CN103820344B - Produce the Saccharomyces cerevisiae gene engineering bacteria of miltirone diene and construction method thereof and application - Google Patents
Produce the Saccharomyces cerevisiae gene engineering bacteria of miltirone diene and construction method thereof and application Download PDFInfo
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- CN103820344B CN103820344B CN201110453388.7A CN201110453388A CN103820344B CN 103820344 B CN103820344 B CN 103820344B CN 201110453388 A CN201110453388 A CN 201110453388A CN 103820344 B CN103820344 B CN 103820344B
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- 150000001299 aldehydes Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses and produce the Saccharomyces cerevisiae gene engineering bacteria of miltirone diene and construction method thereof and application.Genetic engineering bacterium provided by the present invention is to build according to the method that comprises the following steps: introduce Radix Salviae Miltiorrhizae Ke Baji pyrophosphate synthase gene (SmCPS) of external source in the saccharomyces cerevisiae that sets out and Radix Salviae Miltiorrhizae miltirone diene synthase gene (SmKSL) obtains the recombinant Saccharomyces cerevisiae of production miltirone diene, be denoted as recombinant Saccharomyces cerevisiae ZD T 000 and ZD T 010.On this basis, improve the activity of one or more albumen in 3 hydroxyl 3 methyl glutaryl coenzyme A reductases, terpenoid regulatory factor albumen UPC2, cattle cattle base pyrophosphate synthase, farnesyl pyrophosphate synthase, structure obtains 24 strains (ZD T 001～ZD T 008, ZD T 011～ZD T 018, ZD T 021～ZD T 028) the recombinant Saccharomyces cerevisiae bacterial strain of high yield miltirone diene.Saccharomyces cerevisiae ZD Tans 001 constructed by the present invention, under the conditions of supporting oxygen well, can produce the miltirone diene of 487.91mg/L when fermenting 6 days.
Description
Technical field
The present invention relates to produce the Saccharomyces cerevisiae gene engineering bacteria of miltirone diene and construction method thereof and application.
Background technology
Miltirone diene (Miltiradiene) is the fat-soluble Diterpene extracted in important Salvia miltiorrhiza Bge, for
Precursor substance (Wei Gao et al., 2009, Organic Letters, the 11:5170-that tanshinone compound is common
5173).This compounds (such as cryptotanshinone, tanshinone IIA, tanshinone ⅡB, dihydrotanshinone), in antiinflammatory, cardiovascular and cerebrovascular vessel, resists
The aspects such as tumor have extraordinary application prospect, in current relative product, and existing FUFANG DANSHEN PIAN, FUFANG DANSHEN DIWAN
The trump launch of several hundred million RMB etc. in sales volume.Radix Salviae Miltiorrhizae for injection ketone IIA sodium sulfonate be widely used in treatment coronary heart disease,
The diseases such as myocardial infarction, from the point of view of the raw materials market situation of current tanshinone IIA, 4500 yuan/kg of content 20%；Content 50%
8000 yuan/kg；Content is the up to 3.6 ten thousand yuan/kg of 98%.The visible whole industry relevant to tanshinone compound has huge
The big market demand, economic benefit is the most considerable.The main source of tanshinone compound is by Chinese crude drug Radix Salviae Miltiorrhizae at present
Middle extracting directly, but along with reclaiming wasteland, the growing environment of deserted mountain deforestation Radix Salviae Miltiorrhizae wild resource is seriously damaged, Radix Salviae Miltiorrhizae resource
Increasingly reduce；Also the factors such as variety deterioration, substantial amounts of soil and human cost are run into during artificial growth.Tanshinone chemical combination
Produce amount far from the demand of satisfied society, has had a strong impact in the middle of clinical practice and the tanshinone pharmaceutical raw material of Radix Salviae Miltiorrhizae
The development and application of body, it would be highly desirable to need to provide new resources approach.
Currently with the principle of synthetic biology, design and transform microbial strains and recognize to produce the natural product Yi Bei world
For being a kind of most potential method, reach 1000mg/L as produced the precursor Ramulus et folium taxi cuspidatae diene of paclitaxel in escherichia coli
(Parayil Kumaran Ajikumar et al.,2010,Science,330:70-74)；Bilobalide-like
(Ginkgolides) precursor sinistral corean pine diene (Levopimaradiene), reaches in improved colibacillus engineering
The yield (Effendi Leonard et al., 2010, PNAS, 107 (31): 13,654 13659) of 700mg/L；In yeast work
The precursor artelinic acid (Artemisinic acid) producing arteannuin (Artemisinin) in journey bacterium is up to 100mg/L
(Dae-Kyun Ro et al.,2006,Nature,440:940-943)；The most domestic divide at the medicine such as arteannuin and paclitaxel
The biosynthesis aspect of son has correlational study.
Miltirone diene is the product of terpenoid route of synthesis in plant Radix Salviae Miltiorrhizae body, by Radix Salviae Miltiorrhizae cell Mitochondria, cell
Mevalonic acid (MVA Pathway) metabolic pathway that solute and endoplasmic reticulum exist is sweet with acetone acid/phosphoric acid present in plastid
Oil aldehyde (MEP Pathway) metabolic pathway synthesizes jointly.Wherein precursor substance cattle base cattle base pyrophosphoric acid (GGPP) is
Isoprenoid (IPP) and dimethylallylpyrophosphate (DMAPP) are through cattle base cattle base pyrophosphate synthase
(GGPS) catalysis obtains, and GGPP can be by Radix Salviae Miltiorrhizae Ke Baji pyrophosphate synthase (SmCPS) and miltirone diene synthase (SmKSL)
Co-catalysis is miltirone diene, Fig. 1.Microorganism Escherichia coli exists acetone acid/glyceraldehyde phosphate approach, above
In research, Gao Wei et al. research strategy have employed the escherichia expression system of document report, with commercial dual-expression vector
PACYCDuet co expression gene SmCPS and SmKSL, but effect is undesirable, can only obtain the miltirone diene of milligram level
(Wei Gao et al.,2009,Organic Letters,11:5170-5173).But, as normal in traditional zymotic technique
With bacterial strain: saccharomyces cerevisiae, at its internal mevalonate pathway that there is production ter penoids, wherein produce ter penoids ergosterol
(Ergosterol) can reach Biomass 4.6% (Arnezeder, C.et al., 1990, Biotechnol lett., 12:
277-282)；Diterpene Herba Pelargonii Graveolentis acyl Herba Pelargonii Graveolentis acyl alcohol (GGOH) also can reach 283mg/L (Tokuhiro, K.et al., 2009, Appl
Environ Microbiol., 75:5536-5543), thus utilize the mevalonate pathway of the optimization of yeast to produce miltirone
Diene has the biggest potentiality.
Summary of the invention
It is an object of the present invention to provide high yield miltirone diene saccharomyces cerevisiae engineered yeast strain ZD-Tans-001.
Saccharomyces cerevisiae engineered yeast strain ZD-Tans-001 provided by the present invention, its preserving number is CGMCC No.5296.
It is a further object to provide the construction method of a kind of genetic engineering bacterium.
The construction method of genetic engineering bacterium provided by the present invention, comprises the following steps:
In the saccharomyces cerevisiae that sets out, introduce Ke Baji pyrophosphate synthase and the miltirone diene synthase of external source, recombinated
Saccharomyces cerevisiae, is denoted as recombinant Saccharomyces cerevisiae I.
Described method is further comprising the steps of:
3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor is improved in described recombinant Saccharomyces cerevisiae I
The activity of albumen UPC2, obtains recombinant Saccharomyces cerevisiae, is denoted as recombinant Saccharomyces cerevisiae II.
Cattle cattle base pyrophosphate synthase and farnesyl pyrophosphate synthase is improved in described recombinant Saccharomyces cerevisiae I
Activity, obtains recombinant Saccharomyces cerevisiae, is denoted as recombinant Saccharomyces cerevisiae III.
Described method is further comprising the steps of:
In described recombinant Saccharomyces cerevisiae III improve 3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulation and control because of
The activity of sub-albumen UPC2, obtains recombinant Saccharomyces cerevisiae, is denoted as recombinant Saccharomyces cerevisiae IV.
Described method is further comprising the steps of:
Functional gene in clone and artificial reconstructed terpenoid route of synthesis: saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-coenzyme A
Reductase gene 1 (tHMG1), saccharomyces cerevisiae terpenoid regulatory factor albumen UPC2 gene (UPC2.1), saccharomyces cerevisiae cattle
Cattle base pyrophosphate synthase gene (BTS1) and saccharomyces cerevisiae farnesyl phosphate synthase gene (ERG20)；Full synthetic addicted to
The cattle cattle base pyrophosphate synthase gene (SaGGPS of acid heat sulfolobus solfataricussyn).Clone's auxotroph selection markers:
Leucine auxotrophy screening-gene element LEU2, histidine auxotrophy screening-gene element HIS3, uracil auxotrophy
Screening-gene element URA3；Antibiotic type riddled basins element (G418) resistance gene element KanMX and hygromycin resistance base
Because of function element such as element hphNT1.
Described method is further comprising the steps of:
Clone's glycerol 3-phosphate kinase promoter (PGK1) of controlling element saccharomyces cerevisiae, the translation of saccharomyces cerevisiae extend
The factor 1 promoter (TEF1), the ethanol dehydrogenase 1 promoter (ADH1) of saccharomyces cerevisiae, the galactokinase 1 of saccharomyces cerevisiae start
Son (GAL1), S. cerevisiae cytochrome C terminator (CYC1t) and the ethanol dehydrogenase 1 terminator (ADH1t) of saccharomyces cerevisiae.
The Ke Baji pyrophosphate synthase of described introducing external source and miltirone diene synthase are to carry external source by being transformed into
Ke Baji pyrophosphate synthase and the plasmid of miltirone diene synthase gene, or on S. cerevisiae chromosomal, integrate external source Ke's bar
Base pyrophosphate synthase and miltirone diene synthase gene.
The activity of described raising 3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor albumen UPC2 is
3-hydroxy-3-methylglutaryl-coenzyme A reductase and the plasmid of terpenoid regulatory factor albumen UPC2 gene is carried by being transformed into,
Or on S. cerevisiae chromosomal, integrate 3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor albumen UPC2
Gene.
The described activity improving cattle cattle base pyrophosphate synthase and farnesyl pyrophosphate synthase is by being transformed into
Carry cattle cattle base pyrophosphate synthase and the plasmid of farnesyl phosphate synthase gene, or on S. cerevisiae chromosomal
Integrate cattle cattle base pyrophosphate synthase and farnesyl phosphate synthase gene.
Described on S. cerevisiae chromosomal integrator gene be to be incorporated into δ DNA site.
The described plasmid carrying Ke Baji pyrophosphate synthase and miltirone diene synthase gene, replication site is
2MICRON, selection markers is uracil auxotrophy selection markers URA3, and Ke Baji pyrophosphate synthase and miltirone diene close
The promoter of enzyme gene is galactokinase 1 promoter GAL1 of saccharomyces cerevisiae.
The described matter carrying 3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor albumen UPC2 gene
Grain, replication site is the one in 2MICRON and CEN6/ARSH4, and selection markers is leucine auxotrophy selection markers LEU2
With the one in axoneme (G418) resistance screening labelling KanMX, 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene
Promoter is ethanol dehydrogenase 1 promoter ADH1 of saccharomyces cerevisiae, and the promoter of terpenoid regulatory factor albumen UPC gene is wine brewing
Translation elongation factor 1 promoter TEF1 of yeast.
Described carry cattle cattle base pyrophosphate synthase and the plasmid of farnesyl phosphate synthase gene, replication site
For the one in 2MICRON and CEN6/ARSH4, selection markers is histidine auxotrophy selection markers HIS3 and hygromycin sieve
Select the one in labelling hphNT1.A kind of cattle cattle base pyrophosphate synthase gene and farnesyl phosphate synthase gene melt
Being combined, promoter is ethanol dehydrogenase 1 promoter ADH1 of saccharomyces cerevisiae, and another kind of cattle cattle base pyrophosphoric acid closes
The promoter of enzyme gene is the glycerol 3-phosphate kinase promoter PGK1 of saccharomyces cerevisiae.
Described Ke Baji pyrophosphate synthase is the Ke Baji pyrophosphate synthase of Radix Salviae Miltiorrhizae.
Described miltirone diene synthase is the miltirone diene synthase of Radix Salviae Miltiorrhizae.
Described terpenoid regulatory factor albumen UPC2 is the sudden change of saccharomyces cerevisiae sterol regulatory factor protein UPC2.
Described 3-hydroxy-3-methylglutaryl-coenzyme A reductase is the 3-hydroxy-3-methyl glutaryl coenzyme of saccharomyces cerevisiae
A reductase 1 intercepts the functional protein of 5 ' partial sequences.
A kind of cattle cattle base pyrophosphate synthase for saccharomyces cerevisiae of described cattle cattle base pyrophosphate synthase；
Another kind is the cattle cattle base pyrophosphate synthase of codon optimized sulfolobus acidocaldarius, and its encoding gene is sequence
DNA shown in sequence 1 in table.Described farnesyl pyrophosphate synthase is the farnesyl pyrophosphate synthase of saccharomyces cerevisiae.
The described saccharomyces cerevisiae that sets out is Saccharomyces Cerevisiae in S accharomyces cerevisiae BY4742.
Built, by described method, the recombinant Saccharomyces cerevisiae obtained and fall within protection scope of the present invention.
The application in producing miltirone diene of described saccharomyces cerevisiae ZD-Tans-001 or the described saccharomyces cerevisiae is also
Belong to protection scope of the present invention.
A further object of the present invention is to provide a kind of method producing miltirone diene.
The method of production miltirone diene provided by the present invention, comprises the following steps:
Ferment described saccharomyces cerevisiae ZD-Tans-001 or described saccharomyces cerevisiae, obtains miltirone diene.
The temperature of described fermentation is 25 DEG C-37 DEG C or 25 DEG C or 30 DEG C or 32 DEG C or 37 DEG C；
The pH value of the system of described fermentation is 3.0-8.0 or 3.0 or 4.0 or 5.0 or 6.0 or 7.0 or 8.0；
The time of described fermentation be 24-168 hour or 24 hours or 48 hours or 72 hours or 96 hours or 120 hours or
144 hours or 168 hours；
The percent by volume of the inoculum concentration of described fermentation is 0.01%-10% or 0.01% or 0.3% or 1% or 10%；
The culture medium of described fermentation consists of the following composition:
Yeast extract, peptone, glucose, axoneme (G418), hygromycin
Above composition concentration in described fermentation medium is respectively as follows:
Yeast extract 1-20g/L, peptone 1-40g/L, glucose 5-50g/L, G418 10-500mg/L, hygromycin 10-
600mg/L。
Saccharomyces cerevisiae ZD-Tans-001 constructed by the present invention, under the conditions of supporting oxygen well, can produce when fermenting 6 days
487.91mg/L miltirone diene.
Accompanying drawing explanation
Fig. 1 miltirone diene biosynthesis pathway
Fig. 2 carries the plasmid schematic diagram (auxotroph selection markers) of Genetic elements
Fig. 3 carries the plasmid schematic diagram (antibiotic type selection markers) of Genetic elements
Fig. 4 miltirone diene GC-MS analysis chart A: miltirone diene standard substance TIC schemes (Rt=19.922min)；B:
Sample TIC schemes (Rt=19.919min)；C: miltirone diene standard substance MS figure；D: miltirone diene sample MS figure
Fig. 5 strain fermentation result: with ZD-T-000 related strain
Fig. 6 strain fermentation result: with ZD-T-010 and proceed to auxotrophy selection markers type function plasmid related strain
Fig. 7 strain fermentation result: with ZD-T-010 and proceed to antibiotic-screening marking type function plasmid related strain
Fig. 8 ZD-Tans-001 bacterial strain high density fermentation result
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1, the clone of Genetic elements
The clone of Genetic elements is divided into following five steps:
(1) cerevisiae dna extracts
Picking bacterial plaque (Saccharomyces cerevisiae BY4742) is in YPD fluid medium (formula: 1%
Yeast Extract (yeast extract), 2%Peptone (peptone), in 2%Dextrose (glucose), 30 DEG C, 200rpm, training
Support 24h.10000g, within 5 minutes, collection thalline is in 1.5ml centrifuge tube, and water cleans twice, and thalline is resuspended in yeast lysate
(25ul yeast wall breaking enzyme, 470ul sorbitol buffer, 5u l β-ME), centrifugal after 30 DEG C of temperature bath 1h；Thalline 500ul
TENTS buffer (10mM Tris-HCl, pH 7.5；1mM EDTA,pH8.0；100mM NaAc；2%triton-100；1%
SDS) resuspended, 60 DEG C of water-bath 1h；Phenol/chloroform 2 times；Supernatant adds the EtOH of 3 times of volumes, the 3M of 1/10 times of volume
NaAc, 2h placed by-20 DEG C of refrigerators；13000g, 4 DEG C, centrifugal 10min, outwell supernatant, precipitation 70%EtOH, lotion precipitates 2 times
After dry up, distilled water dissolve ,-20 DEG C save backup.
(2) PCR amplification and clone
With pastoris genomic dna as template, with primer in list of primers 1, expand tHMG1, UPC2, δ DNA1, δ DNA1；With
Plasmid YES2.0DNA is template amplification auxotroph selection markers URA3；Lack with plasmid pRS313DNA for template amplification nutrition
Swaged selection markers HIS3；With plasmid pRS425DNA for template amplification auxotroph selection markers LEU2；With plasmid
PRS41KDNA is template amplification antibiotic type selection markers KanMX；Sieve with plasmid pRS42HDNA for template amplification antibiotic type
Select labelling hphNT1.Amplification system is: NewEngland Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM
Each dNTP) 1ul, DNA profiling 20ng, primer (10uM) each 1ul, Phusion High-Fidelity DNA
Polymerase (2.5U/ul) 0.5ul, add distilled water to cumulative volume 50ul.
List of primers 1
Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation)；(annealing temperature is shown in and draws for 98 DEG C of degeneration 10 seconds, annealing 10 seconds
Thing list 1), 72 DEG C extend all with 2 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation).Extension amplification outcome is arrived
On pEASY-Blunt cloning vehicle (purchased from Beijing Quanshijin Biotechnology Co., Ltd).Clone's system is: 1ul PCR expands
Product, 1ul pEASY-Blunt cloning vehicle, be gently mixed, room temperature reaction adds 50ul Trans10 competence after 10 minutes
(purchased from Beijing Quanshijin Biotechnology Co., Ltd) in cell, ice bath 30 minutes.42 DEG C of heat shocks 30 seconds, are immediately placed on ice 2
Minute.Add 250ul LB culture medium, 100rpm, hatch 1 hour for 37 DEG C.Take 200ul bacterium solution to be coated in containing ampicillin
On LB flat board, after incubated overnight, PCR screens 5 positive single bacterium colonies, positive colony carries out liquid culture, extracts positive colony
Plasmid carries out sequence verification, and sequencing result shows to insert purpose fragment on carrier pEASY-Blunt.
(3) UPC2 gene mutation
UPC2 is to regulate and control a key transcription factor gene of terpenoid level in yeast body, and UPC2 gene 3 ' is held
(2669bp) G to A sudden change, can make the aminoacid of corresponding positions point become Aspartate from Glycine, under aerobic conditions
Can improve the content of intracellular terpenoid, we use the point mutation process of gene Quickchange by this site mutation: first exist
Design homologous complementary containing lid mutational site district and G changed into the primer UPC (G-A)-F and UPC (G-A)-R (list of primers 2) of A,
Utilizing the pEASY-Blunt-UPC2 plasmid obtained in second step is template, and amplification system is: 10 × PfuUltra II
Reaction buffer 5ul, dNTP (10mM each dNTP) 1ul, DNA profiling 20ng, primer (10uM) each 1ul,
Stratagene PfuUltra II fusion HS DNA polymerase1ul, add distilled water to cumulative volume 50ul.Expand
Increasing condition is 95 DEG C of denaturations 2 minutes (1 circulation)；95 DEG C of degeneration 30 seconds, annealing 30 seconds (annealing temperature is shown in list of primers 1),
72 DEG C extend all with 6 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation).After product purification, after DpnI ferment treatment,
Proceeding to large intestine competence Trans10, verify through resistance and PCR after coated plate, the gene of successfully sudden change is identified in order-checking, it is thus achieved that sudden change
Gene UPC2.1.
List of primers 2
(4) fusion of BTS1 and ERG20 gene
The cattle cattle base pyrophosphate synthase of saccharomyces cerevisiae BTS1 gene expression can be catalyzed saccharomyces cerevisiae ERG20 gene
The product farnesyl pyrophosphate (FPP) of the farnesyl pyrophosphate synthase expressed is cattle cattle base pyrophosphoric acid (GGPP).Two
Enzyme fusion physically can increase FPP and be converted into the catalysis activity of GGPP, and we use GGGS peptide by BTS1 and ERG20 two
Functional protein merges, to improve its catalytic efficiency.The DNA extracted in the first step, as template, with primer in list of primers 3, expands
Increasing system is: NewEngland Biolabs Phusion 5Xbuffer10ul, dNTP (10mM each dNTP) 1ul, DNA
Template 20ng, primer (10uM) each 1ul, Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul,
Add distilled water to cumulative volume 50ul.Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation)；98 DEG C of degeneration 10 seconds, annealing 10
Second (annealing temperature is shown in list of primers 3), 72 DEG C of extensions are all with 2 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation).Cut
Two purpose fragments of glue purification, each 50ng adds PCR system: NewEngland Biolabs Phusion5Xbuffer 10ul,
DNTP (10mM each dNTP) 1ul, DNA profiling 20ng, add primer Asc1-FPS and SexA-Bst/DPP (10uM) each 1ul,
Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul, add distilled water to cumulative volume 50ul.Expand
Increasing condition is 98 DEG C of denaturations 2 minutes (1 circulation)；98 DEG C of degeneration are annealed 10 seconds, 72 DEG C and are extended all with 2 minutes for 10 seconds, 58 DEG C
(32 circulations)；72 DEG C extend 8 minutes (1 circulation)；Target sizes fragment amplification product cloning to pEASY-Blunt is cloned
Carrier.Convert, sequence verification (method is with step 2).
List of primers 3
(5)SaGGPSSyn.Gene codon optimizes and total gene synthesis
The cattle cattle base pyrophosphate synthase energy of sulfolobus acidocaldarius (Sulfolobus acidocaldarius)
Direct catalysis IPP and DMAPP is converted into GGPP, and (common cattle cattle base pyrophosphate synthase, such as saccharomyces cerevisiae, catalysis
FPP and IPP synthesizes GGPP).We by its gene optimization be yeast be suitable for codon, G/C content be 38.93%, 5 ' hold enzyme-added
Cutting site SexA1,3 ' ends add total gene synthesis gene order 1 after restriction enzyme site Asc1.It is saved in carrier (pUC57), obtains
SaGGPSSyn.Gene.
Embodiment 2, the clone of controlling element
With genes of brewing yeast group DNA as template, with primer in list of primers 4, amplification PGK1 (750bp), TEF1
(450bp) with ADH1 (1500bp) promoter, and terminator ADH1t (158bp).Amplification system is: NewEngland
Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM each dNTP) 1ul, DNA profiling 20ng, primer (10uM)
Each 1ul, Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul, add distilled water to cumulative volume
50ul.Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation)；98 DEG C of degeneration 10 seconds, within 10 seconds, (annealing temperature is shown in primer in annealing
List 4), 72 DEG C extend 1.5 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation).Extension amplification outcome is arrived
On pEASY-Blunt cloning vehicle.Convert, sequence verification (method is with embodiment 1 step 2).
List of primers 4
Embodiment 3, carry the plasmid construction of Genetic elements
The plasmid construction carrying Genetic elements is divided into following two step
(1) plasmid construction of tHMG1-UPC2.1 gene is carried
Carry the plasmid construction of tHMG1-UPC2.1 gene, total following 8 steps:
The first step: the structure of pRS406-δ DNA-URA3 plasmid.
With the plasmid pEASY-Blunt-δ DNA1 obtained in BstE1 and Sac1 enzyme action embodiment 1 step 2；BstE1 and
The plasmid pEASY-Blunt-δ DNA2 obtained in Kpn1 enzyme action embodiment 1 step 2.Rubber tapping two purpose fragments of purification, each 50ng
Addition linked system: 2ul 10XT4ligation Buffer (NEB company), 1ul T4ligase (NEB company, 400,
000cohesive end units/ml), supplementary distilled water is to 20ul, and room temperature reaction obtains connecting product for 2 hours, takes 1ul even
Thing of practicing midwifery addition PCR system: NewEngland Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM each
DNTP) 1ul, DNA profiling 20ng, add primer Sac1-A-δ DNA1 and Kpn-A-δ DNA2 (10uM) each 1ul, Phusion High-
Fidelity DNA Polymerase (2.5U/ul) 0.5ul, add distilled water to cumulative volume 50ul.Amplification condition be 98 DEG C pre-
Degeneration 1.5 minutes (1 circulation)；98 DEG C of degeneration are annealed 10 seconds, 72 DEG C and are extended all with 1 minute (32 circulations) for 10 seconds, 58 DEG C；72
DEG C extend 8 minutes (1 circulation)；By target sizes fragment amplification product cloning to pEASY-Blunt cloning vehicle, convert, survey
Sequence checking (method is with embodiment 1 step 2).
Sac1 and Kpn1 double digestion plasmid pEASY-Blunt-δ DNA and pRS406, rubber tapping recovery purpose fragment: δ DNA
(100ng) with pRS406 (30ng), (NEB is public to add 2ul 10XT4ligation Buffer (NEB company), 1ul T4ligase
Department, 400,000cohesive end units/ml), supplementary distilled water is to 20ul, and room temperature reaction obtains connecting product for 2 hours.
Proceed to Trans10 competent cell, sequence verification (method is with embodiment 1 step 2), obtain pRS406-δ DNA plasmid.
BstE1 digested plasmid pRS406-δ DNA and pEASY-Blunt-URA3, purpose fragment: URA3 is reclaimed in rubber tapping
(100ng) with pRS406-δ DNA (50ng), connect, convert, sequence verification (method is with embodiment 3 step 1 first step).Obtain
PRS406-δ DNA-URA3 plasmid.
Second step: the structure of pRS406-δ DNA-URA3-ADH1-tHMG1-ADH1t plasmid
With the plasmid pEASY-Blunt-ADH1 obtained in SexA1 and Sac11 enzyme action embodiment 2；SexA1 and Asc1 enzyme action
The plasmid pEASY-Blunt-tHMG1 obtained in embodiment 1 step 2, two purpose fragments of purification of tapping rubber, each 50ng adds connection
System: 2ul 10XT4ligation Buffer (NEB company), 1ul T4ligase (NEB company, 400,000cohesive
End units/ml), supplementary distilled water is to 20ul, and room temperature reaction obtains connecting product for 2 hours, takes 1ul and connects product addition PCR
System: NewEngland Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM each dNTP) 1ul, DNA profiling
20ng, add primer Sac11-Pac1-ADH-2 and Asc1-HMG1 (10uM) each 1ul, Phusion High-Fidelity DNA
Polymerase (2.5U/ul) 0.5ul, add distilled water to cumulative volume 50ul.Amplification condition is 98 DEG C of denaturations 2 minutes (1
Circulation)；98 DEG C of degeneration are annealed 10 seconds, 72 DEG C and are extended all with 1.5 minutes (32 circulations) for 10 seconds, 58 DEG C；72 DEG C extend 8 minutes (1
Individual circulation)；By target sizes fragment amplification product cloning to pEASY-Blunt cloning vehicle, converting, (method is same for sequence verification
Embodiment 1 step 2).Obtain pEASY-Blunt-ADH1-tHMG1 plasmid.
Sac11 and Asc1 double digestion plasmid pEASY-Blunt-ADH1-tHMG1；Matter in Asc1 and Pme1 enzyme action embodiment 2
Grain pEASY-Blunt-ADH1t；Rubber tapping two purpose fragments of purification, each 50ng adds linked system, linked system ibid, room temperature
React within 2 hours, obtain connect product, take 1ul connect product add PCR system: NewEngland Biolabs Phusion
5Xbuffer 10ul, dNTP (10mM each dNTP) 1ul, DNA profiling 20ng, add primer Sac11-Pac1-ADH-2 and
Sac11-Pme-ADHt (10uM) each 1ul, Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul,
Add distilled water to cumulative volume 50ul.Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation)；98 DEG C of degeneration are moved back for 10 seconds, 58 DEG C
Fire extends all with 2.5 minutes (32 circulations) for 10 seconds, 72 DEG C；72 DEG C extend 8 minutes (1 circulation)；Target sizes fragment is expanded
Volume increase thing is cloned into pEASY-Blunt cloning vehicle, converts, sequence verification (method is with embodiment 1 step 2).Obtain pEASY-
Blunt-ADH1-tHMG1-ADH1t plasmid.
Sac11 digested plasmid pEASY-Blunt-ADH1-tHMG1-ADH1t and pRS406-δ DNA-URA3 respectively, rubber tapping
Reclaiming purpose fragment: ADH1-tHMG1-ADH1t (100ng) and pRS406-δ DNA-URA3 (30ng), connect, convert, order-checking is tested
Card (method is with embodiment 3 step 1 first step).Obtain pRS406-δ DNA-URA3-ADH1-tHMG1-ADH1t plasmid.
3rd step: the structure of pHUra-δ DNA-tHMG1-UPC2.1 plasmid
SexA1 and Pac1 double digestion plasmid pRS406-δ DNA-URA3-ADH1-tHMG1-ADH1t and pEASY-Blunt-
TEF1, rubber tapping recovery purpose fragment: pRS406-δ DNA-URA3-//-tHMG1-ADH1t (100ng) and TEF1 (30ng), even
Connect, convert, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid: pRS406-δ DNA-URA3-TEF1-
tHMG1-ADH1t。
SexA1 and Asc1 double digestion plasmid pRS406-δ DNA-URA3-TEF1-tHMG1-ADH1t and pEASY-Blunt-
UPC2.1, rubber tapping recovery purpose fragment: pRS406-δ DNA-URA3-TEF1-//-ADH1t (100ng) and UPC2.1 (30ng),
Connect, convert, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid: pRS406-δ DNA-URA3-TEF1-
UPC2.1-ADH1t。
Sac11-Pme-ADHt and pme-pTEF1 is primer (list of primers 4,5), plasmid pRS406-δ DNA-URA3-
TEF1-UPC2.1-ADH1t is template, and PCR obtains DNA fragmentation: TEF1-UPC2.1-ADH1t.
Pme1 digested plasmid pRS406-δ DNA-URA3-ADH1-tHMG1-ADH1t and DNA fragmentation TEF1-UPC2.1-
ADH1t, rubber tapping recovery purpose fragment: pRS406-δ DNA-URA3-ADH1-tHMG1-ADH1t (100ng) and TEF1-UPC2.1-
ADH1t (30ng), connects, and converts, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid: pHUra-δ DNA-
tHMG1-UPC2.1。
4th step: the structure of pLHis-tHMG1-UPC2.1 plasmid
Sac11 digested plasmid pHUra-δ DNA-tHMG1-UPC2.1 and pRS313, purpose fragment: ADH1-is reclaimed in rubber tapping
THMG1-ADH1t-TEF1-UPC2.1-ADH1t (100ng) and pRS313 (30ng), connects, and converts, and (method is same for sequence verification
Embodiment 3 step 1 first step).Obtain plasmid: pLHis-tHMG1-UPC2.1.
5th step: the structure of pHLeu-tHMG1-UPC2.1 plasmid
Sac11 digested plasmid pHUra-δ DNA-tHMG1-UPC2.1 and pRS425, purpose fragment: ADH1-is reclaimed in rubber tapping
THMG1-ADH1t-TEF1-UPC2.1-ADH1t (100ng) and pRS425 (30ng), connects, and converts, and (method is same for sequence verification
Embodiment 3 step 1 first step).Obtain plasmid: pHLeu-tHMG1-UPC2.1.
6th step: the structure of pLKanMX-tHMG1-UPC2.1 plasmid
The Genetic elements KanMX that Example 1 obtains, takes 30ng pcr amplification product after purification, adds
2ul10XT4ligation Buffer (NEB company), 1ul T4Polynucleotide kinase (NEB company), supplement and steam
Distilled water to 20ul, 37 DEG C of phosphorylations 60 minutes；With with plasmid pLHis-tHMG1-UPC2.1 as template, V313-to-R and V313-
To-F is that Maker reversely part (100ng) is gone in primer PCR acquisition, and T4 ligase connects, and converts, and (method is same to be implemented sequence verification
Example 3 step 1 first step).Obtain plasmid: pLKanMX-tHMG1-UPC2.1.
7th step: the structure of pHKanMX-tHMG1-UPC2.1 plasmid
The Genetic elements KanMX that Example 1 obtains, takes 30ng pcr amplification product after purification, adds
2ul10XT4ligation Buffer (NEB company), 1ul T4Polynucleotide kinase (NEB company), supplement and steam
Distilled water to 20ul, 37 DEG C of phosphorylations 60 minutes；With with plasmid pHLeu-tHMG1-UPC2.1 as template, V-425-to-R and V-
425-to-F is that primer PCR acquisition pRS425 goes Maker reversely part (100ng), connects, converts, and (method is same real for sequence verification
Execute example 3 step 1 first step).Obtain plasmid: pHKanMX-tHMG1-UPC2.1.
8th step: the structure of pLLeu-tHMG1-UPC2.1 plasmid
The Genetic elements LEU2 element that Example 1 obtains, takes 30ng pcr amplification product after purification, adds
2ul10XT4ligation Buffer (NEB company), 1ul T4Polynucleotide kinase (NEB company), supplement and steam
Distilled water to 20ul, 37 DEG C of phosphorylations 60 minutes；With with plasmid pLHis-tHMG1-UPC2.1 as template, V313-to-R and V313-
To-F is that Maker reversely part (100ng) is gone in primer PCR acquisition, connects, converts, and (method is with embodiment 3 step 1 for sequence verification
The first step).Obtain plasmid: pLLeu-tHMG1-UPC2.1.
(2) ERG20, BTS1 and SaGGPS are carriedSyn.The plasmid construction of gene
Carry ERG20, BTS1 and SaGGPSSyn.The plasmid construction of gene, total following 7 steps:
The first step: pHUra-δ DNA-ERG20/BTS1-SaGGPSSyn.The structure of plasmid
SexA1 and Asc1 double digestion pRS406-δ DNA-URA3-ADH1-tHMG1-ADH1t and plasmid
PEASY-Blunt-ERG20/BTS1, rubber tapping recovery purpose fragment:
PRS406-δ DNA-URA3-ADH1-//-ADH1t (100ng) and ERG20/BTS1 (30ng), connects, and converts, and surveys
Sequence checking (method is with embodiment 3 step 1 first step).Obtain plasmid:
pRS406-δDNA-URA3-ADH1-ERG20/BTS1-ADH1t。
SexA1 and Pac1 double digestion pRS406-δ DNA-URA3-ADH1-tHMG1-ADH1t plasmid and pEASY-Blunt-
PGK1, rubber tapping recovery purpose fragment: pRS406-δ DNA-URA3-//-tHMG1-ADH1t (100ng) and PGK1 (30ng), even
Connect, convert, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid: pRS406-δ DNA-URA3-PGK1-
tHMG1-ADH1t。
SexA1 and Asc1 double digestion pRS406-δ DNA-URA3-PGK1-tHMG1-ADH1t and embodiment 1 step 5 obtain
Plasmid pUC57-SaGGPSSyn., rubber tapping recovery purpose fragment:
PRS406-δ DNA-URA3-PGK1-//-ADH1t (100ng) and SaGGPSSyn.(30ng), connect, convert, order-checking
Checking (method is with embodiment 3 step 1 first step).Obtain plasmid: pRS406-δ DNA-URA3-PGK1-SaGGPSSyn.-
ADH1t.Sac11-Pme-ADHt and pme-pPGK1 is primer, plasmid
pRS406-δDNA-URA3-PGK1-SaGGPSSyn.-ADH1t is that template PCR obtains:
PGK1-SaGGPSSyn.-ADH1t。
Pme1 digested plasmid pRS406-δ DNA-URA3-ADH1-ERG20/BTS1-ADH1t and PGK1-SaGGPSSyn.-
ADH1t, rubber tapping recovery purpose fragment: pRS406-δ DNA-URA3-ADH1-ERG20/BTS1-ADH1t (100ng) and PGK1-
SaGGPSSyn.-ADH1t (30ng), connects, and converts, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid:
pHUra-δDNA-ERG20/BTS1-SaGGPSSyn.。
Second step: pLUra-ERG20/BTS1-SaGGPSSyn.The structure of plasmid
Sac11 digested plasmid pHUra-δ DNA-ERG20/BTS1-SaGGPSSyn.And pRS316, purpose sheet is reclaimed in rubber tapping
Section: ADH1-ERG20/BTS1-ADH1t-PGK1-SaGGPSSyn.-ADH1t (100ng) and pRS316 (30ng), connects, and converts,
Sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid:
pLUra-ERG20/BTS1-SaGGPSSyn.。
3rd step: pHUra-ERG20/BTS1-SaGGPSSyn.The structure of plasmid
Sac11 digested plasmid pHUra-δ DNA-ERG20/BTS1-SaGGPSSyn.And pRS426, purpose sheet is reclaimed in rubber tapping
Section: ADH1-ERG20/BTS1-ADH1t-PGK1-SaGGPSSyn.-ADH1t (100ng) and pRS426 (30ng), connect, and converts,
Sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid:
pHUra-ERG20/BTS1-SaGGPSSyn.。
4th step: pLhphNT1-ERG20/BTS1-SaGGPSSyn. the structure of plasmid
The Genetic elements hphNT1 that Example 1 obtains, takes 30ng pcr amplification product after purification, adds 2ul
10XT4ligation Buffer (NEB company), 1ul T4Polynucleotide kinase (NEB company), supplement distilled water
To 20ul, 37 DEG C of phosphorylations 60 minutes；With with plasmid pLUra-ERG20/BTS1-SaGGPSSyn.For template, V316-to-R and
V316-to-F is that Maker reversely part (100ng) is gone in primer PCR acquisition, connects, converts, and (method is with embodiment 3 for sequence verification
Step 1 first step).Obtain plasmid:
pLhphNT1-ERG20/BTS1-SaGGPSSyn.。
5th step: pHhphNT1-ERG20/BTS1-SaGGPSSyn. the structure of plasmid
The Genetic elements hphNT1 that Example 1 obtains, takes 30ng pcr amplification product after purification, adds 2ul
10XT4ligation Buffer (NEB company), 1ul T4Polynucleotide kinase (NEB company), supplement distilled water
To 20ul, 37 DEG C of phosphorylations 60 minutes；With with plasmid pHUra-ERG20/BTS1-SaGGPSSyn. for template, V316-to-R and
V316-to-F is that Maker reversely part (100ng) is gone in primer PCR acquisition, connects, converts, and (method is with embodiment 3 for sequence verification
Step 1 first step).Obtain plasmid:
pHhphNT1-ERG20/BTS1-SaGGPSSyn.。
6th step: pLHis-ERG20/BTS1-SaGGPSSyn.The structure of plasmid
The Genetic elements HIS3 that Example 1 obtains, takes 30ng pcr amplification product after purification, adds
2ul10XT4ligation Buffer (NEB company), 1ul T4Polynucleotide kinase (NEB company), supplement and steam
Distilled water to 20ul, 37 DEG C of phosphorylations 60 minutes；With with plasmid pLhphNT1-ERG20/BTS1-SaGGPSSyn.For template, V316-
To-R and V316-to-F is that Maker reversely part (100ng) is gone in primer PCR acquisition, connects, converts, and (method is same for sequence verification
Embodiment 3 step 1 first step).Obtain plasmid:
pLHis-ERG20/BTS1-SaGGPSSyn.。
7th step: pHHis-ERG20/BTS1-SaGGPSSyn.The structure of plasmid
The Genetic elements HIS3 that Example 1 obtains, takes 30ng pcr amplification product after purification, adds
2ul10XT4ligation Buffer (NEB company), 1ul T4Polynucleotide kinase (NEB company), supplement and steam
Distilled water to 20ul, 37 DEG C of phosphorylations 60 minutes；With with plasmid pHhphNT1-ERG20/BTS1-SaGGPSSyn.For template, V316-
To-R and V316-to-F is that Maker reversely part (100ng) is gone in primer PCR acquisition, connects, converts, and (method is same for sequence verification
Embodiment 3 step 1 first step).Obtain plasmid:
pHHis-ERG20/BTS1-SaGGPSSyn.。
Primer table 5
Embodiment 4, the structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-000
The structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-000 is divided into following two step:
(1) structure of SmCPS and SmKSL gene plasmid is carried
Plasmid template pUC19-SmCPS, primer Kpn-CPS-2 and CPS-Not, expand SmCPS；Plasmid template pUC19-
SmKSL, primer 2-pvul-KSL and KSL-Pme1, expand SmKSL；Plasmid template YES2/CT, primer Kpn-GAL1 and 2-
GAL1-pvul, expands GAL1；Primer Not1-CYCt and CYCt-Kpn1 expands CYC1t, sees list of primers 6.Amplification system is:
NewEngland Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM each dNTP) 1ul, DNA profiling 20ng,
Primer (10uM) each 1ul, Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul, add distilled water
To cumulative volume 50ul.Amplification condition is 98 DEG C of denaturations 1.5 minutes (1 circulation)；98 DEG C of degeneration 10 seconds, annealing (annealing in 10 seconds
Temperature is shown in list of primers 6), 72 DEG C extend all with 2 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation), and product is through cutting
Glue reclaims and preserves.
By Kpn 1 and Not 1 double digestion plasmid YES2/CT and fragment SmCPS, purpose fragment: YES2/CT is reclaimed in rubber tapping
(100ng) with SmCPS (30ng), connect, convert, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid:
YES2/CT-GAL1-SmCPS-CYC1t
With Kpn 1 and pvul enzyme action genetic fragment SmKSL, GAL1, CYC1t, tap rubber after reclaiming purpose fragment respectively,
SmKSL (100ng), GAL1 (30ng) and CYC1t (30ng) mixing add linked system, and ibid, room temperature reaction 2 is little for linked system
Time obtain connect product, take 1ul connect product add PCR system: NewEngland Biolabs Phusion 5Xbuffer
10ul, dNTP (10mM each dNTP) 1ul, DNA profiling 20ng, to add primer Not1-CYCt and KSL-Pme1 (10uM) each
1ul, Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul, add distilled water to cumulative volume
50ul.Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation)；98 DEG C of degeneration are annealed 10 seconds, 72 DEG C and are extended all for 10 seconds, 58 DEG C
With 2 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation)；By target sizes fragment amplification product cloning to pEASY-
Blunt cloning vehicle, pEASY-Blunt-CYC1t-GAL1-SmKSL, convert, sequence verification (method is with embodiment 1 step 2).
Not 1 and Pme1 double digestion plasmid YES2/CT-GAL1-SmCPS-CYC1t and pEASY-Blunt-CYC1t-
GAL1-SmKSL, purpose fragment YES2/CT-GAL1-SmCPS-CYC1t (50ng) and CYC1t-GAL1-SmKSL are reclaimed in rubber tapping
(100ng), connect, convert, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid: pHUra-SmCPS-
SmKSL。
List of primers 6
(2) Plastid transformation carrying SmCPS and SmKSL gene enters saccharomyces cerevisiae
Set out bacterium Saccharomyces Cerevisiae in S accharomyces cerevisiae BY4742 incubated overnight in YPD, take 1ml (OD
About 0.6-1.0) be dispensed in 1.5ml EP pipe, 4 DEG C, 10000g be centrifuged 1min, abandon supernatant, precipitation sterilized water (4 DEG C) washing,
It is centrifuged under similarity condition, abandons supernatant.Thalline adds 1ml treatment fluid (10mM LiAc；10mM DTT；0.6M sorbitol；10mM
Tris-HCl (pH7.5), treatment fluid just adds DTT when using), place 20min at 25 DEG C.Centrifugal, abandon supernatant, thalline adds
1ml 1M sorbitol (it is degerming that 0.22um water system film crosses film) is resuspended, centrifugal, abandons supernatant (with the resuspended secondary of 1M sorbitol),
It is about 90 μ l to final volume.Proceed to, after adding 5ug Transformation plasmid: pHUra-SmCPS-SmKSL mixing, the cup (0.2cm) that shocks by electricity
In, ice bath 5min., 3kv, 25 μ F, 200 Ω, electric shock is once.1mL 1M sorbitol it is added immediately, in 30 after sucking-off after electric shock
DEG C cultivate 1h.Bacterium solution is all coated in after processing by centrifugation on selection flat board, formula: 0.8% yeast Selective agar medium SD-Ura-
Trp-His (general Jino, Beijing (functional genome) Science and Technology Ltd.), 2% glucose, 0.005%His., 0.01%Trp..
30 degree, cultivate more than 36h.PCR identifies correct positive colony, named bacterial strain ZD-T-000
Embodiment 5, the structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-001, ZD-T-002, ZD-T-003 and ZD-T-004
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to plasmid
pLLeu-tHMG1-UPC2.1.The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-Trp-Leu-His,
2% glucose, 0.005%His., 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies
Correct positive colony, named bacterial strain ZD-T-001.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to plasmid
pHLeu-tHMG1-UPC2.1.The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-Trp-Leu-His,
2% glucose, 0.005%His., 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies
Correct positive colony, named bacterial strain ZD-T-002.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to plasmid
pLHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-Trp-
His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correct sun
Sex clone, named bacterial strain ZD-T-003.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to respectively
Plasmid pHHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-
Trp-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correctly
Positive colony, named bacterial strain ZD-T-004.
Embodiment 6, the structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-005, ZD-T-006, ZD-T-007 and ZD-T-008
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to plasmid
PLLeu-tHMG1-UPC2.1 and pLHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-005.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to plasmid
PLLeu-tHMG1-UPC2.1 and pHHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-006.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to plasmid
PHLeu-tHMG1-UPC2.1 and pLHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-007.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-000 competent cell, proceed to plasmid
PHLeu-tHMG1-UPC2.1 and pHHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-008.
Embodiment 7, use Saccharomyces cerevisiae gene engineering bacteria ZD-T-000, ZD-T-001, ZD-T-002, ZD-T-003, ZD-T-
004, ZD-T-005, ZD-T-007, ZD-T-007 and ZD-T-008 produce miltirone diene
The engineered strain that picking activates in solid screening and culturing flat board, cultivate in fluid medium (30 DEG C,
250rpm, 16 hours), prepare fermentation seed liquid.Centrifugal collection thalline, is transferred in 20ml fermentation triangular flask, adjusts OD to 0.1,
The same screening culture medium of the corresponding fermentation medium of each bacterial strain (2% glucose replaces with 2% galactose), 30 DEG C, 250rpm/
Min., shaken cultivation 4 days, check OD600, and the content of miltirone diene.
Fermentation liquid 8000g collects thalline, adds a small amount of quartz sand, 500ul acetonitrile, the Votex30 second, ultrasonic 30min in frozen water,
7000g is centrifuged 5min, takes supernatant (extracting three times, merge supernatant)；Supernatant crosses 0.22um organic membrane, and GC-MS measures: sample introduction
Mouth temperature 300 DEG C, sampling volume 1ul, do not shunt, solvent time delay 5min.；Chromatographic column: HP-5ms (30m*0.25*0.5um)；Color
Spectral condition: 50 DEG C, 2min；20 DEG C min-1 to 250 DEG C insulation 8min；MS condition: SIM:148,272；Rt :～12.90min, mark
Directrix curve quantitative analysis, is shown in Fig. 4.In the case of increasing SmCPS-SmKSL gene expression with high copy number plasmid abduction delivering
(plasmid pHUra-SmCPS-SmKSL), the netic module tHMG1, UPC2.1 (pHLeu-tHMG1-UPC2.1) of high copy；Low copy
The netic module BTS1/ERG20, SaGGPS of shellfishsyn.(pLHis-ERG20/BTS1-SaGGPSSyn.) the engineered strain ZD-that obtains
T-007, yield is 12.5 times of reference strain ZD-T-000, reaches 14.4mg/L, sees Fig. 5.
Embodiment 8, the structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-010
The structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-010 is divided into following two step
(1): the structure of δ DNA-URA3-TEF1-SmCPS-ADH1t-PGK1-SmKSL-ADH1t functional module
With plasmid template pUC19-SmCPS, SexA-CPS/KS and ASC1-CPS for primer, expand SmCPS；Plasmid template
PUC19-SmKSL, SexA1-KSL and Asc1-CPS/KS are primer, expand SmKSL.Amplification system is: NewEngland
Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM each dNTP) 1ul, DNA profiling 20ng, primer (10uM)
Each 1ul, Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul, add distilled water to cumulative volume
50ul.Amplification condition is 98 DEG C of denaturations 2 minutes (1 circulation)；98 DEG C of degeneration 10 seconds, within 10 seconds, (annealing temperature is shown in primer in annealing
List 7), 72 DEG C extend all with 2 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation), after product cleaning with SexA1 with
Asc1 double digestion, and recovery of tapping rubber.
SexA1 and Asc1 double digestion plasmid pRS406-δ DNA-URA3-TEF1-UPC2.1-ADH1t and pRS406-δ DNA-
URA3-PGK1-tHMG1-ADH1t, and connect with corresponding endonuclease bamhi SmCPS and SmKSL, converting, (method is same for sequence verification
Embodiment 3 step 1 first step).Obtain plasmid: pRS406-δ DNA-URA3-TEF1-SmCPS-ADH1t and pRS406-δ DNA-
URA3-PGK1-SmKSL-ADH1t
Sac11-Pme-ADHt and pme-pPGK1 is primer, plasmid pRS406-δ DNA-URA3-PGK1-SmKSL-ADH1t
Obtain for template PCR, Genetic elements: PGK1-SmKSL-ADH1t.
Pme1 digested plasmid pRS406-δ DNA-URA3-TEF1-SmCPS-ADH1t and PGK1-SmKSL-ADH1t.Rubber tapping
Recovery purpose fragment: pRS406-δ DNA-URA3-TEF1-SmCPS-ADH1t (50ng) and PGK1-SmKSL-ADH1t
(100ng), connect, convert, sequence verification (method is with embodiment 3 step 1 first step).Obtain plasmid: pHUra-δ DNA-
SmCPS-SmKSL。
Being primer with plasmid template pHUra-δ DNA-SmCPS-SmKSL, δ DNA-F and δ DNA-R-2, amplification system is:
NewEngland Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM each dNTP) 1ul, DNA profiling 20ng,
Primer (10uM) each 1ul, Phusion High-Fidelity DNA Polymerase (2.5U/ul) 0.5ul, add distilled water
To cumulative volume 50ul.Amplification condition is 98 DEG C of denaturation 3 clocks (1 circulation)；98 DEG C of degeneration 10 seconds, 10 seconds (annealing temperatures of annealing
See list of primers 7), 72 DEG C extend all with 2 minutes (32 circulations)；72 DEG C extend 8 minutes (1 circulation), and product rubber tapping is reclaimed
Standby.
List of primers 7
(2) δ DNA-URA3-TEF1-SmCPS-ADH1t-PGK1-SmKSL-ADH1t functional module is integrated into saccharomyces cerevisiae
Chromosome
Functional module δ DNA-URA3-TEF1-SmCPS-ADH1t-PGK1-SmKSL-ADH1t electric shock proceeds to bacterium wine brewing of setting out
In yeast Saccharomyces cerevisiae BY4742, convert and screening technique is with embodiment 4 step 2.
PCR filters out positive colony bacterial strain, randomly selects 10 strains, ferments (carbon source is 2% glucose), and product detection is same
Embodiment 7.Choose the bacterial strain that miltirone diene yield is the highest, named ZD-T-010.
Embodiment 9, the structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-011, ZD-T-012, ZD-T-013, ZD-T-014
(auxotroph)
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
pLLeu-tHMG1-UPC2.1.The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-Trp-Leu-His,
2% glucose, 0.005%His., 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies
Correct positive colony, named bacterial strain ZD-T-011.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
pHLeu-tHMG1-UPC2.1.The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-Trp-Leu-His,
2% glucose, 0.005%His., 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies
Correct positive colony, named bacterial strain ZD-T-012.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
pLHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-Trp-
His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correct sun
Sex clone, named bacterial strain ZD-T-013.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to respectively
Plasmid pHHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast Selective agar medium SD-Ura-
Trp-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correctly
Positive colony, named bacterial strain ZD-T-014.
Embodiment 10, the structure of Saccharomyces cerevisiae gene engineering bacteria ZD-T-015, ZD-T-016, ZD-T-017, ZD-T-018
(auxotroph)
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PLLeu-tHMG1-UPC2.1 and pLHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-015.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PLLeu-tHMG1-UPC2.1 and pHHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-016.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PHLeu-tHMG1-UPC2.1 and pLHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-017.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PHLeu-tHMG1-UPC2.1 and pHHis-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 0.8% yeast choosing
Select culture medium SD-Ura-Trp-Leu-His, 2% glucose, 0.01%Trp.；The condition of screening and culturing is: 30 degree, cultivates 36h
Above.PCR identifies correct positive colony, named bacterial strain ZD-T-018.
Embodiment 11, use Saccharomyces cerevisiae gene engineering bacteria ZD-T-010, ZD-T-011, ZD-T-012, ZD-T-013, ZD-
T-014, ZD-T-015, ZD-T-016, ZD-T-017 and ZD-T-018 produce miltirone diene (auxotroph)
The engineered strain that picking activates in solid screening and culturing flat board, cultivate in fluid medium (30 DEG C,
250rpm, 16 hours), prepare fermentation seed liquid.Centrifugal collection thalline, is transferred in 20ml fermentation triangular flask, adjusts OD to 0.1,
The same screening culture medium of the corresponding fermentation medium of each bacterial strain, 30 DEG C, 250rpm/min., shaken cultivation 4 days, check OD600, and
The content of miltirone diene.
Fermentation liquid 8000g collects thalline, adds a small amount of quartz sand, 500ul acetonitrile, the votex30 second, ultrasonic 30min in frozen water,
7000g is centrifuged 5min, takes supernatant (extracting three times, merge supernatant)；Supernatant crosses 0.22um organic membrane, and GC-MS measures: sample introduction
Mouth temperature 300 DEG C, sampling volume 1ul, do not shunt, solvent time delay 5min.；Chromatographic column: HP-5ms (30m*0.25*0.5um)；Color
Spectral condition: 50 DEG C, 2min；20 DEG C min-1 to 250 DEG C insulation 8min；MS condition: SIM:148,272；Rt :～12.90min, mark
Directrix curve quantitative analysis.
δ DNA-URA3-TEF1-SmCPS-is integrated in Saccharomyces Cerevisiae in S accharomyces cerevisiae BY4742
In the case of ADH1t-PGK1-SmKSL-ADH1t gene, netic module tHMG1, the UPC2.1 (pHLeu-tHMG1-of high copy
UPC2.1)；The netic module BTS1/ERG20, SaGGPS of high copysyn.(pHHis-ERG20/BTS1-SaGGPSSyn.) obtain
Engineered strain ZD-T-018, yield is 2.7 times of reference strain ZD-T-000, reaches 11.39mg/L, sees Fig. 6.
Embodiment 12, Saccharomyces cerevisiae gene engineering bacteria ZD-T-021, the structure of ZD-T-022, ZD-T-023, ZD-T-024
(antibiotic type)
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
pLKanMX-tHMG1-UPC2.1.The culture medium of screening and culturing is: 1%Yeast Extract (yeast extract), 2%Peptone
(peptone), 2%Dextrose (glucose), 200mg/L G418；The condition of screening and culturing is: 30 degree, cultivates more than 36h.
PCR identifies correct positive colony, named bacterial strain ZD-T-021.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
pHKanMX-tHMG1-UPC2.1.The culture medium of screening and culturing is: 1%Yeast Extract (yeast extract), 2%Peptone
(peptone), 2%Dextrose (glucose), 200mg/L G418；The condition of screening and culturing is: 30 degree, cultivates more than 36h.
PCR identifies correct positive colony, named bacterial strain ZD-T-022.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
pLhphNT1-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 1%Yeast Extract (yeast extract), 2%
Peptone (peptone), 2%Dextrose (glucose), 300mg/L hygromycin；The condition of screening and culturing is: 30 degree, cultivates
More than 36h.PCR identifies correct positive colony, named bacterial strain ZD-T-023.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to respectively
Plasmid pHhphNT1-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 1%Yeast Extract (yeast
Cream), 2%Peptone (peptone), 2%Dextrose (glucose), 300mg/L hygromycin；The condition of screening and culturing is: 30
Degree, cultivates more than 36h.PCR identifies correct positive colony, named bacterial strain ZD-T-024.
Embodiment 13, Saccharomyces cerevisiae gene engineering bacteria ZD-T-025, the structure of ZD-T-026, ZD-T-027, ZD-T-028
(antibiotic type)
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PLKanMX-tHMG1-UPC2.1 and pLhphNT1-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 1%
Yeast Extract (yeast extract), 2%Peptone (peptone), 2%Dextrose (glucose), 200mg/L G418,
300mg/L hygromycin；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correct positive colony, name
For bacterial strain ZD-T-025.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PLKanMX-tHMG1-UPC2.1 and pHhphNT1-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 1%
Yeast Extract (yeast extract), 2%Peptone (peptone), 2%Dextrose (glucose), 200mg/L G418,
300mg/L hygromycin；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correct positive colony, name
For bacterial strain ZD-T-026, have another name called ZD-Tans-001.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PHKanMX-tHMG1-UPC2.1 and pLhphNT1-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 1%
Yeast Extract (yeast extract), 2%Peptone (peptone), 2%Dextrose (glucose), 200mg/L G418,
300mg/L hygromycin；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correct positive colony, name
For bacterial strain ZD-T-027.
Use the method identical with in embodiment 4 to carry out preparation and the conversion of ZD-T-010 competent cell, proceed to plasmid
PHKanMX-tHMG1-UPC2.1 and pHhphNT1-ERG20/BTS1-SaGGPSSyn..The culture medium of screening and culturing is: 1%
Yeast Extract (yeast extract), 2%Peptone (peptone), 2%Dextrose (glucose), 200mg/L G418,
300mg/L hygromycin；The condition of screening and culturing is: 30 degree, cultivates more than 36h.PCR identifies correct positive colony, name
For bacterial strain ZD-T-028.
Embodiment 14, use Saccharomyces cerevisiae gene engineering bacteria ZD-T-010, ZD-T-021, ZD-T-022, ZD-T-023, ZD-
T-024, ZD-T-025, ZD-T-026, ZD-T-027 and ZD-T-028 produce miltirone diene (antibiotic type)
The engineered strain that picking activates in solid screening and culturing flat board, preparation fermentation in YPD liquid screening medium
Seed liquor (30 DEG C, 250rpm, 16 hours)；Centrifugal collection thalline, is transferred in 20ml fermentation triangular flask, adjusts OD to 0.1, each bacterium
The same screening culture medium of the corresponding fermentation medium of strain, 30 DEG C, 250rpm/min. shaken cultivation 4 days, check OD600, and secondary Radix Salviae Miltiorrhizae
The content of ketone diene.
Fermentation liquid 8000g collects thalline, adds a small amount of quartz sand, 500ul acetonitrile, the votex30 second, ultrasonic 30min in frozen water,
7000g is centrifuged 5min, takes supernatant (extracting three times, merge supernatant)；Supernatant crosses 0.22um organic membrane, and GC-MS measures: sample introduction
Mouth temperature 300 DEG C, sampling volume 1ul, do not shunt, solvent time delay 5min.；Chromatographic column: HP-5ms (30m*0.25*0.5um)；Color
Spectral condition: 50 DEG C, 2min；20 DEG C min-1 to 250 DEG C insulation 8min；MS condition: SIM:148,272；Rt :～12.90min, mark
Directrix curve quantitative analysis.
δ DNA-URA3-TEF1-SmCPS-is integrated in Saccharomyces Cerevisiae in S accharomyces cerevisiae BY4742
In the case of ADH1t-PGK1-SmKSL-ADH1t function element, netic module tHMG1, the UPC2.1 (pLKanMX-of low copy
tHMG1-UPC2.1)；The netic module BTS1/ERG20, SaGGPS of high copysyn.(pHhphNT1-ERG20/BTS1-
SaGGPSSyn.) the engineered strain ZD-T-026 that obtains, yield is 11.5 times of bacterium ZD-T-010 of setting out, and reaches 61.8mg/L, sees figure
7。
Embodiment 15, with Saccharomyces cerevisiae gene engineering bacteria ZD-Tans-001 (ZD-T-026) produce miltirone diene join
Culture medium processed:
Culture medium 1: fluid medium, formula: 1%Yeast Extract (yeast extract), 2%Peptone (peptone), 1
～2%Dextrose (glucose), 200mg/L G418,300mg/L hygromycin；Solid medium need to add 2% agar powder again.
Culture medium feed supplement 2:20g/L Yeast Extract, 40g/L peptone, 400g/L Glucose, 200mg/L
G418,300mg/L hygromycin.
Fermentation culture conditions: the engineered strain ZD-Tans-001 that picking brings back to life in solid medium 1 flat board, in liquid
In culture medium 1, fermentation seed liquid (30 DEG C, 250rpm, 16 hours) is prepared in cultivation；Centrifugal collection thalline, is transferred to 5L fermentation tank
In, adjust OD to 0.5.High density fermentation condition: temperature, 30 DEG C；PH, 5.5 (regulation and control of 5M ammonia)；Rotating speed, 600rpm/min；Air
Flow velocity 2L/min, feed supplement is by dissolved oxygen feedback control；Every 24h takes a sample to check OD600, and the content of miltirone diene.
Result: saccharomyces cerevisiae ZD-Tans-001, under the conditions of supporting oxygen well, utilizes fluid medium 1, can give birth to when fermenting 6 days
Produce the miltirone diene of 487.91mg/L, see Fig. 8.
Claims (9)
1. saccharomyces cerevisiae (Saccharomyces cerevisiae) ZD-Tans-001, its preserving number is CGMCC No.5296.
2. a construction method for genetic engineering bacterium, comprises the following steps:
In the saccharomyces cerevisiae that sets out, introduce Ke Baji pyrophosphate synthase and the miltirone diene synthase of external source, obtain restructuring wine brewing
Yeast, is denoted as recombinant Saccharomyces cerevisiae I；
Cattle cattle base pyrophosphate synthase and the work of farnesyl pyrophosphate synthase is improved in described recombinant Saccharomyces cerevisiae I
Property, obtain recombinant Saccharomyces cerevisiae, be denoted as recombinant Saccharomyces cerevisiae III；
3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor egg is improved in described recombinant Saccharomyces cerevisiae III
The activity of white UPC2, obtains recombinant Saccharomyces cerevisiae, is denoted as recombinant Saccharomyces cerevisiae IV；
Described Ke Baji pyrophosphate synthase is the Ke Baji pyrophosphate synthase of Radix Salviae Miltiorrhizae；
Described miltirone diene synthase is the miltirone diene synthase of Radix Salviae Miltiorrhizae；
Described terpenoid regulatory factor albumen UPC2 is the sudden change of saccharomyces cerevisiae sterol regulatory factor protein UPC2, wherein, sudden change
Site is the sudden change that UPC2 gene 3 ' holds G to the A of 2669bp, and the aminoacid in corresponding site becomes sky from glycine Glycine
Aspartic Acid Aspartate；
Described 3-hydroxy-3-methylglutaryl-coenzyme A reductase be the 3-hydroxy-3-methylglutaryl-coenzyme A of saccharomyces cerevisiae also
Protoenzyme 1 intercepts the functional protein of 5 ' partial sequences, and wherein, described functional protein is that application is drawn with genes of brewing yeast as template
Thing to for:
SexA-HMG1 TC Gcg accwggtaaaacaATGGCTGCAGACCAATTGG,
Asc1-HMG 1TC GcggcgcgccTTAGGATTTAATGCAGGTGACGGAC
The albumen of the gene fragment expression obtained after expanding；
A kind of cattle cattle base pyrophosphate synthase for saccharomyces cerevisiae of described cattle cattle base pyrophosphate synthase；Another
Planting the cattle cattle base pyrophosphate synthase for codon optimized sulfolobus acidocaldarius, its encoding gene is in sequence table
DNA shown in sequence 1；Described farnesyl pyrophosphate synthase is the farnesyl pyrophosphate synthase of saccharomyces cerevisiae；
The described saccharomyces cerevisiae that sets out is Saccharomyces Cerevisiae in S accharomyces cerevisiae BY4742.
Method the most according to claim 2, it is characterised in that:
The Ke Baji pyrophosphate synthase of described introducing external source and miltirone diene synthase are to carry external source Ke's bar by being transformed into
Base pyrophosphate synthase and the plasmid of miltirone diene synthase gene, or it is burnt to integrate external source Ke's Buckie on S. cerevisiae chromosomal
Phosphate synthase and miltirone diene synthase gene；
The activity of described raising 3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor albumen UPC2 is to pass through
It is transformed into and carries 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the plasmid of terpenoid regulatory factor albumen UPC2 gene, or
3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor albumen UPC2 gene is integrated on S. cerevisiae chromosomal；
The described activity improving cattle cattle base pyrophosphate synthase and farnesyl pyrophosphate synthase is to carry by being transformed into
Cattle cattle base pyrophosphate synthase and the plasmid of farnesyl phosphate synthase gene, or integrate on S. cerevisiae chromosomal
Cattle cattle base pyrophosphate synthase and farnesyl phosphate synthase gene.
Method the most according to claim 3, it is characterised in that:
Described on S. cerevisiae chromosomal integrator gene be to be incorporated into δ DNA site；
The described plasmid carrying Ke Baji pyrophosphate synthase and miltirone diene synthase gene, replication site is 2MICRON, sieve
Choosing is labeled as opening of uracil auxotrophy selection markers URA3, Ke Baji pyrophosphate synthase and miltirone diene synthase gene
Mover is galactokinase 1 promoter GAL1 of saccharomyces cerevisiae；
The described plasmid carrying 3-hydroxy-3-methylglutaryl-coenzyme A reductase and terpenoid regulatory factor albumen UPC2 gene, multiple
Site processed is the one in 2MICRON and CEN6/ARSH4, and selection markers is leucine auxotrophy selection markers LEU2 and base
One in factor G418 resistance screening labelling KanMX, the promoter of 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene
For ethanol dehydrogenase 1 promoter ADH1 of saccharomyces cerevisiae, the promoter of terpenoid regulatory factor albumen UPC gene is saccharomyces cerevisiae
Translation elongation factor 1 promoter TEF1；
Described carrying cattle cattle base pyrophosphate synthase and the plasmid of farnesyl phosphate synthase gene, replication site is
One in 2MICRON and CEN6/ARSH4, selection markers is histidine auxotrophy selection markers HIS3 and hygromycin selection
One in labelling hphNT1；A kind of cattle cattle base pyrophosphate synthase gene and farnesyl phosphate synthase gene merge
Together, promoter is ethanol dehydrogenase 1 promoter ADH1 of saccharomyces cerevisiae, another kind of cattle cattle base pyrophosphate synthase
The promoter of gene is the glycerol 3-phosphate kinase promoter PGK1 of saccharomyces cerevisiae.
5. the recombinant Saccharomyces cerevisiae obtained is built by the method described in claim 4.
6. recombinant Saccharomyces cerevisiae described in saccharomyces cerevisiae described in claim 1 or claim 5 is in producing miltirone diene
Application.
7. the method producing miltirone diene, comprises the following steps: the ferment saccharomyces cerevisiae described in claim 1 or power
Profit requires recombinant Saccharomyces cerevisiae described in 5, obtains miltirone diene.
Method the most according to claim 7, it is characterised in that:
The temperature of described fermentation is 25 DEG C-37 DEG C；
The pH value of the system of described fermentation is 3.0-8.0；
The time of described fermentation is 24-168 hour or 24 hours；
The percent by volume of the inoculum concentration of described fermentation is 0.01%-10%；
The culture medium of described fermentation consists of the following composition:
Yeast extract, peptone, glucose, axoneme G418, hygromycin
Above composition concentration in described fermentation medium is respectively as follows:
Yeast extract 1-20g/L, peptone 1-40g/L, glucose 5-50g/L, G418 10-500mg/L, hygromycin 10-600mg/
L。
Method the most according to claim 8, it is characterised in that:
The temperature of described fermentation is 25 DEG C or 30 DEG C or 32 DEG C or 37 DEG C；
The pH value of the system of described fermentation is 3.0 or 4.0 or 5.0 or 6.0 or 7.0 or 8.0；
The time of described fermentation is 24 hours or 48 hours or 72 hours or 96 hours or 120 hours or 144 hours or 168 little
Time；
The percent by volume of the inoculum concentration of described fermentation is 0.01% or 0.3% or 1% or 10%.
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| CN101538576A (en) * | 2008-07-10 | 2009-09-23 | 中国中医科学院中药研究所 | Gene for generating related diterpene synthase together with tanshinone type compound as well as encoding product and application thereof |
| CN101928716A (en) * | 2009-06-30 | 2010-12-29 | 中国中医科学院中药研究所 | SmFPS (Salviamiltiorrhizabge Farnesyl Pyrophosphate Synthase) gene as well as coded protein and application thereof |
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