ambergris alcohol.

The ninth object of the invention is to provide the use of the third recombinant Saccharomyces cerevisiae for producing ambroxol by fermentation.

The technical scheme of the invention is summarized as follows:

a construction method of recombinant saccharomyces cerevisiae for producing ambergris alcohol comprises the following steps: introducing a site-directed mutagenesis coding gene delta SHC of a hopene cyclase and a coding gene BmeTC of a tetraphenyl-beta-curcumene synthase into Saccharomyces cerevisiae (Saccharomyces cerevisiaeW-1 a) to obtain recombinant bacteria 1;

the nucleotide sequence of the site-directed mutagenesis patchoulene cyclase coding gene delta SHC is shown as SEQ ID No. 2.

The nucleotide sequence of the tetraphenyl-beta-curcumene synthase encoding gene BmeTC is shown as SEQ ID No. 3.

Recombinant saccharomyces cerevisiae for producing ambergris alcohol constructed by the method.

The application of the recombinant saccharomyces cerevisiae in producing ambergris alcohol by fermentation.

The second construction method of the recombinant saccharomyces cerevisiae for producing ambergris alcohol comprises the following steps: introducing truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 into recombinant bacterium 1 to obtain recombinant bacterium 2;

the nucleotide sequence of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 is shown as SEQ ID No. 4.

Recombinant saccharomyces cerevisiae for producing ambergris constructed by the second method.

The use of the second recombinant saccharomyces cerevisiae for producing ambergris by fermentation.

The third construction method of the recombinant saccharomyces cerevisiae for producing ambergris alcohol comprises the following steps: introducing a farnesyl pyrophosphate synthase encoding gene Erg20 into the recombinant bacterium 1 to obtain a recombinant bacterium 3;

the nucleotide sequence of the farnesyl pyrophosphate synthase encoding gene Erg20 is shown as SEQ ID No. 5.

Recombinant saccharomyces cerevisiae for producing ambergris constructed by the third method.

The third recombinant saccharomyces cerevisiae is used for producing ambergris alcohol by fermentation.

Experiments prove that the recombinant saccharomyces cerevisiae for producing ambergris can ferment and produce ambergris. Lays a foundation for artificially synthesizing ambroxol.

Drawings

FIG. 1 is a schematic representation of plasmid PXP 320-. DELTA.SHC.

FIG. 2 is a schematic representation of plasmid PXP-BmeTC.

FIG. 3 is a GC-MS detection chart of ambroxol.

Detailed Description

The invention is further illustrated by the following examples.

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

Saccharomyces cerevisiae Saccharomyces cerevisiae W303-1a (American ATCC: 208352) useful in the present invention,

purchase time, 2016.6 website: https:// www.atcc.org-

The Saccharomyces cerevisiae is disclosed to enable one skilled in the art to better understand the present invention, but is not limited thereto. Other yeasts may also be used in the present invention.

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

Example 1 construction method of plasmid PXP 320-. DELTA.SHC

TABLE 1 primer sequences

According to Table 1, a fragment ΔSHC was amplified using an artificially synthesized ΔSHC gene fragment (SEQ ID No.2, site-directed mutant patchoulene cyclase-encoding gene ΔSHC) as a template, spe1-SHC-F (SEQ ID No. 6) as a forward primer, and SHC-Xho1-R (SEQ ID No. 7) as a backward primer.

Empty plasmid PXP320 (purchased from Addgene) stored in escherichia coli was extracted using a plasmid miniprep kit, the extraction procedure being as follows:

1. inoculating Escherichia coli containing empty plasmid PXP320 into LB liquid medium containing antibiotics (ampicillin) for culturing for 12 hours, collecting 3mL of bacterial liquid, centrifuging at 12000rpm for 1min, and discarding supernatant (the supernatant is removed as much as possible);

2. balancing the adsorption column with 500 mu L BL balance liquid, centrifuging at 12000rpm, and pouring out waste liquid in the collecting pipe;

3. adding 250 mu L of P1 solution into the centrifuge tube with the bacterial precipitate obtained in the step 1, and completely suspending the bacterial precipitate;

4. then 250 mu L of P2 lysis solution is added into the centrifuge tube, the centrifuge tube is gently turned over for 6-7 times, so that the thalli are fully lysed, and the bacterial liquid becomes clear at the moment;

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

6. centrifuging the centrifuge tube 12000rpm after the operation of the step 5 for 10min, thoroughly centrifuging the precipitate to the bottom, taking supernatant, adsorbing for 5min in a refrigerator at the temperature of-20 ℃ in an adsorption column balanced by BL balance liquid in the step 2, centrifuging for 1min at 12000rpm, and pouring out waste liquid in a collecting tube;

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

8. 100. Mu.L of deionized water was added to the column, the column was allowed to dissolve in water at 37℃for 10min, the column was placed in a centrifuge tube and centrifuged at 12000rpm for 2min, and empty plasmid PXP320 was collected.

The amplified fragment Δshc and the extracted plasmid PXP320 were subjected to double cleavage with restriction enzymes SpeI and XhoI in the following manner:

and (3) placing the prepared enzyme digestion system in a water bath kettle at 37 ℃ for enzyme digestion for 1h, purifying and recovering the digested plasmids and fragments, and carrying out a connection reaction.

The connection system is as follows:

the prepared connection system is placed at 22 ℃ for connection for 1h. The constructed plasmid PXP 320.DELTA.SHC was then validated by transformation with E.coli competent cells Trans1-T1 (competent cells for short) purchased from the full gold company. The conversion steps are as follows: taking out competent cells from a refrigerator at-80 ℃, placing on ice, melting, taking 50 mu L of competent cells into a centrifuge tube, adding 5 mu L of connected product (PXP 320-delta SHC), gently mixing, and standing on ice for 30min; transferring the centrifuge tube into a water bath kettle with the temperature of 42 ℃, performing heat shock for 30S, immediately taking out, placing on ice, standing for 2min, ensuring stable movement process, and not severely shaking the centrifuge tube; adding 500 mu L of LB medium without antibiotics into the centrifuge tube in the step 2, and resuscitating for 1 hour in a shaking table at 37 ℃; centrifuging at 4000rpm, removing the supernatant LB culture solution, adding 100 mu L of sterile water, re-suspending cells, coating the bacterial solution on an LB plate culture medium containing an antibiotic Amp, culturing at 37 ℃, growing single colonies, extracting plasmids, and performing enzyme digestion verification to verify that the construction is correct.

Example 2 construction method of plasmid PXP218-BmeTC

The construction method of the plasmid PXP-BmeTC was the same as in example 1, and the fragment BmeTC was amplified by PCR using the artificially synthesized BmeTC gene fragment (SEQ ID No. 3) as a template, spe1-BmeTC-F as a forward primer (SEQ ID No. 8), xho1-BmeTC-R (SEQ ID No. 9) as a backward primer. Empty plasmid PXP218 (purchased from Addgene) stored in e.coli was extracted.

The amplified fragment BmeTC and the extracted plasmid PXP218 were digested with restriction enzymes SpeI and XhoI, prepared according to the digestion system in example 1, and the prepared digestion system was placed in a 37℃water bath for digestion for 1 hour, and the digested plasmid and fragment were purified and recovered for ligation reaction. The prepared connection system is placed at 22 ℃ for connection for 1h. The constructed plasmid (PXP-BmeTC) was transformed into E.coli competent cells Trans1-T1 according to the method of example 1, and after single colony had developed, the plasmid was extracted for enzyme digestion verification, and the construction was successful.

Example 3 construction method of Saccharomyces cerevisiae recombinant 1

A site-directed mutagenesis of the patchoulene cyclase encoding gene DeltaSHC and the tetraphenyl-. Beta. -curcumene synthase encoding gene BmeTC was introduced into Saccharomyces cerevisiae (Saccharomyces cerevisiaeW-303-1 a) to obtain recombinant bacterium 1. The nucleotide sequence of the site-directed mutagenesis patchoulene cyclase coding gene delta SHC is shown as SEQ ID No. 2; the nucleotide sequence of the tetraphenyl-beta-curcumene synthase encoding gene BmeTC is shown as SEQ ID No. 3.

Delta SHC (SEQ ID No. 2) is a site-directed mutant hopene cyclase obtained by mutating 377 th amino acid of hopene cyclase SHC (SEQ ID No. 1) from aspartic acid to cysteine, wherein the hopene cyclase (SHC) is derived from alicyclobacillus acidocaldarius (Alicyclobacillus acidocaldarius) and the tetraphenyl-beta-curcumene synthase (BmeTC) is derived from bacillus megaterium (Bacillus megaterium), is synthesized by Wohan Jin Kairui biological engineering Co-Ltd through a chemical synthesis method, is subjected to codon optimization for Saccharomyces cerevisiae, is connected to an E.coli plasmid, and is stored in E.coli.

The plasmid PXP 320-. DELTA.SHC constructed in example 1 was transformed into a strain of Saccharomyces cerevisiae by the following method:

1. inoculating the parent strain Saccharomyces cerevisiae Saccharomyces cerevisiae W-1 a into a test tube YPD, and culturing overnight at 30deg.C;

2. transferring overnight cultured Saccharomyces cerevisiae into new test tube YPD liquid culture medium at volume ratio of 1/10, and shake culturing at 30deg.C for 4 hr to achieve logarithmic phase;

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

4. the washed Saccharomyces cerevisiae was resuspended in 1mL 100mM LiAc in water and allowed to stand for 5min;

centrifuging at 5.5000rpm for 3min, removing LiAc aqueous solution, and retaining bottom saccharomycetes;

6. preparing a conversion system in a centrifuge tube containing saccharomycetes, and specifically adding the following reagents in sequence:

( The transformed plasmid is PXP 320-delta SHC, and is more than 300ng; wherein salmon sperm DNA is boiled in boiling water for 5min to be melted, and then rapidly transferred to ice bath for yeast transformation; )

7. Blowing and sucking the prepared conversion system by a pipettor, or vibrating the conversion system on a vortex oscillator for 1min to fully and uniformly mix the system, and putting the system into a water bath kettle at 42 ℃ for heat shock for 30min;

8. centrifuging the heat-shocked yeast, removing supernatant with a pipettor, adding 1mLYPD medium, and resuscitating in a shaker at 30deg.C for 2 hr;

centrifuging at 9.5000rpm for 3min, removing YPD liquid medium, and washing with sterile water for 2 times;

10. yeast cells were resuspended by adding 100. Mu.L of sterile water and plated onto SC selective solid medium lacking histidine His and incubated in an incubator at 30℃for 2d.

11. After single colonies were grown, colony PCR was performed.

The invention adopts a freeze thawing method to roughly extract saccharomyces cerevisiae genome, firstly, single colony is picked into 10 mu L of NaOH solution with the concentration of 10mM, boiled in boiling water for 10min, then placed into a refrigerator with the temperature of minus 20 ℃ to be cooled for 10min, repeatedly freeze thawing for three times, namely, saccharomyces cerevisiae cells are broken, the saccharomyces cerevisiae cells can be directly used as templates, colony verification is carried out, and the strain with correct colony verification is the saccharomyces cerevisiae containing plasmid PXP-delta SHC.

Colony PCR verifies the correct strain for the next transformation.

The plasmid PXP-BmeTC constructed in example 2 was transformed into a Saccharomyces cerevisiae strain containing plasmid PXP-DeltaSHC, and after transformation, the strain was spread on SC selective solid medium lacking uracil Ura and histidine His for selection, colony PCR was performed after single colony had developed, and correct bacteria were confirmed to be recombinant bacteria 1.

Example 4 preparation of plasmid pRS304-tHMG1

According to Table 2, fragment P was amplified by PCR using Saccharomyces cerevisiae genome (Saccharomyces cerevisiaeW-1 a) as a template, apaI-Tdh3P-F (SEQ ID No. 10) as a forward primer, tdh3P-R-tHMG1 (SEQ ID No. 11) as a backward primer _Tdh3 ；

PCR amplified fragment tHMG1 (SEQ ID No. 4) using Saccharomyces cerevisiae genome (Saccharomyces cerevisiae W303-1 a) as a template, tdh3p-tHMG1-F (SEQ ID No. 12) as a forward primer, tHMG1-R-Cyc1t (SEQ ID No. 13) as a backward primer (the sequence encoding the N-terminal amino acid of HMG1 was truncated, and only the sequence encoding 503 amino acids of the C-terminal end of HMG1 protein was retained);

PCR amplification of fragment T using Saccharomyces cerevisiae genome (Saccharomyces cerevisiae W303.303-1 a) as template, tHMG1-Cyc1T-F (SEQ ID No. 14) as forward primer, cyc1T-R-Pts1 (SEQ ID No. 15) as backward primer _Cyc1 . Fragment P _Tdh3 、tHMG1、T _Cyc1 Fusion to expression frame P by fusion PCR _Tdh3 -tHMG1-T _Cyc1 。

The PCR enzyme used in the invention is Nanjinouzan biotechnology Co., ltd

Max Super-Fidelity polymerase. The 50. Mu.L PCR amplification system was as follows: DNA template, 1. Mu.L; 2. Mu.L each of the front primer (10. Mu.M) and the rear primer (10. Mu.M); dNTP (10 mM), 1. Mu.L; 2X Phanta Max Buffer, 25. Mu.L; />

Max Super-Fidelity polymerase, 1. Mu.L; finally, 50. Mu.L of the mixture was supplemented with double distilled water. An amplification program was set on the PCR instrument. The amplification conditions were 95℃for 4min of pre-denaturation (1 cycle); denaturation at 95℃for 15sec, annealing at 60℃for 15sec, extension at 72℃for 1min (34 cycles); extension at 72℃for 5min (1 cycle).

The fusion PCR system used in the invention is as follows: the total amount of DNA fragments is 800ng, and the molar ratio is 1:1; dNTP (10 mM), 1. Mu.L; 2X Phanta Max Buffer, 25. Mu.L;

max Super-Fidelity polymerase, 1. Mu.L; finally, 50. Mu.L of the mixture was supplemented with double distilled water. An amplification program was set on the PCR instrument. The amplification conditions were 95℃for 4min of pre-denaturation (1 cycle); denaturation at 95℃for 15sec, annealing at 60℃for 30sec, extension at 72℃for 1min (11 cycles), extension at 72℃for 5min (1 cycle).

Expression of fusionFrame P _Tdh3 -tHMG1-T _Cyc1 And empty vector pRS304 (purchased from Biovector plasmid vector cell Gene collection NTCC) were ligated by cleavage according to the method of example 1 to construct expression vector pRS304-tHMG1, the restriction enzymes selected here being ApaI and PstI. The constructed plasmid pRS304-tHMG1 was transformed into E.coli as in example 1, and after single colonies were grown, the plasmid was extracted for enzyme digestion to verify that the construction was correct.

TABLE 2 primer sequences

Example 5 construction method of Saccharomyces cerevisiae recombinant 2

And introducing truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 into the recombinant bacterium 1 to obtain recombinant bacterium 2, wherein the nucleotide sequence of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene tHMG1 is shown as SEQ ID No. 4.

The plasmid pRS304-tHMG1 constructed in example 4 was transformed into recombinant bacterium 1 as follows:

1. inoculating recombinant bacterium 1 into a test tube YPD, and culturing overnight at 30 ℃ in a shaking table;

2. transferring the recombinant bacteria 1 cultured overnight into a new test tube YPD liquid culture medium at a volume ratio of 1/10, and shake culturing at 30deg.C for 4 hr to reach logarithmic phase;

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

4. the washed recombinant bacterium 1 was resuspended in 1mL of 100mM LiAc aqueous solution and allowed to stand for 5min;

centrifuging at 5.5000rpm for 3min, removing LiAc aqueous solution, and retaining bottom saccharomycetes;

6. preparing a conversion system in a centrifuge tube containing saccharomycetes, and specifically adding the following reagents in sequence:

( The transformed plasmid was pRS304-tHMG1, greater than 300ng; wherein salmon sperm DNA is boiled in boiling water for 5min to be melted, and then rapidly transferred to ice bath for yeast transformation; )

7. Blowing and sucking the prepared conversion system by a pipettor, or vibrating the conversion system on a vortex oscillator for 1min to fully and uniformly mix the system, and putting the system into a water bath kettle at 42 ℃ for heat shock for 30min;

8. centrifuging the heat-shocked yeast, removing supernatant with a pipettor, adding 1mLYPD medium, and resuscitating in a shaker at 30deg.C for 2 hr;

centrifuging at 9.5000rpm for 3min, removing YPD liquid medium, and washing with sterile water for 2 times;

10. yeast cells were resuspended by adding 100. Mu.L of sterile water and plated onto SC selective solid medium lacking tryptophan and incubated in an incubator at 30℃for 2d.

11. After single colonies were grown, colony PCR was performed.

The invention adopts a freeze thawing method to roughly extract saccharomyces cerevisiae genome, firstly, single colony is picked into 10 mu L of NaOH solution with the concentration of 10mM, boiled in boiling water for 10min, then placed into a refrigerator with the temperature of minus 20 ℃ to be frozen for 10min, repeatedly freeze thawing for three times, namely, saccharomyces cerevisiae cells are broken, the saccharomyces cerevisiae cells can be directly used as templates, colony verification is carried out, and the correct bacterial strain is recombinant bacteria 2.

Example 6 preparation of plasmid pRS405-Erg20

According to Table 3, fragment P was amplified by PCR using Saccharomyces cerevisiae genome (Saccharomyces cerevisiae W-1 a) as a template, pst1-Pgk P-F (SEQ ID No. 16) as a forward primer, pgk P-R-Erg20 (SEQ ID No. 17) as a backward primer _Pgk1 The method comprises the steps of carrying out a first treatment on the surface of the Using Saccharomyces cerevisiae genome (Saccharomyces cerevisiae W303-1 a) as a template, pgk p-Erg20-F (SEQ ID No. 18) as a forward primer, erg20-R-Cyc1t (SEQ ID No. 19) as a backward primer, and PCR amplifying to obtain a fragment Erg20 (SEQ ID No. 5); PCR amplification of fragment T using Saccharomyces cerevisiae genome (Saccharomyces cerevisiae W303.303-1 a) as template, erg20-Cyc1T-F (SEQ ID No. 20) as forward primer, cyc1T-R-BamHI (SEQ ID No. 21) as backward primer _Cyc1 . Fragment P _Pgk1 、Erg20、T _Cyc1 Fusion PCR method to form expression frame P _Pgk1 -Erg20-T _Cyc1 。

The PCR enzyme used in the invention is Nanjinouzan biotechnology Co., ltd

Max Super-Fidelity polymerase. The 50. Mu.L PCR amplification system was as follows: DNA template, 1. Mu.L; 2. Mu.L each of the front primer (10. Mu.M) and the rear primer (10. Mu.M); dNTP (10 mM), 1. Mu.L; 2X Phanta Max Buffer, 25. Mu.L; />

Max Super-Fidelity polymerase, 1. Mu.L; finally, 50. Mu.L of the mixture was supplemented with double distilled water. An amplification program was set on the PCR instrument. The amplification conditions were 95℃for 4min of pre-denaturation (1 cycle); denaturation at 95℃for 15sec, annealing at 60℃for 15sec, extension at 72℃for 1min (34 cycles); extension at 72℃for 5min (1 cycle).

The fusion PCR system used in the invention is as follows: the total amount of DNA fragments is 800ng, and the molar ratio is 1:1; dNTP (10 mM), 1. Mu.L; 2X Phanta Max Buffer, 25. Mu.L;

max Super-Fidelity polymerase, 1. Mu.L; finally, 50. Mu.L of the mixture was supplemented with double distilled water. An amplification program was set on the PCR instrument. The amplification conditions were 95℃for 4min of pre-denaturation (1 cycle); denaturation at 95℃for 15sec, annealing at 60℃for 30sec, extension at 72℃for 1min (11 cycles), extension at 72℃for 5min (1 cycle).

Fusion of the resulting expression cassette P _Pgk1 -Erg20-T _Cyc1 And empty vector pRS405 (purchased from biological wind) were ligated by cleavage in the same manner as in example 1 to construct expression vector pRS405-Erg20. The restriction enzymes used were Pst1 and BamHI, which had asterisk activity, and were digested for 30min. The constructed plasmid pRS405-Erg20 was transformed into E.coli as in example 1, and after single colonies were grown, the plasmid was extracted and subjected to enzyme digestion to verify that the construction was correct.

TABLE 3 primer sequences

Example 7 construction method of Saccharomyces cerevisiae recombinant 3

And introducing a farnesyl pyrophosphate synthase encoding gene Erg20 into the recombinant bacterium 1 to obtain a recombinant bacterium 3, wherein the nucleotide sequence of the farnesyl pyrophosphate synthase encoding gene Erg20 is shown as SEQ ID No. 5.

The plasmid pRS405-Erg20 constructed in example 6 was transformed into recombinant bacterium 1 as follows:

1. inoculating recombinant bacterium 1 into a test tube YPD, and culturing overnight at 30 ℃ in a shaking table;

2. transferring the recombinant bacteria 1 cultured overnight into a new test tube YPD liquid culture medium at a volume ratio of 1/10, and shake culturing at 30deg.C for 4 hr to reach logarithmic phase;

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

4. the washed recombinant bacterium 1 was resuspended in 1mL of 100mM LiAc aqueous solution and allowed to stand for 5min;

centrifuging at 5.5000rpm for 3min, removing LiAc aqueous solution, and retaining bottom saccharomycetes;

6. preparing a conversion system in a centrifuge tube containing saccharomycetes, and specifically adding the following reagents in sequence:

( The transformed plasmid is pRS405-Erg20, which is more than 300ng; wherein salmon sperm DNA is boiled in boiling water for 5min to be melted, and then rapidly transferred to ice bath for yeast transformation; )

7. Blowing and sucking the prepared conversion system by a pipettor, or vibrating the conversion system on a vortex oscillator for 1min to fully and uniformly mix the system, and putting the system into a water bath kettle at 42 ℃ for heat shock for 30min;

8. centrifuging the heat-shocked yeast, removing supernatant with a pipettor, adding 1mLYPD medium, and resuscitating in a shaker at 30deg.C for 2 hr;

centrifuging at 9.5000rpm for 3min, removing YPD liquid medium, and washing with sterile water for 2 times;

10. yeast cells were resuspended by adding 100. Mu.L of sterile water and plated onto leucine-deficient SC selective solid medium and incubated for 2d in a 30℃incubator.

11. After single colonies were grown, colony PCR was performed.

The invention adopts a freeze thawing method to roughly extract saccharomyces cerevisiae genome, firstly, single colony is picked into 10 mu L of NaOH solution with the concentration of 10mM, boiled in boiling water for 10min, then placed into a refrigerator with the temperature of minus 20 ℃ for freezing for 10min, repeatedly freeze thawing for three times, namely, saccharomyces cerevisiae cells are crushed, the saccharomyces cerevisiae cells can be directly used as templates, colony verification is carried out, and the correct bacterial strain is recombinant bacteria 3.

Example 8 production of ambroxol by fermentation with recombinant bacteria

(1) Recombinant bacterium culture and product extraction

First, recombinant bacterium 1, recombinant bacterium 2, recombinant bacterium 3 obtained in examples 3, 5 and 7, together with ATCC208352, were activated on an SC selective solid medium; then inoculating each recombinant strain into test tube YPD liquid culture medium, and culturing overnight until OD ₆₀₀ Up to about 4.0, inoculated into shake flasks containing 30mLYPD medium to give an initial OD ₆₀₀ After fermentation at 220rpm for 5d at 0.05, 30℃the ambroxol yield was measured.

The extraction method of ambroxol comprises the following steps: for extracellular ambroxol extraction, 5mL of the fermentation broth was mixed with 500. Mu.L of n-hexane, shaken with a vortex shaker for 30min, and then centrifuged at 12000g for 5min to collect the n-hexane layer. The intracellular ambroxol is extracted as follows: after separating the supernatant, the cells were mixed with 0.5mL of n-hexane and 0.25mL of glass beads having a diameter of 0.5mm, vortexed for 30min to break the cells, and then 12000g was centrifuged for 5min to collect an n-hexane layer.

(2) GC-MS detection of ambroxol

Ambroxol detection consisted of Agilent 7890A gas chromatograph and 5975C insert XLEI/CIMSD mass spectrometer.

The GC-MS detection method of ambroxol comprises the following steps: the chromatographic column HP-5ms, helium flow rate 1mL/min, sample inlet temperature 290 ℃, column initial column temperature 220 ℃,2 ℃/min rise to 290 ℃, scanning range 40-500m/z.

(3) Detection result

A. Saccharomyces cerevisiae W303-1aATCC208352 did not detect ambroxol;

B. recombinant bacterium 1: extracting a fermentation product of recombinant bacterium 1, and detecting that the ambroxol content is 5.1mg/L;

C. recombinant bacterium 2: extracting a recombinant bacterium 2 fermentation product, and detecting that the content of ambroxol is 8.3mg/L;

D. recombinant bacterium 3: and (3) extracting a recombinant bacterium 3 fermentation product, and detecting that the ambroxol content is 15.4mg/L.

TABLE 4 ability of recombinant strains to produce ambroxol

Bacterial name	Ambergris alcohol (mg/L)
		Saccharomyces cerevisiae W303-1aATCC208352	0
Recombinant bacterium 1	5.1
		Recombinant bacterium 2	8.3
Recombinant bacterium 3	15.4

SC selective solid medium formulation: 6.7g/L Yeast Nitrogen Base (YNB), 20g/L glucose, 20g/L agar powder; 2g/L of the corresponding default amino acid mixture.

The corresponding default amino acid mixture components include:

YPD medium: 20g/L peptone, 10g/L yeast extract powder, 20g/L glucose, and 2% agar powder added to the solid medium.

Sequence listing

<110> university of Tianjin

<120> recombinant Saccharomyces cerevisiae for producing ambergris and construction method thereof

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attcagccgc cttggtttta cgcgctcatc gcgctcaaga ttctcgacat gacgcagcat 840

ccggcgttca tcaagggctg ggaaggtcta gagctgtacg gcgtggagct ggattacgga 900

ggatggatgt ttcaggcttc catctcgccg gtgtgggaca cgggcctcgc cgtgctcgcg 960

ctgcgcgctg cggggcttcc ggccgatcac gaccgcttgg tcaaggcggg cgagtggctg 1020

ttggaccggc agatcacggt tccgggcgac tgggcggtga agcgcccgaa cctcaagccg 1080

ggcgggttcg cgttccagtt cgacaacgtg tactacccgg acgtggacga cacggccgtc 1140

gtggtgtggg cgctcaacac cctgcgcttg ccggacgagc gccgcaggcg ggacgccatg 1200

acgaagggat tccgctggat tgtcggcatg cagagctcga acggcggttg gggcgcctac 1260

gacgtcgaca acacgagcga tctcccgaac cacatcccgt tctgcgactt cggcgaagtg 1320

accgatccgc cgtcagagga cgtcaccgcc cacgtgctcg agtgtttcgg cagcttcggg 1380

tacgatgacg cctggaaggt catccggcgc gcggtggaat atctcaagcg ggagcagaag 1440

ccggacggca gctggttcgg tcgttggggc gtcaattacc tctacggcac gggcgcggtg 1500

gtgtcggcgc tgaaggcggt cgggatcgac acgcgcgagc cgtacattca aaaggcgctc 1560

gactgggtcg agcagcatca gaacccggac ggcggctggg gcgaggactg ccgctcgtac 1620

gaggatccgg cgtacgcggg taagggcgcg agcaccccgt cgcagacggc ctgggcgctg 1680

atggcgctca tcgcgggcgg cagggcggag tccgaggccg cgcgccgcgg cgtgcaatac 1740

ctcgtggaga cgcagcgccc ggacggcggc tgggatgagc cgtactacac cggcacgggc 1800

ttcccagggg atttctacct cggctacacc atgtaccgcc acgtgtttcc gacgctcgcg 1860

ctcggccgct acaagcaagc catcgagcgc aggtga 1896

<210> 2

<211> 1896

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggcagaac aattagttga agctccagca tacgctagaa ctttggatag agctgttgaa 60

tatttgttgt cttgtcaaaa ggatgaaggt tactggtggg gtccattgtt gtcaaacgtt 120

acaatggaag ctgaatacgt tttgttgtgt catatcttgg atagagttga tagagataga 180

atggaaaaga ttagaagata tttgttgcat gaacaaagag aagatggtac ttgggcttta 240

tacccaggtg gtccaccaga tttggatact acaatcgaag catacgttgc tttgaagtac 300

atcggcatgt ctagagatga agaaccaatg caaaaagctt tgagattcat tcaatcacaa 360

ggtggtatcg aatcttcaag agtttttaca agaatgtggt tggctttagt tggtgaatat 420

ccatgggaaa aagttccaat ggttccacca gaaatcatgt tcttgggtaa aagaatgcca 480

ttgaacatct atgaattcgg ttcttgggca agagctactg ttgttgcatt gtctattgtt 540

atgtcaagac aaccagtttt tccattacca gaaagagcta gagttccaga attgtatgaa 600

acagatgttc caccaagaag aagaggtgca aaaggtggtg gtggttggat ttttgatgca 660

ttagatagag ctttgcatgg ttaccaaaag ttgtctgttc atccttttag aagagctgca 720

gaaattagag cattggattg gttgttagaa agacaagctg gtgacggttc atggggtggt 780

attcaaccac catggttcta cgcattgatc gctttgaaga tcttggatat gactcaacat 840

ccagctttta ttaaaggttg ggaaggtttg gaattatacg gtgttgaatt agattatggt 900

ggttggatgt ttcaagcttc tatttcacca gtttgggata ctggtttggc agttttggct 960

ttaagagctg caggtttacc agcagatcat gatagattgg ttaaagctgg tgaatggttg 1020

ttagatagac aaattacagt tccaggtgac tgggcagtta aaagaccaaa tttgaaacca 1080

ggtggtttcg ctttccaatt cgataacgtt tactacccag atgttgattg tactgcagtt 1140

gttgtttggg ctttgaatac attgagatta ccagatgaaa gaagaagaag agatgctatg 1200

actaaaggtt ttagatggat tgttggtatg caatcttcaa atggtggttg gggtgcttat 1260

gatgttgata acacatctga tttgccaaac catatcccat tctgtgattt cggtgaagtt 1320

actgatccac catctgaaga tgttacagct catgttttag aatgtttcgg ttcattcggt 1380

tatgatgatg catggaaagt tattagaaga gctgttgaat acttgaagag agaacaaaaa 1440

ccagatggtt cttggtttgg tagatggggt gttaattatt tgtacggtac tggtgctgtt 1500

gtttcagcat tgaaagctgt tggtatcgat acaagagaac catacatcca aaaagcattg 1560

gattgggttg aacaacatca aaatccagat ggtggttggg gtgaagattg tagatcttac 1620

gaagatccag cttatgctgg taaaggtgct tctactccat cacaaacagc atgggcttta 1680

atggcattga ttgctggtgg tagagcagaa tcagaagctg caagaagagg tgttcaatat 1740

ttggttgaaa cacaaagacc agatggtggt tgggatgaac catattacac tggtacaggt 1800

tttccaggtg acttctattt gggttacact atgtacagac atgtttttcc aacattggca 1860

ttaggtagat acaaacaagc tattgaaaga agataa 1896

<210> 3

<211> 1878

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atgatcatat tgctaaagga agttcagcta gagattcagc gaagaatcgc ctatctgcgc 60

ccaacacaaa aaaatgacgg gtcatttcgc tactgttttg aaacaggcgt tatgcctgat 120

gcgtttttaa ttatgcttct tcgcaccttt gatttagata aagaagtgtt gattaaacaa 180

ttaaccgaac ggatcgtttc ccttcaaaat gaagatggtc tttggacgtt gtttgatgat 240

gaagaacata acttatccgc cactattcaa gcttatacag ctcttttata ttcagggtat 300

taccaaaaaa acgaccggat tttgcgaaaa gcagaaagat atattataga ttcgggaggc 360

atttcgcgcg ctcattttct tacaagatgg atgctttctg ttaacggttt atacgagtgg 420

ccaaagctat tttacctccc gctttctctt ttgctcgtgc ctacctatgt accgcttaac 480

ttttatgaat taagcaccta tgccagaatt cacttcgttc cgatgatggt agcaggaaac 540

aaaaaatttt cacttacttc taggcataca ccttctcttt ctcatttaga tgtaagagaa 600

cagaaacagg aatcggagga aactactcaa gaatcacgcg cttctatttt tttagtcgac 660

catttaaaac agctggcttc tttaccttct tacatccaca agcttggtta tcaagcagcg 720

gagcgttaca tgctagaaag aattgaaaaa gacggaacac tctacagcta cgccacctct 780

acttttttta tgatttacgg tcttttggct cttggctata aaaaagattc atttgtgatc 840

caaaaagcaa ttgacggtat ttgttcacta cttagtacat gcagcggcca cgtgcacgta 900

gaaaactcca cgtcaaccgt ttgggatacc gcgctgctat cttacgctct acaggaagca 960

ggtgtaccgc agcaagatcc tatgattaaa ggcacaactc gctacttaaa gaaaagacag 1020

catacaaagc ttggagattg gcagtttcat aacccaaata cagcacctgg aggctggggg 1080

ttttccgata ttaatacgaa taaccctgac ttagacgata cgtctgctgc tatcagagct 1140

ctttctagaa gagcacaaac cgatacagat tatttggagt cttggcaaag aggcattaac 1200

tggctgctgt ccatgcaaaa caaagacggg ggttttgctg catttgaaaa aaatactgac 1260

tctattttat ttacttatct cccgcttgaa aatgcaaaag atgcagcgac ggatccggct 1320

actgccgatt taaccggtcg agtgcttgag tgcctcggaa actttgctgg tatgaataaa 1380

tcccaccctt cgattaaagc tgcagtaaaa tggctgtttg atcatcagtt ggataacggg 1440

agctggtacg gccggtgggg agtttgctac atttacggaa cgtgggccgc tattacagga 1500

cttcgtgctg taggggtttc tgcttctgat ccgcgtatca tcaaagctat caactggctc 1560

aaaagcattc aacaagaaga cggtggattc ggagaatcat gctatagcgc ttctttaaaa 1620

aaatatgtgc cactatcgtt tagcacccct tctcaaacgg cttgggctct cgatgcttta 1680

atgacaatat gtccattaaa agatcgatcc gttgaaaaag gaattaaatt tttactgaat 1740

ccaaatctta cagagcagca aactcattac cccacgggaa ttggtcttcc tggacaattt 1800

tatattcagt accacagcta caatgatatt tttcctcttc ttgcacttgc tcactacgca 1860

aaaaaacatt cttcgtaa 1878

<210> 4

<211> 1512

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

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> 5

<211> 1059

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 5

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 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> 6

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ggactagtca tggcagaaca attagttgaa g 31

<210> 7

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ccctcgaggt tatcttcttt caatagctt 29

<210> 8

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ggactagtca tgatcatatt gctaaagga 29

<210> 9

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ccctcgaggt tacgaagaat gtttttttgc 30

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

aagggcccat actagcgttg aatgttagcg 30

<210> 11

<211> 50

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ctgttttatt ggttaaaact ggcattttgt ttgtttatgt gtgtttattc 50

<210> 12

<211> 50

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gaataaacac acataaacaa acaaaatgcc agttttaacc aataaaacag 50

<210> 13

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cgacaaagga aaaggggcct gtttaggatt taatgcaggt gacgg 45

<210> 14

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ccgtcacctg cattaaatcc taaacaggcc ccttttcctt tgtcg 45

<210> 15

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tgcctgcaga agcagacgct actaaggaaa ac 32

<210> 16

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

taactgcagt attttagatt cctgacttca actc 34

<210> 17

<211> 49

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ctaatttctt tttctgaagc cattgtttta tatttgttgt aaaaagtag 49

<210> 18

<211> 49

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ctacttttta caacaaatat aaaacaatgg cttcagaaaa agaaattag 49

<210> 19

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

cgacaaagga aaaggggcct gtctatttgc ttctcttgta aactttg 47

<210> 20

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

caaagtttac aagagaagca aatagacagg ccccttttcc tttgtcg 47

<210> 21

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

cgggatccaa gcagacgcta ctaaggaaaa c 31

Claims (3)

1. The construction method of the recombinant saccharomyces cerevisiae for producing ambergris alcohol is characterized by comprising the following steps: introducing a farnesyl pyrophosphate synthase encoding gene Erg20 into the recombinant bacterium 1 to obtain a recombinant bacterium 3;

the nucleotide sequence of the farnesyl pyrophosphate synthase encoding gene Erg20 is shown in SEQ ID No. 5;

the recombinant bacterium 1 is constructed by the following method: to Saccharomyces cerevisiaeSaccharomyces cerevisiaeW303-1aIntroducing a site-directed mutagenesis coding gene delta SHC of the patchoulene cyclase and a coding gene BmeTC of the tetraphenyl-beta-curcumene synthase to obtain recombinant bacteria 1;

the nucleotide sequence of the site-directed mutagenesis patchoulene cyclase coding gene delta SHC is shown as SEQ ID No. 2;

the nucleotide sequence of the tetraphenyl-beta-curcumene synthase encoding gene BmeTC is shown as SEQ ID No. 3.

2. A recombinant saccharomyces cerevisiae constructed by the method of claim 1 that produces ambergris.

3. Use of the recombinant saccharomyces cerevisiae of claim 2 for the fermentative production of ambergris.

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