CN106244615A - A kind of engineering bacteria and construction method thereof and the application in preparing geraniol - Google Patents
A kind of engineering bacteria and construction method thereof and the application in preparing geraniol Download PDFInfo
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Abstract
The present invention relates to genetic engineering field, particularly relate to a kind of engineering bacteria and construction method thereof and the application in preparing geraniol.The engineering bacteria that the present invention provides is expressed IDI1 gene, tHMG gene and engineered GES gene.Relative to bacterial strain of the prior art, the yield that the engineering bacteria that the present invention provides produces geraniol is higher, and ferment 120h, and the concentration of geraniol is up to 360.06mg/L.
Description
Technical field
The present invention relates to genetic engineering field, particularly relate to a kind of engineering bacteria and construction method thereof and in preparing geraniol
Application.
Background technology
Geraniol (Geraniol, 3,7-dimethyl-2,6-octadiene-1-alcohol, C10H18O, molecular weight is 154.2493),
Have another name called geraniol, be a kind of acyclic monoterpene compounds found from plant volatile oil.Owing to geraniol has gentleness
Flos Rosae Rugosae taste, is widely used in floral type daily essence, and GB2760-1996 specifies, geraniol is the edible perfume (or spice) allowing to use
Material, mainly in order to prepare the essence such as Fructus Mali pumilae, Fructus Persicae, Fructus Pruni, Lee, Fructus Fragariae Ananssae, Fructus Citri Limoniae, Rhizoma Zingiberis Recens, Cortex Cinnamomi, Semen Myristicae.It is by as Flos Rosae Rugosae system perfume
The host of essence, is indispensable perfumery raw material in various floral perfume, can be used for cosmetics, soap etc..Geraniol is also raw
Producing the raw material of citronellol, ionoionone, vitamin A etc., the ester that geraniol is formed also is good spice.Except being used for perfume (or spice)
Essence, geraniol also has certain pharmacologically active, may be used for the clinical treatment of antibacterial, anthelmintic and chronic bronchitis, also
It is proved to can be used for the treatment of cancer.Additionally, due to the characteristic that geraniol energy is high, hygroscopicity is low, volatility is relatively weak, it
It is also considered to be a kind of gasoline succedaneum being better than ethanol.But the geraniol limits throughput of natural production, chemosynthesis is deposited again
In safety problem, in the face of the ever-increasing market demand, a Microbial cell factories excellent geraniol of can yet be regarded as produces flat
Platform.Microbe synthesis has that low cost, yield is high and product safety and the advantage such as easily operated.At present, at Microbe synthesis
In the research of geraniol, the host used focuses primarily upon escherichia coli, saccharomyces cerevisiae and methanosarcina.
The method of micro-organisms geraniol comes across 2013 the earliest, and the industrial bio science and technology laboratory of Southern Yangtze University leads to
The method crossing reinforcement precursor supply achieves the production of geraniol in saccharomyces cerevisiae.Subsequently, constantly there is micro-organisms fragrant
The successful case of folic alcohol occurs, but mostly being that escherichia coli are as host.Saccharomyces cerevisiae is as the generally acknowledged micro-life of safe mode
Thing, genetic background understands, genetic manipulation simple, can carry out large scale fermentation production.Comparing escherichia coli, its thalline dimension is raw
Element, protein content are high, can make edible medicinal and feedstuff, and sweat need not add antibiotic agents.Therefore, real
Existing geraniol high yield in saccharomyces cerevisiae will represent great competitiveness in geraniol micro-organisms industrialization.So
And, the report utilizing saccharomyces cerevisiae synthesis geraniol so far is less and yield is the lowest.Compared to geraniol escherichia coli
In yield, geraniol yield in saccharomyces cerevisiae is the most relatively low, can not reach far away industrialized requirement.Therefore, urgently
The recombinant Saccharomyces cerevisiae of high yield geraniol to be developed.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of engineering bacteria and construction method thereof and preparing
Application in geraniol, the bacterial strain that the present invention provides can ferment generation geraniol, produce the maximum concentration of geraniol up to
360mg/L。
The invention provides engineered GES gene, its nucleotide sequence is shown in as any one of SEQ ID NO:1～7；
Or obtain for nucleotide sequence shown in SEQ ID NO:1～7 any one is substituted, lacks or adds one or more nucleotide
Nucleotide sequence.
The GES gene that the present invention provides is respectively from 7 kinds of different plants, wherein:
Shown in SEQ ID NO:1, nucleotide sequence alterations is from the GES gene of Cinnamomum tenuipilum (Cinnamomum tenuipile)
(CtGES)；
Nucleotide sequence alterations shown in SEQ ID NO:2 is from the GES gene (LdGES) of lippia aulcis (Lippia dulcis)；
Nucleotide sequence alterations shown in SEQ ID NO:3 is from the GES gene (VvGES) of Fructus Vitis viniferae (Vitis vinifera)；
Shown in SEQ ID NO:4, nucleotide sequence alterations is from the GES base of Folium Perillae eucalyptus citriodora (Perilla citriodora)
Because of (PcGES)；
Shown in SEQ ID NO:5, nucleotide sequence alterations is from the GES gene of Herba Catharanthi Rosei (Catharanthus roseus)
(CrGES)；
SEQ ID NO:6, nucleotide sequence alterations shown in 7 are from the GES gene of Rhizoma et radix valerianae (Valeriana officinalis)
(VoGES)；Wherein, only have one between GES albumen expressed by SEQ ID NO:6 (VoGES1) and SEQ ID NO:7 (VoGES2)
Individual amino acid whose difference；.
Experiments indicate that, in the yeast of conversion of saccharomycetes such as IDIl gene and tHMG gene, proceed to further
The GES gene of nucleotide sequence shown in SEQ ID NO:1～7, it is possible to make yeast produce geraniol.At SEQ ID NO:1～7
Nucleotide sequence shown in any one is substituted, lacks or adds the nucleotide sequence that one or more nucleotide obtains, and
The protein DNA molecule with GES enzymatic activity can be encoded, also within protection scope of the present invention.
According to Analysis of test results, GES is positioned chloroplast, but does not has chloroplast in saccharomyces cerevisiae, may result in
GES can not fold well thus affect its activity, so its positioning sequence is carried out truncate.The film positioning sequence of GES albumen
It is positioned at N end, therefore, 5 ' ends of GES gene order is carried out truncate.The length of the film positioning sequence of GES albumen about 14～88
Aminoacid.Effect after LdGES, CrGES, VoGES1, VoGES2 truncate is verified by the embodiment of the present invention, cuts at LdGES
28 aminoacid of short N end, CrGES 14, truncate N end, 28,43 aminoacid respectively, and VoGES1, VoGES2 truncate N respectively
Hold 58 and 88 aminoacid, proceed to yeast with the gene of GES albumen after expressing above-mentioned truncate, it is possible to further improve perfume (or spice)
The yield of folic alcohol.
The sequence of the engineered GES gene that the present invention provides is nucleotide sequence shown in SEQ ID NO:1～7 any one
5 ' the one or more nucleotide of the disappearance held.
The plurality of is 42～264, specially 42,69,84,129,174,264.
In the embodiment of the present invention, engineered GES gene order is as shown in SEQ ID NO:8～15 any one.
In some specific embodiments, at 5 ' end 69 nucleotide (SEQ of disappearance of nucleotide sequence shown in SEQ ID NO:2
ID NO:8)；
5 ' end disappearance 42 nucleotide (SEQ ID NO:9), 84 nucleoside at nucleotide sequence shown in SEQ ID NO:5
Acid (SEQ ID NO:10), 129 nucleotide (SEQ ID NO:11)；
Nucleotide sequence shown in SEQ ID NO:6 5 ' end 174 nucleotide (SEQ ID NO:12) of disappearance, 264
Nucleotide (SEQ ID NO:13)；
Nucleotide sequence shown in SEQ ID NO:7 5 ' end 174 nucleotide (SEQ ID NO:14) of disappearance, 264
Nucleotide (SEQ ID NO:15).
Experiment shows, after LdGES 84 nucleotide of gene 5 ' end truncate, LdGES enzyme N end is truncated 28 aminoacid,
After being transformed into yeast, the yield of geraniol is improved to 127.68mg/L by 4.53mg/L, improves about 28 times；
After CrGES 42 nucleotide of gene 5 ' end truncate, CrGES enzyme N end is truncated 14 aminoacid, is transformed into yeast
After bacterium, the yield of geraniol is improved to 283.36mg/L by 42.63mg/L, improves about 7 times；
After CrGES 84 nucleotide of gene 5 ' end truncate, CrGES enzyme N end is truncated 28 aminoacid, is transformed into yeast
After bacterium, the yield of geraniol is improved to 295.62mg/L by 42.63mg/L, improves about 7 times；
After CrGES 129 nucleotide of gene 5 ' end truncate, CrGES enzyme N end is truncated 43 aminoacid, is transformed into ferment
After female bacterium, the yield of geraniol is improved to 360.06mg/L by 42.63mg/L, improves about 8 times；
174 nucleotide VoGES1 enzyme N ends of VoGES1 gene 5 ' end truncate are truncated 58 aminoacid, are transformed into yeast
After bacterium, the yield of geraniol is improved to 102.46mg/L by 2.73mg/L, improves about 37.53 times；
After VoGES1 264 nucleotide of gene 5 ' end truncate, VoGES1 enzyme N end is truncated 88 aminoacid, is transformed into
After yeast, the yield of geraniol is improved to 12.41mg/L by 2.73mg/L, improves about 5 times；
After VoGES2 174 nucleotide of gene 5 ' end truncate, VoGES2 enzyme N end is truncated 58 aminoacid, is transformed into
After yeast, the yield of geraniol is improved to 241.28mg/L by 9.51mg/L, improves about 25 times；
After VoGES2 264 nucleotide of gene 5 ' end truncate, VoGES2 enzyme N end is truncated 88 aminoacid, is transformed into
After yeast, the yield of geraniol is improved to 98.54mg/L by 9.51mg/L, improves about 10 times.
Visible, converting nucleotide sequence shown in SEQ ID NO:11 can be by the output increased of geraniol to 360.06mg/L.
Converting nucleotide sequence shown in SEQ ID NO:9～11, SEQ ID any one of NO:14 can be by the yield of geraniol
Improve to more than 200mg/L.
Converting nucleotide sequence shown in SEQ ID NO:8～12, SEQ ID any one of NO:14 can be by the yield of geraniol
Improve to more than 100mg/L.
Converting nucleotide sequence shown in SEQ ID NO:8～12, SEQ ID NO:14～15 any one can be by geraniol
Output increased is to more than 98.5mg/L.
The engineered GES gene that the present invention provides application in building the engineering bacteria producing geraniol.
Present invention also offers a kind of engineering bacteria, it expresses IDI1 gene, tHMG gene and engineered GES gene；Institute
State the sequence of IDI1 gene as shown in SEQ ID NO:16；Described tHMG gene order is as shown in SEQ ID NO:17.
Biosynthesis pathway (Fig. 1) according to geraniol, HMG CoA is formed after HMGR, IDIl, ERG20, GES effect
Geraniol.By the present invention in that chassis bacterium expresses IDI1 gene, tHMG gene and engineered GES gene, it is achieved thereby that
High yield geraniol in engineering bacteria.Engineering bacteria of the present invention is selected from algae, yeast, mycete or antibacterial.Wherein, yeast is excellent
Choosing solves fat and belongs to yeast, Crewe dimension genus yeast or saccharomyces cerevisiae；Described saccharomyces cerevisiae is selected from CEN.PK series, BY series；Described mould
The preferred streptomycete of bacterium；The preferred escherichia coli of antibacterial or bacillus subtilis.
In embodiments of the present invention, its initial strain is yeast CEN.PK2-1C.
Described IDIl gene and tHMG gene are from yeast CEN.PK2-1 genome.
Described IDIl gene and tHMG gene integration are at the GAL80 gene location of yeast CEN.PK2-1C, yeast
The GAL80 gene of CEN.PK2-1C is knocked.
Described engineered GES gene uses centromere plasmid to import.
The genotype of the engineering bacteria that the present invention provides is CEN.PK2-1C, Δ gal80::tHMG-PGAL1,10-IDI1-
His3, pRS415-GPM1t-PGAL7-GES-GPDt。
Wherein, the gene order of GES is as shown in SEQ ID NO:1～7 any one, or is SEQ ID NO:1～7 any one
Shown nucleotide sequence is substituted, lacks or adds the nucleotide sequence that one or more nucleotide obtains.The present invention implements
In example, engineered GES gene order is as shown in SEQ ID NO:8～15 any one.
The engineering bacteria that the present invention provides application in producing geraniol.
The engineering bacteria using the present invention to provide can make the output increased of geraniol to 360.1mg/L.
In order to build the engineering bacteria that the present invention provides, present invention also offers one and knock out box, including be linked in sequence
The left homology arm of gal80, ADH1t terminator, GAL1/10 promoter, TDH2t terminator, His nutritional labeling, the right homology of gal80
Arm.
The a length of 400bp of the right homology arm of a length of 400bp, gal80 of the left homology arm of described gal80.
As preferably, the skeleton knocking out box is pRS425K.
Wherein, the left homology arm of gal80, the right homology arm of gal80, ADH1t terminator, GAL1/10 promoter, TDH2t terminate
Sub from yeast CEN.PK2-1.His nutritional labeling is from yeast S288C.
The primer of the amplification left homology arm of gal80 is to as shown in SEQ ID NO:20～21.
The primer of amplifying ADH 1t terminator is to as shown in SEQ ID NO:22～23.
The primer of amplification GAL1,10 promoter is to as shown in SEQ ID NO:24～25.
The primer of amplification TDH2t terminator is to as shown in SEQ ID NO:26～27.
The primer of the amplification right homology arm of gal80 is to as shown in SEQ ID NO:28～29.
The primer of amplification His nutritional labeling is to as shown in SEQ ID NO:30～31.
The construction method knocking out box that the present invention provides includes:
Step 1: by left for gal80 homology arm, ADH1t terminator, GAL1/10 promoter, TDH2t terminator, His nutrition mark
Signing, the right homology arm of gal80 is sequentially spliced obtained fragment gal80up-ADH1t-P by OE-PCR methodGAL1/10-TTDH2t-His-
gal80down；
Step 2: by fragment gal80up-ADH1t-PGAL1/10-TTDH2t-His-gal80down and multicopy plasmid
PRS425K is connected by NotI restriction enzyme site, obtains Δ gal80::ADH1t-PGAL1,10-TDH2t-His3 knocks out box.
Described OE-PCR is with the primer such as SEQ ID NO:20 as forward primer, under with the primer such as SEQ ID NO:31 being
Trip primer.
Further, IDIl and tHMG is connected into knock out box, it is thus achieved that knock out box Δ gal80::ADH1t-IDI1-
PGAL1/10-tHMG-TTDH2t-His3, method particularly includes:
Step 1: tHMG fragment is passed through BsmBI restriction enzyme site and knocks out box Δ gal80::ADH1t-PGAL1,10-TDH2t-
His3 reclaims after NotI enzyme action after connecting and knocks out box, transfers to obtain on pLD2 carrier striking containing tHMG gene by knocking out box
Except box；
Step 2: be connected to knock out on box containing IDIl gene by BsaI restriction enzyme site by IDI1 fragment, it is thus achieved that
pLD2-gal80up-ADH1t-IDI1-PGAL1/10-tHMG-TTDH2t-His-gal80down。
What the present invention provided knocks out box Δ gal80::ADH1t-IDI1-PGAL1/10-tHMG-TTDH2t-His3 is building product perfume
Application in the engineering bacteria of folic alcohol.
In order to build the engineering bacteria producing geraniol, present invention also offers a kind of containing the centromere of GES gene after transformation
Plasmid, its construction method and plasmid map are as shown in Figure 3.This centromere plasmid, with pRS415 plasmid as skeleton, connects including order
GPM1t, GAL7 promoter of connecing, GES gene, GPDt.
The structure of this centromere plasmid is with expression cassette pLD2-GPM1t-PGAL7-GPDt is initial, by engineered GES base
PLD2-GPM1t-P is obtained because being connected in expression cassette by BsaI enzyme actionGAL7-GES-GPDt.Utilize NotI restriction enzyme site, by table
Reach box to transfer to singly copy on plasmid pRS415.
The preparation method of GES genetic fragment is not limited by the present invention, in order to GES gene is connected into expression cassette, at GES
5 ' ends of gene add sequence gcggccgcggtctcca (SEQ ID NO:18)；3 ' add sequence
taaaggagaccgcggccgc(SEQ ID NO:19)。
The construction method of the engineering bacteria that the present invention provides, by IDI1 gene, tHMG gene and engineered GES gene transformation
Enter starter bacteria.
Concrete, the construction method of the engineering bacteria that the present invention provides includes:
Step 1: to knock out box Δ gal80::ADH1t-IDI1-PGAL1/10-tHMG-TTDH2t-His3 by IDI1 gene and
THMG gene transformation enters starter bacteria and obtains chassis bacterium；
Step 2: chassis bacterium will be entered containing the centromere Plastid transformation of GES gene after transformation, it is thus achieved that the work that the present invention provides
Journey bacterium.
In the present invention, starter bacteria is yeast, and preferred yeast is CEN.PK2-1C.
Convert described in step 1, step 2 and use lithium acetate Efficient Conversion method.
Present invention also offers the preparation method of a kind of geraniol, the engineering bacteria that the fermentation present invention provides.
Described fermentation is biphase fermentation；The organic facies of described biphase fermentation uses isopropyl myristate.
The present invention uses biphase fermentation method to reduce the geraniol injury to brewing yeast cell as far as possible.
The culture medium of described fermentation includes: synthetic yeast nitrogen source YNB 6.7g/L；Glucose 20g/L；Lack leucic mixed
Close powder of amino acids 2g/L.
Described isopropyl myristate volume is the 20% of fermentation medium.
The temperature of described fermentation is 30 DEG C, and 200rpm shakes.
Described fermentation is particularly as follows: with initial OD600=0.2 is inoculated in fresh 5ml fermentation medium, in 30 DEG C,
Under the conditions of 250rpm, cultivation about 12h is to thalli growth to mid-log phase, with initial OD600=0.2 is inoculated in 50ml fermentation culture
In base, be simultaneously introduced isopropyl myristate that volume fraction is 20% 30 DEG C, carry out biphase fermentation, 20h under the conditions of 200rpm
Time add 10g/L dehydrated alcohol, after 120h terminate fermentation.
Use the present invention provide engineering bacterium fermentation, due in initial medium because with the presence of glucose, transcribing of GES is subject to
Suppress to glucose；Along with the carrying out of fermentation, glucose is gradually consumed, and fermentation about 19h glucose exhausts, and glucose suppresses
Effect releases, P in the case of GAL80 has knocked outGALPromoter automatically turns on, and GES starts to transcribe and translate, thus gradually
Accumulation geraniol.Utilize PGALThalli growth and product are produced be able to separately, thus reduce product toxicity cell growth
Adverse effect, and then make promoting further of geraniol yield be possibly realized.
After described fermentation ends, also include the step extracted.
Described it is extracted as: take isopropyl myristate layer, except obtaining the solution containing geraniol after water.
The invention provides a kind of engineering bacteria and construction method thereof and the application in producing geraniol, the present invention provides
Engineering bacteria is expressed IDI1 gene, tHMG gene and engineered GES gene.Relative to bacterial strain of the prior art, the present invention
The engineering bacteria provided produces the yield higher fermentation 120h of geraniol, and the concentration of geraniol is up to 360.06mg/L.
Accompanying drawing explanation
Fig. 1 shows the pathway figure utilizing recombinant Saccharomyces cerevisiae synthesis geraniol；
Fig. 2 shows that chassis bacterial strain knocks out box structure figure；
Fig. 3 shows GES expression casette structure figure；
Fig. 4 shows the growth curve chart of cell under different fermentations environment.
Detailed description of the invention
The invention provides a kind of engineering bacteria and construction method thereof and the application in preparing geraniol, people in the art
Member can use for reference present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change
Apparent to those skilled in the art, they are considered as being included in the present invention.The method of the present invention and application
Being described by preferred embodiment, related personnel substantially can be right in without departing from present invention, spirit and scope
Methods herein and application are modified or suitably change and combine, and realize and apply the technology of the present invention.
In the present invention, synthesis geraniol precursor substance be GPP, GPP geraniol synthase (geraniol synthase,
GES) geraniol is synthesized under effect.The GES gene source of present invention employing and sequence such as table 1.
In the present invention, the implication that english abbreviation represents is as follows:
GES geraniol synthase
THMG 3-hydroxy-3-methylglutaryl-coenzyme A reductase encoding gene
IDI1 Isopentenyl diphosphate isomerase
The left homology arm of gal80up GAL
The right homology arm of gal80down GAL
PGAL1GAL1 promoter
TTDH2tTDH2t terminator
PGAL1/10GAL1/10 promoter
After △ ga80l::Gal80 is knocked, quilt:: sequence below replaces
Glucose glucose
Acetyl-CoA S-acetyl-coenzyme-A
HMG-CoA 3-hydroxyl-3-methylglutaryl coenzyme A
Mevalonate mevalonic acid
IPP Isoprenoid
DMAPP dimethyl propylene thiazolinyl diphosphonic acid-three ammonium salt
GPP cattle base pyrophosphoric acid
Geraniol geraniol
ALD gene aldehyde dehydrogenase gene
OE-PCR Overlap extension PCR
The biomaterial such as plasmid used in saccharomyces cerevisiae engineered yeast strain that the present invention provides and construction method, application
Being buied by market, primer sequence all can be synthesized by biotech firm.
Below in conjunction with embodiment, the present invention it is expanded on further:
Embodiment 1 builds chassis bacterial strain
With CEN.PK2-1C as initial strains, build and knock out gene GAL80 simultaneously at GAL80 position integration idi1 (SEQ
ID NO:16) and the Wine brewing yeast strain CEN.PK2-1C, Δ gal80::tHMG-of tHMGR (SEQ ID NO:17) gene
PGAL1,10-IDI1-His3, obtains final chassis bacterial strain SyBE_Sc020201.Concrete building process is as follows:
The primer sequence used in building process such as table 1:
Table 1: primer sequence
| The amplification left homology arm of gal80 | SEQ ID NO:20～21 |
| Amplifying ADH 1t terminator | SEQ ID NO:22～23 |
| Amplification GAL1,10 promoter | SEQ ID NO:24～25 |
| Amplification TDH2t terminator | SEQ ID NO:26～27 |
| The amplification right homology arm of gal80 | SEQ ID NO:28～29 |
| Amplification His nutritional labeling | SEQ ID NO:30～31 |
| Amplification OE-PCR | SEQ ID NO:20、31 |
| Amplification gene IDI1 | SEQ ID NO:35～36 |
| Amplification gene tHMG | SEQ ID NO:37～38 |
| Bacterium colony PCR | SEQ ID NO:35-38 |
With saccharomyces cerevisiae CEN.PK2-1 genome as template, design upstream and downstream primer PCR amplification gal80 upstream and downstream
400bp homology arm, ADH1t terminator, PGAL1,10Promoter, TDH2t terminator, His nutritional labeling, then by gal80 upstream
Homology arm, ADH1t terminator, PGAL1,10Promoter, TDH2t terminator, His nutritional labeling, gal80 downstream homology arm pass through
OE-PCR method is stitched together, and must arrive two ends and comprise NotI restriction enzyme site and at PGAL1Wrap between promoter and TDH2t terminator
Containing two BsaI restriction enzyme sites, at PGAL10Fragment gal80up-of two BsmBI is comprised between promoter and ADH1t terminator
ADH1t-PGAL1/10-TTDH2t-His-gal80down, is connected by NotI restriction enzyme site with multicopy plasmid pRS425K, obtains
Δgal80::ADH1t-PGAL1,10-TDH2t-His3 knocks out box.With saccharomyces cerevisiae CEN.PK2-1 genome as template, in design
Downstream primer amplification gene IDI1 and tHMG, the tHMG first amplification obtained respectively pass through BsmBI restriction enzyme site and knock out box
Connecting, proceed in E. coli competent TransT1, bacterium colony PCR screens, and upgrading grain carries out NotI digestion verification and order-checking is tested
Card obtains correct plasmid pRS425K-gal80up-ADH1t-PGAL1/10-tHMG-TTDH2t-His-gal80down, by this plasmid
Reclaim after NotI enzyme action and knock out box, will knock out box and transfer on pLD2 carrier, verify through blue white macula screening, bacterium colony PCR and
NotI digestion verification obtains correctly clones, and its plasmid is pLD2-gal80up-ADH1t-PGAL1/10-tHMG-TTDH2t-His-
gal80down.Then IDI1 amplification obtained is connected in plasmid obtained in the previous step by BsaI restriction enzyme site and then obtains
To pLD2-gal80up-ADH1t-IDI1-PGAL1/10-tHMG-TTDH2t-His-gal80down, also passes through order-checking and enzyme action
Checking obtains correct clone later.
Knock out after box built, carry out enzyme action with NotI restriction endonuclease, obtain fragment gal80up-ADH1t-IDI1-
PGAL1/10-tHMG-TTDH2t-His-gal80down, uses lithium acetate Efficient Conversion method that the fragment obtained individually is converted wine brewing ferment
Mother strains CEN.PK2-1C, by about GAL80 homologous sequence and on Yeast genome GAL80 site occur homologous recombination and whole
It is bonded in genes of brewing yeast group.SC-drop solid medium (synthetic yeast nitrogen source YNB 6.7g/L, Fructus Vitis viniferae is used after conversion
Sugar 20g/L, lacks tryptophan, leucine, histidine and the kilnitamin powder 2g/L of uracil, the agar powder of 2%) sieve
Choosing, the transformant obtained is extracted Yeast genome after carrying out the pure and mild enrichment culture of line point and is carried out PCR checking, correct to checking
Recombinant bacterial strain preserves glycerol stock named SyBE_Sc020201 (CEN.PK2-1C, Δ gal80::tHMG-PGAL1,10-IDI1-
His3)。
Embodiment 2 produces the structure of geraniol Wine brewing yeast strain
1, the acquisition of external source functional gene element
Exogenous gene includes the geraniol synthase gene GES for synthesizing geraniol: choose the function of 9 kinds of separate sources
The gene source (such as table 2) that genescreen synthesis geraniol is optimum, the present invention relates to the exogenous gene of 10 synthesis geraniol altogether.
Said gene is codon optimized through saccharomyces cerevisiae and after suitably evading conventional restriction enzyme site, adds at gene 5 ' end
Add gcggccgcggtctcca (SEQ ID NO:18), 3 ' interpolations taaaggagaccgcggccgc (SEQ ID NO:19) are passed through
Synthetic obtains.
Table 2 engineered CrtZ gene source and sequence
The wherein amino acid whose difference of only one of which between the albumen of VoGES1 and VoGES2 coding.Obtaining of 5 ' end deletion fragments
The mode of obtaining is: the film by website http://www.cbs.dtu.dk/index.shtml prediction LdGES, CrGES, VoGES is fixed
Bit sequence, and carry out gradient truncate according to predicting the outcome.Method for truncating is as follows: with gene LdGES, CrGES, VoGES of synthesis
For template, design upstream and downstream primer (table 3) amplification obtains truncate and the two ends tGES with BsaI restriction enzyme site.LdGES truncate N
Hold 23 aminoacid, CrGES 14, truncate N end, 28,43 aminoacid respectively, and VoGES1, VoGES2 truncate N end respectively
58 and 88 aminoacid.
Table 3 expands the upstream and downstream primer of truncated sequence
| Purpose fragment | Primer sequence |
| SEQ ID NO:8 | SEQ ID NO:46、SEQ ID NO:47 |
| SEQ ID NO:9 | SEQ ID NO:39、SEQ ID NO:42 |
| SEQ ID NO:10 | SEQ ID NO:40、SEQ ID NO:42 |
| SEQ ID NO:11 | SEQ ID NO:41、SEQ ID NO:42 |
| SEQ ID NO:12 | SEQ ID NO:43、SEQ ID NO:45 |
| SEQ ID NO:13 | SEQ ID NO:44、SEQ ID NO:45 |
| SEQ ID NO:14 | SEQ ID NO:43、SEQ ID NO:45 |
| SEQ ID NO:15 | SEQ ID NO:44、SEQ ID NO:45 |
2, the structure of geraniol Wine brewing yeast strain is produced
Utilize the existing expression cassette pLD2-GPM1t-P of laboratoryGAL7-GPDt (its sequence is as shown in SEQ ID NO:48), will
The GES of 10 kinds of synthetic is connected in expression cassette by BsaI enzyme action and obtains pLD2-GPM1t-PGAL7-GES-GPDt.Utilize
NotI restriction enzyme site, transfers to expression cassette singly copy on plasmid pRS415, converts escherichia coli TransT1, through blue white macula
Obtaining correct clone after screening, bacterium colony PCR checking and sequence verification, its plasmid is pRS415-GPM1t-PGAL7-GES-
GPDt.By lithium acetate Efficient Conversion method by the plasmid obtained respectively proceed to optimize after Wine brewing yeast strain SyBE_
In Sc020201, (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L lack color ammonia to use SC-drop solid panel after conversion
The kilnitamin powder 2g/L of acid, leucine, histidine and uracil, the agar powder of 2%) screen, the conversion obtained
Son carries out bacterium colony PCR checking after carrying out the pure and mild enrichment culture of line point, the recombinant bacterial strain that checking is correct is preserved glycerol stock and divides
Not Ming Ming: strain name with proceed to sequence corresponding relation such as table 4:
Table 4 strain is named and proceeds to GES gene order
| SyBE_Sc020202 | SEQ ID NO:1 |
| SyBE_Sc020204 | SEQ ID NO:32 |
| SyBE_Sc020205 | SEQ ID NO:33 |
| SyBE_Sc020206 | SEQ ID NO:2 |
| SyBE_Sc020207 | SEQ ID NO:3 |
| SyBE_Sc020208 | SEQ ID NO:4 |
| SyBE_Sc020209 | SEQ ID NO:5 |
| SyBE_Sc020210 | SEQ ID NO:6 |
| SyBE_Sc020211 | SEQ ID NO:7 |
| SyBE_Sc020212 | SEQ ID NO:34 |
| SyBE_Sc020215 | SEQ ID NO:8 |
| SyBE_Sc020216 | SEQ ID NO:9 |
| SyBE_Sc020217 | SEQ ID NO:10 |
| SyBE_Sc020218 | SEQ ID NO:11 |
| SyBE_Sc020219 | SEQ ID NO:12 |
| SyBE_Sc020220 | SEQ ID NO:13 |
| SyBE_Sc020221 | SEQ ID NO:14 |
| SyBE_Sc020222 | SEQ ID NO:15 |
The preparation of embodiment 3 geraniol
Test material: the production geraniol Wine brewing yeast strain that embodiment 2 builds
Test method:
Seed culture medium: synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, lacks leucic kilnitamin powder
End 2g/L
Fermentation medium: synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, lacks leucic kilnitamin powder
End 2g/L
From solid streak plate, picking list colony inoculation is in 5ml SC-Leu fluid medium, at 30 DEG C, 250rpm
Under the conditions of cultivate about 20h to thalli growth reach mid-log phase when, with initial OD600=0.2 is inoculated in fresh 5ml
In SC-Leu fluid medium, in 30 DEG C, cultivate about 12h under the conditions of 250rpm to thalli growth to mid-log phase, with initially
OD600It is inoculated in 50ml fermentation medium Sc-Leu, is simultaneously introduced the isopropyl myristate of 20% 30 DEG C, 200rpm condition
Under carry out biphase fermentation, add 10g/L dehydrated alcohol during 20h, after 120h terminate fermentation.
Geraniol extracting method: being poured into by fermentation liquid in 50ml centrifuge tube, 12000rpm is centrifuged 5min, with pipettor by upper
Layer isopropyl myristate is transferred in 2ml centrifuge tube, and the anhydrous sodium sulfate being added thereto to dry in right amount spins upside down the most also
Stand about 10min to carry out, except water, after diluting finally by the 0.22 organic membrane filtration of μm and normal hexane, utilizing GC-MS to enter
Row detection is analyzed.Testing result such as table 5:
Table 5 each strain geraniol yield
Experimental result: because with the presence of glucose, transcribing of GES is suppressed by glucose in initial medium；Along with fermentation
Carrying out, glucose is gradually consumed, fermentation about 19h glucose exhaust, glucose depression effect release, at GAL80
P in the case of knocking outGALPromoter automatically turns on, and GES starts to transcribe and translate, thus gradually accumulates geraniol.Utilize PGALMake
Obtain thalli growth and product produces and is able to separately, thus reduce the adverse effect of product toxicity cell growth, and then make perfume (or spice)
Promoting further of folic alcohol yield is possibly realized.
As shown in Table 4, after not carrying out the Sequence Transformed yeast of truncate, the geraniol yield of SyBE_Sc020209 is the highest,
It is significantly better than other yeast converting non-truncated sequence (p < 0.01).Although SyBE_Sc0202010 and SyBE_Sc020211
But only one of which amino acid whose difference yield but differs close to 4 times.It follows that seek the gene source of optimum, use PGAL
Promoter has positive meaning for efficiently producing geraniol.
After converting the sequence of truncate, the geraniol yield of most of yeast strains is substantially improved, wherein, and SyBE_
The geraniol yield up to 360mg/L of Sc020218 is significantly better than other bacterial strain (p < 0.01).
For the CrGES of three kinds of different truncates, geraniol yield is obviously improved, and will position sequence according to predicting the outcome
Arrange the geraniol yield obtained by complete truncate the highest, be significantly better than the bacterial strain (p < 0.01) of part truncate；And LdGES,
After VoGES1, VoGES2 truncate, geraniol yield is all improved to some extent, but for VoGES1 and VoGES2 according in advance
Survey result by the effect of complete for positioning sequence truncate be not as good as part truncate.
The impact of embodiment 4 biphase fermentation cell growth
1, the structure of control strain
Plasmid pRS415 is passed through lithium acetate Efficient Conversion method the plasmid obtained is proceeded to respectively the saccharomyces cerevisiae after optimizing
In bacterial strain SyBE_Sc020201, after conversion, use SC-Leu solid panel (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/
L, lacks leucic kilnitamin powder 2g/L, the agar powder of 2%) to screen, it is pure that the transformant obtained carries out line point
Carry out bacterium colony PCR checking after cultivation, the recombinant bacterial strain that checking is correct is preserved glycerol stock and is respectively designated as SyBE_
Sc020214。
2, the impact that relatively biphase fermentation and aqueous phase fermentation cell growth and geraniol produce
Test material: SyBE_Sc020209, SyBE_Sc020214
Test method:
Seed culture medium: synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, lacks leucic kilnitamin powder
End 2g/L
Fermentation medium: synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, lacks leucic kilnitamin powder
End 2g/L
From solid streak plate, picking list colony inoculation is in 5ml SD-Leu fluid medium, at 30 DEG C, 250rpm
Under the conditions of cultivate about 20h to thalli growth reach mid-log phase when, with initial OD600=0.2 be inoculated in fresh
In 5mlSC-Leu fluid medium, in 30 DEG C, cultivate about 12h under the conditions of 250rpm to thalli growth to mid-log phase, with just
Beginning OD600Be inoculated in 50ml fermentation medium Sc-Leu, 30 DEG C, ferment under the conditions of 200rpm.For SyBE_
The isopropyl myristate or the n-decane that are separately added into 20% during Sc020209 inoculation carry out biphase fermentation, for SyBE_
The isopropyl myristate adding 20% during Sc020214 inoculation carries out biphase fermentation.Additionally, for SyBE_Sc020209 also simultaneously
Single-phase fermentation has been carried out with aqueous phase.Add 10g/L dehydrated alcohol during 19h, after 120h, terminate fermentation.
Experimental result: as shown in Figure 4, the SyBE_Sc020209 matched group being not added with isopropyl myristate is raw after 19h
Long being substantially less than experimental group, illustrate that about 19h glucose has gradually consumed, geraniol starts accumulation, to cell generation toxicity ten
Four isopropyl propionates can well alleviate the geraniol toxicity to cell；The experimental group cell growth adding n-decane is substantially not so good as
Other organize cell, illustrate that n-decane has bigger toxicity to cell；Add the SyBE_Sc020214 matched group of isopropyl myristate
The SyBE_Sc020209 experimental group growth grown and add isopropyl myristate is basically identical, further illustrates tetradecylic acid isopropyl
Ester can well alleviate the cytotoxicity that geraniol produces.It follows that use isopropyl myristate to carry out as organic facies
Biphase fermentation production efficient for saccharomyces cerevisiae geraniol has positive meaning.
Below it is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
Saying, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
- The most engineered GES gene, its nucleotide sequence is shown in as any one of SEQ ID NO:1～7；Or be SEQ ID NO: Nucleotide sequence shown in 1～7 any one is substituted, lacks or adds the nucleotide sequence that one or more nucleotide obtains.
- Engineered GES gene the most according to claim 1, it is characterised in that its sequence is SEQ ID NO:1～7 The one or more nucleotide of disappearance that the 5 ' of one shown nucleotide sequence are held.
- Engineered GES gene the most according to claim 1, it is characterised in that its sequence such as SEQ ID NO:8～15 Shown in one.
- 4. the application in building the engineering bacteria producing geraniol of the engineered GES gene described in any one of claims 1 to 3.
- 5. an engineering bacteria, it is characterised in that it is expressed described in IDI1 gene, tHMG gene and any one of claims 1 to 3 Engineered GES gene；The sequence of described IDI1 gene is as shown in SEQ ID NO:16；Described tHMG gene order such as SEQ ID Shown in NO:17.
- Engineering bacteria the most according to claim 5, it is characterised in that its initial strain is yeast CEN.PK2-1C.
- 7. the application in producing geraniol of the engineering bacteria described in any one of claim 5～6.
- 8. the construction method of the engineering bacteria described in any one of claim 5～6, it is characterised in that by IDI1 gene, tHMG gene Starter bacteria is entered with the engineered GES gene transformation described in claim 1.
- 9. the preparation method of a geraniol, it is characterised in that fermentation engineering bacteria described in any one of claim 5～6.
- Preparation method the most according to claim 9, it is characterised in that described fermentation is biphase fermentation；Described biphase fermentation Organic facies use isopropyl myristate.
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| CN109777745A (en) * | 2017-11-15 | 2019-05-21 | 中国科学院青岛生物能源与过程研究所 | A kind of genetic engineering bacterium synthesizing sabinene and its construction method and application |
| CN112391327A (en) * | 2019-08-14 | 2021-02-23 | 中国农业科学院烟草研究所 | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof |
| CN114517170A (en) * | 2022-03-18 | 2022-05-20 | 广西优比特生物科技有限公司 | Bacillus subtilis for degrading vomitoxin and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109777745A (en) * | 2017-11-15 | 2019-05-21 | 中国科学院青岛生物能源与过程研究所 | A kind of genetic engineering bacterium synthesizing sabinene and its construction method and application |
| CN109777745B (en) * | 2017-11-15 | 2020-04-24 | 中国科学院青岛生物能源与过程研究所 | Genetic engineering bacterium for synthesizing sabinene and construction method and application thereof |
| CN112391327A (en) * | 2019-08-14 | 2021-02-23 | 中国农业科学院烟草研究所 | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof |
| CN112391327B (en) * | 2019-08-14 | 2022-08-05 | 中国农业科学院烟草研究所 | Engineering bacterium for co-production of geraniol and nerol and construction method and application thereof |
| CN114517170A (en) * | 2022-03-18 | 2022-05-20 | 广西优比特生物科技有限公司 | Bacillus subtilis for degrading vomitoxin and application thereof |
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