CN106893726A

CN106893726A - A kind of promoter and restructuring yeast strains

Info

Publication number: CN106893726A
Application number: CN201710090101.6A
Authority: CN
Inventors: 周晓; 陈艳; 王颖; 肖文海; 姚明东; 李博; 刘宏; 元英进
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2017-02-20
Filing date: 2017-02-20
Publication date: 2017-06-27
Anticipated expiration: 2037-02-20
Also published as: CN106893726B

Abstract

The present invention relates to gene engineering technology field, a kind of promoter and restructuring yeast strains are disclosed.Promoter of the present invention carries out following one or more for the treatment of on the basis of yeast strain ALD6 promoter full length sequences：(1) conserved sequence in one or more snippets ALD6 promoter is knocked out；(2) sequence comprising conserved sequence in one or more snippets ALD6 promoter is knocked out；(3) conserved sequence in one or more snippets ALD6 promoter is mutated；(4) conserved sequence in one or more snippets ALD6 promoter is replaced；(5) sequence comprising conserved sequence in one or more snippets ALD6 promoter is replaced.The present invention is by the further investigation to yeast strain ALD6 promoters, it was found that the situation such as the missing of its conserved sequence, displacement, mutation can improve yield of the terpenoid production with yeast strain, the more excellent recombination engineering of production capacity can be constructed as the optimization means of Yeast engineering bacteria synthesis path.

Description

A kind of promoter and restructuring yeast strains

Technical field

The present invention relates to gene engineering technology field, more particularly to a kind of promoter and recombinant yeast Strain.

Background technology

Terpenoid is the general name of all isoprene copolymers and its derivative, and isoamyl two is included according to its molecule The number of alkene unit can be divided into：Monoterpene, sequiterpene, diterpene, triterpene, tetraterpene.Terpene generally existing in plant kingdom, with important Medical value and economic worth, such as antitumor drug paclitaxel, anti-malaria medicaments qinghaosu, powerful antioxidant carotenoid, Also lycopene, geraniol, sweet wormwood diene etc..Yet with plant resources it is rare, activity substance content is low, chemical synthesis The factor such as difficulty is big, limits extensive use of the terpene in medicine and other fields.

Synthesising biological cell factory is carried out targetedly for host cell (microorganism etc.) and target organism synthesis path Transformation produces direct benefit, can reduce production cost, shorten the production cycle, therefore efficiently be given birth to synthesising biological cell factory Above-claimed cpd is produced, with very wide application prospect.Saccharomyces cerevisiae is the pattern microorganism of generally recognized as safe, its growth cycle Short and easy High Density Cultivation, can eat, bakers' yeast, be the very outstanding host of above-mentioned substance synthesis.

The key scientific problems that current synthesising biological cell factory is present are between host cell and heterologous organisms synthesis path Adaptation.One side heterologous organisms synthesis path metabolic flux in itself determines the yield of target product, it is therefore desirable to optimize different Source path, including optimize expression, the screening of gene source, the supply of intracellular precursor substance of heterologous gene etc.；On the other hand come from The intrinsic metabolism of host cell and regulator control system can also influence the production capacity of target organism synthesis path, and this is accomplished by chassis Cell go deep into the optimization of system.

In saccharomyces cerevisiae, Gene A LD6 (being controlled by ALD6 promoters) encoding glyoxylate dehydrogenase, in activated cell matter Acetaldehyde generates acetic acid.The expression of Gene A LD6 can directly affect the accumulation of acetic acid in cell, but not yet have any gene The report of terpenoid yield can be influenceed.

The content of the invention

In view of this, it is an object of the invention to provide a kind of promoter so that the promoter can improve place ferment Various terpenoid yield of mother strains；

Another object of the present invention is to provide a kind of restructuring yeast strains for including above-mentioned promoter sequence, is made Its yield that can improve various terpenoids.

For achieving the above object, the present invention provides following technical scheme：

A kind of promoter, on the basis of yeast strain ALD6 promoter full length sequences, carries out following one or more of place Reason：

(1) conserved sequence in one or more snippets ALD6 promoter is knocked out；

(2) sequence comprising conserved sequence in one or more snippets ALD6 promoter is knocked out；

(3) conserved sequence in one or more snippets ALD6 promoter is mutated；

(4) conserved sequence in one or more snippets ALD6 promoter is replaced；

(5) sequence comprising conserved sequence in one or more snippets ALD6 promoter is replaced.

In terms of the research of current yeast strain ALD6 promoter functions is limited to Acetic Acid Accumulation, and otherwise function There is not relevant report, the present invention is directed to current present situation, ALD6 promoters are studied from many aspects, finds ALD6 promoters The missing of conserved sequence, displacement, mutation can improve the output increased that terpenoid produces yeast strain.

Preferably, promoter of the present invention is on the basis of yeast strain ALD6 promoter full length sequences, for carry out as Promoter after one of lower item treatment：

(1) sequence comprising conserved sequence in one or more snippets ALD6 promoter is knocked out；

(2) conserved sequence in one section of ALD6 promoter is knocked out；

(3) conserved sequence in one section of ALD6 promoter is mutated；

(4) sequence comprising conserved sequence in one or more snippets ALD6 promoter is knocked out, in one section of ALD6 promoter Conserved sequence be mutated；

(5) sequence comprising conserved sequence in one or more snippets ALD6 promoter is replaced using resistance label.

Preferably, yeast strain of the present invention is saccharomyces cerevisiae, CEN.PK series saccharomyces cerevisiaes are may be selected from, such as made wine Yeast CEN.PK2-1C or saccharomyces cerevisiae CEN.PK2-1D or saccharomyces cerevisiae CEN.PK2, also selected from BY series saccharomyces cerevisiae, such as BY4741 saccharomyces cerevisiaes or BY4742 saccharomyces cerevisiaes or BY4743 saccharomyces cerevisiaes.It is all by the saccharomyces cerevisiae of gene order-checking ALD6 promoters possess identical conserved sequence, and also possess essentially identical non-conserved sequences, as a whole, own The ALD6 promoters of saccharomyces cerevisiae are essentially identical.Wherein, above-mentioned saccharomyces cerevisiae CEN.PK2-1C, the saccharomyces cerevisiae for referring to CEN.PK2-1D, BY4741 saccharomyces cerevisiae three possess identical ALD6 promoter sequences.

UCSC Genome Browser (http in utilization promoter conserved sequence prediction website of the invention://genome- asia.ucsc.edu/cgi-bin/hgBlatHgsid=471419258_3MF1PADILg7Od OMuaIJ15FmRtAai＆ Command=start), to BY4741 saccharomyces cerevisiae ALD6 promoters (sequence such as SEQ ID NO:Shown in 1) evolutionary conservatism is pre- Survey, as a result obtain 9 sections of conserved sequences, as shown in Figure 1 in A promoters (ALD6 total lengths promoter), the conserved sequence is (numbering is for 1-377bp (numbering is IX) in ALD6 promoter full length sequences, 630-654bp (numbering is VIII), 701-787bp VII), 846-900bp (numbering is VI), 916-925bp (numbering is V), 941-1116bp (numbering is IV), 1184-1205bp (numbering is III), 1227-1313bp (numbering is II), nine sections of conserved sequences of 1430-1469bp (numbering is I).

Because 9 sections of conserved sequences are the key factors of solve problem, therefore knock out in the present invention comprising conservative sequence Sequence in being listed in can also realize expected purpose, and the sequence comprising conserved sequence can be conserved sequence upstream, under Trip or upstream and downstream position connect adjacent sequence, can there is non-conserved sequences in these adjacent sequences, it is possibility to have other guarantors Keep sequence, or both to have concurrently, can be non-conserved sequences or the full sequence, or partial order of other conserved sequences Row.

As described above, the sequence comprising conserved sequence has diversified forms, optional in the specific embodiment of the invention From 1-629bp sequences, 630-700bp sequences, 701-845bp sequences, 846-915bp sequences, 916-940bp sequences, 941- 1183bp sequences, 1184-1226bp sequences, 1227-1429bp sequences, 1430-1479bp sequences, 378-654bp sequences, 655- 787bp sequences, 787-900bp sequences, 788-900bp sequences, 901-925bp sequences, 926-1116bp sequences, 1117- 1205bp sequences, 1117-1191bp sequences, 1206-1313bp sequences, 1314-1469bp sequences.

In the knockout treatment to correlated series, the present invention preferably enters according to A promoters order from left to right in Fig. 1 Row gradient is knocked out, i.e., according to knock out IX sequences, knock out whole section of adjacent non-conserved sequences of IX sequence+IX downstreams, knock out IX sequences+ Whole section of adjacent non-conserved sequences of whole section of adjacent non-conserved sequences+VIII sequence of IX downstreams, knockout IX sequence+IX downstreams+ The mode of this progressively superposition sequence of whole section of adjacent non-conserved sequences of VIII sequence+VIII downstreams carries out multi-form knockout, reflects In a fairly large number of reason, the gradient that the present invention does not include all modes as described above all herein knocks out form, But those skilled in the art can realize that other unrequited gradients knock out form under the guiding of foregoing description, and this is also this hair Bright protection domain.

In the mutation treatment to conserved sequence, the present invention is mutated to whole section of conserved sequence, and preferably base replacement Mutation, is illustrated in specific implementation process by taking base conversion mutation as an example, and so-called base transition mutations will pyrimidine base Base C and T is exchanged, purine bases A and G are exchanged.

In the replacement Treatment to correlated series, may be selected not influenceing the sequence of yeast strain terpenoid production function Enter line replacement, in specific implementation process of the present invention, the present invention combines the convenient of actual screening and enters line replacement from resistance label, Such as KanMX resistance labels.

For the ease of understanding the technology of the present invention thinking, the present invention is illustrated in the way of illustrating but not limiting, referring to The B-L promoters of Fig. 1, each promoter has carried out various treatment such as knockout, mutation and displacement on the basis of the A promoters of total length Mode obtains the promoter for meeting condition of the present invention.Wherein, A promoters are ALD6 total length promoters, common 1479bp；B starts Son is the promoter of knockout IX sequences；C promoters are the promoter for knocking out the sequence (1-629bp) comprising IX；D promoters are to strike Except the promoter of the sequence (1-629bp, 630-700bp) comprising IX and VIII；E promoters are to carry out base to whole section of sequence of VI The promoter of transition mutations；F promoters are to knock out sequence (1-629bp, 630-700bp, 701- comprising IX, VIII and VII Promoter 845bp)；G promoters are the promoter for carrying out base transition mutations to whole section of sequence of VI on the basis of F promoters；H Promoter is to knock out the sequence (1-629bp, 630-700bp, 701-845bp, 846-915bp) comprising IX, VIII, VII and VI Promoter；I promoters are to knock out sequence (1-629bp, 630-700bp, 701- comprising IX, VIII, VII, VI and V 845bp, 846-915bp, 916-940bp) promoter；J promoters are to knock out the sequence comprising IX, VIII, VII, VI, V and IV Arrange the promoter of (1-629bp, 630-700bp, 701-845bp, 846-915bp, 916-940bp, 941-1183bp)；K starts Son includes sequence (1-629bp, 630-700bp, 701-845bp, 846- of IX, VIII, VII, VI, V, IV and III to knock out 915bp, 916-940bp, 941-1183bp, 1184-1226bp) promoter；L promoters are to be put using KanMX resistance labels Change sequence (787-900bp, 901-925bp, 926-1116bp, 1117- comprising VI, V, IV, part III and part VII Promoter 1191bp).

It is subjects that the present invention uses above-mentioned C, D, E, F, G, H, K, L promoter, and geraniol, sweet wormwood two are replaced respectively Original ADL6 promoters in the production Wine brewing yeast strain such as alkene, trans-Geranylgeraniol, cryptosterol and lycopene, then Carry out yield comparison with original bacterial strain, as a result show, using promoter of the present invention after, each bacterial strain compared to original bacterial strain in spiceleaf Alcohol, sweet wormwood diene, trans-Geranylgeraniol, cryptosterol and yield of lycopene are improved.

Meanwhile, in order to verify whether conserved sequence is major influence factors, retain on the basis of promoter of the present invention Its adjacent non-conserved sequences is contrasted, and is as a result shown, both not notable differences in terpenoid yield, shows to protect Missing, displacement, the mutation of sequence in ALD6 promoters etc. is kept for major influence factors.

Based on above-mentioned beneficial effect, the present invention provide the promoter build terpenoid production with yeast strain with And the application in terpenoid is produced.Wherein, the terpenoid be geraniol, sweet wormwood diene, trans-Geranylgeraniol, One or more in cryptosterol, lycopene.

Additionally, according to result of the test and application, the invention provides a kind of production terpenoid yeast strain of restructuring, Original ALD6 promoters are replaced using promoter of the present invention.During actual implementation, needs can be in advance constructed The promoter, by the autologous recombination and integration of yeast to genome, it is also possible on the basis of original ALD6 promoters The operation such as directly knocked out, replaced, being mutated to realize.The yeast strain is Wine brewing yeast strain, may be selected from CEN.PK systems Row saccharomyces cerevisiae, such as saccharomyces cerevisiae CEN.PK2-1C or saccharomyces cerevisiae CEN.PK2-1D, also selected from BY series saccharomyces cerevisiae, such as BY4741 saccharomyces cerevisiaes.

From above technical scheme, the present invention has found that it is protected by the further investigation to yeast strain ALD6 promoters Keeping the situations such as missing, displacement, the mutation of sequence can improve yield of the terpenoid production with yeast strain, can be used as yeast The optimization means of engineering bacteria synthesis path, construct the more excellent recombination engineering of production capacity.

Brief description of the drawings

Fig. 1 show ALD6 total lengths promoter and promoter schematic diagram of the present invention；Wherein, Roman number is represented Conserved sequence in ALD6 promoters, English alphabet represents each promoter numbering, and the VI conserved sequences in E and G promoters are carried out Base transition mutations；

Fig. 2 show the Genetic elements ideograph of genetic fragment 1；Wherein, two ends TRP1LHA, TRP1RHA represents ferment respectively Female trp1 sites upstream and downstream homologous sequence；

Fig. 3 show the Genetic elements ideograph of genetic fragment 2；Wherein, two ends LEU2LHA, LEU2RHA represents ferment respectively Female leu2 sites upstream and downstream homologous sequence；

Fig. 4 show the Genetic elements ideograph of genetic fragment 3；Wherein, two ends TRP1LHA, TRP1RHA represents ferment respectively Female trp1 sites upstream and downstream homologous sequence；

Fig. 5 show the Genetic elements ideograph of genetic fragment 4；Wherein, two ends LEU2LHA, LEU2RHA represents ferment respectively Female leu2 sites upstream and downstream homologous sequence；

Fig. 6 show the Genetic elements ideograph of genetic fragment 5；Wherein, two ends TRP1LHA, TRP1RHA represents ferment respectively Female trp1 sites upstream and downstream homologous sequence；

Fig. 7 show the Genetic elements ideograph of genetic fragment 6；Wherein, two ends LEU2LHA, LEU2RHA represents ferment respectively Female leu2 sites upstream and downstream homologous sequence；

Fig. 8 show the geraniol yield column diagram of different recombinant bacterial strains；Wherein, A represents the weight containing total length promoter A Group bacterial strain, C, D, E, F, G, H, K, L represent the recombinant bacterial strain containing reference numeral promoter, and abscissa is geraniol yield, single Position mg/L；

Fig. 9 show the sweet wormwood diene yield column diagram of different recombinant bacterial strains；Wherein, A is represented and is contained total length promoter A's Recombinant bacterial strain, C, D, E, F, G, H, K, L represent the recombinant bacterial strain containing reference numeral promoter, and abscissa is produced for sweet wormwood diene Amount, unit mg/L；

Figure 10 show the trans-Geranylgeraniol yield column diagram of different recombinant bacterial strains；Wherein, A is represented and started containing total length The recombinant bacterial strain of sub- A, C, D, E, F, G, H, K, L represent the recombinant bacterial strain containing reference numeral promoter, and abscissa is geranyl Geraniol yield, unit mg/L；

Figure 11 show the cryptosterol yield column diagram of different recombinant bacterial strains；Wherein, A is represented and is contained total length promoter A Recombinant bacterial strain, C, D, E, F, G, H, K, L represent the recombinant bacterial strain containing reference numeral promoter, and abscissa is that cryptosterol is produced Amount, unit mg/g DCW；

Figure 12 show the yield of lycopene column diagram of different recombinant bacterial strains；Wherein, A is represented and is contained total length promoter A Recombinant bacterial strain, C, D, E, F, G, H, K, L represent the recombinant bacterial strain containing reference numeral promoter, and abscissa is that lycopene is produced Amount, unit mg/g DCW.

Specific embodiment

The invention discloses a kind of promoter and restructuring yeast strains, those skilled in the art can be used for reference in herein Hold, be suitably modified technological parameter realization.In particular, all similar replacements and change are to those skilled in the art For be it will be apparent that they are considered as being included in the present invention.Promoter of the present invention and bacterial strain have passed through preferably Embodiment is described, and related personnel can be not substantially being departed from present invention, spirit and scope to startup described herein Son and bacterial strain are modified or suitably change is realized and apply the technology of the present invention with combining.

In the specific embodiment of the invention, for the ease of the carrying out of shake flask fermentation experiment, present invention employs wine brewing ferment Female CEN.PK2-1C, it is quadruple auxotroph (leucine, tryptophan, histidine, uracil) saccharomyces cerevisiae, beneficial to screening Correct bacterial strain, while in order to ensure that saccharomyces cerevisiae does not consume derivant galactolipin, the present invention knocked out gal1 in saccharomyces cerevisiae, Tri- genes of gal7 and gal10, these above-mentioned changes to yeast are intended merely to facilitate the carrying out of checking test, to final effect The realization of fruit is without influence.

Meanwhile, saccharomyces cerevisiae CEN.PK2-1C do not possess in itself production terpenoid ability, lack part enzyme, it is necessary to Foreign gene element is built to import wherein as production bacterial strain.Wherein, the yeast strain of fermenting and producing geraniol need to be included through ferment Following genetic fragment on female autologous recombination and integration to its genome：

Yeast trp1 site upstreams homologous sequence, GAL1 promoters, geraniol synthetase-coding gene GES, PGK1 terminate The genetic fragment 1 that son, yeast trp1 sites downstream homologous sequences are sequentially spliced, schematic diagram is shown in Fig. 2, and geraniol synzyme is compiled Code gene GES derives from catharanthus roseus (Catharanthusroseus)；

Yeast leu2 site upstreams homologous sequence, LEU2 marks, ACT1 terminators, the HMG-CoA reductase gene of truncation The genetic fragment 2 that tHMGR1, GAL10 promoter, yeast leu2 sites downstream homologous sequences are sequentially spliced, schematic diagram is shown in figure 3。

The yeast strain of fermenting and producing sweet wormwood diene need to be included through on yeast autologous recombination and integration to its genome Following genetic fragment：

Yeast leu2 site upstreams homologous sequence, LEU2 marks, ACT1 terminators, the HMG-CoA reductase gene of truncation The genetic fragment 2 that tHMGR1, GAL10 promoter, yeast leu2 sites downstream homologous sequences are sequentially spliced；

Yeast trp1 site upstreams homologous sequence, GAL1 promoters, sweet wormwood diene synthetase-coding gene ADS, PGK1 end The genetic fragment 3 that only son, yeast trp1 sites downstream homologous sequences are sequentially spliced, schematic diagram is shown in Fig. 4, the synthesis of sweet wormwood diene Enzyme coding gene ADS derives from sweet wormwood (Artemisia annua).

The yeast strain of fermenting and producing trans-Geranylgeraniol need to be included through yeast autologous recombination and integration to its genome On following genetic fragment：

Yeast leu2 site upstreams homologous sequence, LEU2 marks, ACT1 terminators, the HMG-CoA reductase gene of truncation THMGR1, GAL10 promoter, GAL1 promoters, trans-Geranylgeraniol synthetase-coding gene GGPPS, GPM1 terminator, yeast The genetic fragment 4 that leu2 sites downstream homologous sequences are sequentially spliced, schematic diagram is shown in Fig. 5, and trans-Geranylgeraniol synzyme is compiled Code gene GGPPS derives from Taxus x media (Taxus x media).

The yeast strain of fermenting and producing cryptosterol need to be included through on yeast autologous recombination and integration to its genome Following genetic fragment：

Yeast leu2 site upstreams homologous sequence, LEU2 marks, ACT1 terminators, the HMG-CoA reductase gene of truncation The genetic fragment 2 that tHMGR1, GAL10 promoter, yeast leu2 sites downstream homologous sequences are sequentially spliced.

The yeast strain of fermenting and producing lycopene need to be included through on yeast autologous recombination and integration to its genome Following genetic fragment：

Genetic fragment 1 in CN105087406A, is named as genetic fragment 5 in the present invention, and schematic diagram is shown in Fig. 6, wherein The source of gene crtB is pantoea agglomerans (Pantoea agglomerans), is designated as PacrtB, and the source of gene crtI is three spores Cloth Laplace mould (Blakeslea trispora), is designated as Bt crtI：

Genetic fragment 2 in CN105087406A, is named as genetic fragment 6 in the present invention, and schematic diagram is shown in Fig. 7, gene The source of crtE is pantoea agglomerans (Pantoea agglomerans), is designated as PacrtE, such as SEQ ID NO:Shown in 5：

If necessary to the above-mentioned plurality of target product of fermenting and producing simultaneously, the genetic fragment for introducing as required is introduced i.e. one by one Can.The genome with Wine brewing yeast strain BY4741 such as above-mentioned each Genetic elements, homologous sequence designs and synthesizes conjunction as template Suitable primer, is expanded by PCR and obtained, and specifically can refer to the record in patent CN105087406A；Heterologous gene be by Codon optimization and suitably being evaded obtained by artificial synthesized after conventional restriction enzyme site, wherein from catharanthus roseus (Catharanthusroseus) geraniol synthetase-coding gene GES sequences such as SEQ ID NO:Shown in 2, from sweet wormwood The sweet wormwood diene synthetase-coding gene ADS such as SEQ ID NO of (Artemisia annua):It is sub- red from graceful ground shown in 3 The trans-Geranylgeraniol synthetase-coding gene GGPPS such as SEQ ID NO of beans China fir (Taxus x media):Shown in 4.

With reference to embodiment, the present invention is expanded on further.

Embodiment 1：Promoter of the present invention

Corresponding promoter is obtained in such a way on the basis of ALD6 total length promoters：

B promoters are the promoter for knocking out IX sequences；

C promoters are the promoter for knocking out the sequence (1-629bp) comprising IX；

D promoters are the promoter for knocking out the sequence (1-629bp, 630-700bp) comprising IX and VIII；

E promoters are the promoter that base transition mutations are carried out to whole section of sequence of VI；

F promoters are to knock out opening for sequence (1-629bp, 630-700bp, 701-845bp) comprising IX, VIII and VII Mover；

G promoters are the promoter for carrying out base transition mutations to whole section of sequence of VI on the basis of F promoters；

H promoters be knock out comprising IX, VIII, VII and VI sequence (1-629bp, 630-700bp, 701-845bp, Promoter 846-915bp)；

I promoters be knock out comprising IX, VIII, VII, VI and V sequence (1-629bp, 630-700bp, 701-845bp, 846-915bp, 916-940bp) promoter；

J promoters are to knock out sequence (1-629bp, 630-700bp, 701- comprising IX, VIII, VII, VI, V and IV 845bp, 846-915bp, 916-940bp, 941-1183bp) promoter；

K promoters be knock out comprising IX, VIII, VII, VI, V, IV and III sequence (1-629bp, 630-700bp, 701-845bp, 846-915bp, 916-940bp, 941-1183bp, 1184-1226bp) promoter；

L promoters are to replace the sequence comprising VI, V, IV, part III and part VII using KanMX resistances label The promoter of (787-900bp, 901-925bp, 926-1116bp, 1117-1191bp).

Each promoter can carry out fully synthetic acquisition knowing in above-mentioned in its sequence basis, can also be expanded by primer and obtained ALD6 promoters, and processed according to conventional knockout technique, method of replacing and mutation method, obtain correspondence promoter.

Embodiment 2：Shake flask fermentation tests (contrast between total length promoter and promoter of the present invention)

1st, the structure of test strain

Basic bacterial strain：

With reference to the record in patent CN105087406A, gal1, gal7, gal10 in saccharomyces cerevisiae CEN.PK2-1C are knocked out Three genes, structure obtains recombinant Saccharomyces cerevisiae strains A (control strain, corresponding to total length promoter A)；

With reference to the record in patent CN105087406A, gal1, gal7, gal10 in saccharomyces cerevisiae CEN.PK2-1C are knocked out Three genes, C, D, E, F, G, H, K, L promoter in embodiment 1 are recombinated by yeast homologous and replace original ALD6 startups Son, structure obtains recombinant Saccharomyces cerevisiae bacterial strain C, D, E, F, G, H, K and L, corresponding to self-editing number of each self-starting；

Spiceleaf alcohol production bacterial strain：

Integrator gene fragment 1 and genetic fragment 2 on the basis of recombinant Saccharomyces cerevisiae strains A, obtain spiceleaf alcohol production bacterial strain A；

Integrator gene fragment 1 and genetic fragment 2, obtain on the basis of recombinant Saccharomyces cerevisiae bacterial strain C, D, E, F, G, H, K and L Spiceleaf alcohol production bacterial strain C, D, E, F, G, H, K and L；

Sweet wormwood diene produces bacterial strain：

Integrator gene fragment 3 and genetic fragment 2 on the basis of recombinant Saccharomyces cerevisiae strains A, obtain sweet wormwood diene production bacterium Strain A；

Integrator gene fragment 3 and genetic fragment 2, obtain on the basis of recombinant Saccharomyces cerevisiae bacterial strain C, D, E, F, G, H, K and L Sweet wormwood diene production bacterial strain C, D, E, F, G, H, K and L；

Geranylgeranyl alcohol production bacterial strain：

The integrator gene fragment 4 on the basis of recombinant Saccharomyces cerevisiae strains A, obtains geranylgeranyl alcohol production strains A；

The integrator gene fragment 4 on the basis of recombinant Saccharomyces cerevisiae bacterial strain C, D, E, F, G, H, K and L, obtains geranylgeranyl Alcohol production bacterial strain C, D, E, F, G, H, K and L；

Cryptosterol produces bacterial strain：

The integrator gene fragment 2 on the basis of recombinant Saccharomyces cerevisiae strains A, obtains cryptosterol production strains A；

The integrator gene fragment 2 on the basis of recombinant Saccharomyces cerevisiae bacterial strain C, D, E, F, G, H, K and L, obtains cryptosterol life Produce bacterial strain C, D, E, F, G, H, K and L；

Lycopene produces bacterial strain：

Integrator gene fragment 5 and 6 on the basis of recombinant Saccharomyces cerevisiae strains A, obtain lycopene production strains A；

Integrator gene fragment 5 and 6, obtains lycopene on the basis of recombinant Saccharomyces cerevisiae bacterial strain C, D, E, F, G, H, K and L Production bacterial strain C, D, E, F, G, H, K and L；

Above-mentioned corresponding gene fragment is transformed into by each bacterial strain using Li-acetate method respectively, by trp1 or leu2 upstream and downstream There is restructuring with trp1 or leu2 sites on Yeast genome and be incorporated on genome in homologous sequence.SD-TRP is used after conversion Or SD-LEU solid panels (the mixing ammonia of synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, single scarce tryptophan or leucine Base acid powder 2g/L, 2% agar powder) screened, the transformant for obtaining extracts Yeast genome after carrying out line point pure culture Enter performing PCR checking, to verifying that correct recombinant bacterial strain preserves glycerol stock and names respectively.

2nd, the structure of genetic fragment

Genetic fragment 1, genetic fragment 3, genetic fragment 5 with reference to patent CN105087406A embodiments 3 method, using right The Genetic elements answered are prepared；

Genetic fragment 2, genetic fragment 4, genetic fragment 6 with reference to patent CN105087406A embodiments 4 method, using right The Genetic elements answered are prepared；

3rd, fermentation process

Seed culture medium：20g/L glucose, 20g/L peptones, 10g/L yeast extracts；

Fermentation medium：20g/L glucose, 20g/L peptones, 10g/L yeast extracts, 10g/L D- galactolipins.

(note：For geraniol, sweet wormwood diene, geranylgeranyl alcohol production bacterial strain, fermentation medium will also add 20% N-dodecane)

Above-mentioned bacterial strains are inoculated in 5mL seed culture mediums, 14-16h is cultivated in 30 DEG C, 250rpm, it is dense with initial thalline Degree OD₆₀₀=0.2 transfers in fresh 25mL seed culture mediums, is cultivated into logarithmic growth under the conditions of 30 DEG C, 250rpm Phase, with initial cell concentration OD₆₀₀=0.5 is inoculated in 50mL fermentation mediums respectively, is cultivated under the conditions of 30 DEG C, 250rpm, Cell density (OD in monitoring fermentation process₆₀₀) and yield.

4th, yield detection

Geraniol, sweet wormwood diene, trans-Geranylgeraniol：After fermentation 48 hours, taken after zymotic fluid 12000g centrifugations 5min Layer organic phase, GC-MS detections are carried out after being diluted with n-hexane.

Cryptosterol：After fermentation 48 hours, the zymotic fluid of two equal portions, 4000g centrifugation 2min collects thallines are taken, and wash two It is secondary.A copy of it thalline is placed in 80 DEG C of drying to constant weight, calculating dry cell weight of weighing；Another thalline is extracted to product, Specific method is：With 1mL 2N NaOH re-suspended cells, it is placed in and 10min is boiled in boiling water bath, immediately after ice bath 3min；Will be broken Broken cell 12000rpm, 4 DEG C of centrifugation 4min abandon supernatant, the methanol solution for adding 300uL to contain 1.5M NaOH, 60 DEG C of saponification 4h, 300uL n-hexane vortex oscillation 10min are added, organic phase is collected by centrifugation；Water mutually adds 300uL n-hexanes vortex to continue to vibrate again 10min, is finally collected by centrifugation organic phase.After the organic phase vacuum freeze drying that will be collected, 100ul derivatization reagents are added 30 DEG C of incubation 2h of MSTFA, GC-MS detections are carried out after finally being diluted with n-hexane.

Lycopene：After fermentation 48 hours, the zymotic fluid of two equal portions, 4000g centrifugation 2min collects thallines are taken, and wash two It is secondary.A copy of it thalline is placed in 80 DEG C of drying to constant weight, calculating dry cell weight of weighing；Another thalline is extracted to product, Specific method is：With 3N HCl re-suspended cells, it is placed in and 2min is boiled in boiling water bath, immediately after ice bath 3min；It is thin by what is crushed Born of the same parents 12000rpm, 4 DEG C of centrifugation 4min abandon supernatant, and acetone, and the 5min that is vortexed are added after washing 2 times；Acetone phase is finally collected by centrifugation, Detect that lycopene Detection wavelength is 471nm with upper ultraviolet liquid phase after 2 μm of membrane filtrations.

5th, result

Result is shown in Fig. 8-Figure 12, it can be clearly seen that, ALD6 total length promoters, i.e. A promoters are contained in original bacterial strain, its It is relatively low in geraniol, sweet wormwood diene, trans-Geranylgeraniol, cryptosterol, each terpene substances yield of lycopene, and replace with After each promoter of the present invention, each terpene substances yield is improved.

Embodiment 3：Shake flask fermentation experiment (removal conserved sequence and removal the sequence promoter containing the conserved sequence it Between contrast)

In order to verify that conserved sequence is major influence factors, the present invention provides whether following several groups differ only in have non-guarantor Keeping the promoter of sequence, and build recombinant bacterial strain according to the mode in embodiment 2 carries out the contrast of each terpene substances yield, as a result It is shown in Table 1.

1st group：Promoter B and promoter C, difference is promoter B has more the non-of one section of 378-629bp than promoter C Conserved sequence；

2nd group：Promoter I and promoter I+926-940bp non-conserved sequences；

3rd group：Promoter J and promoter J+1117-1183bp non-conserved sequences；

Table 1

As can be seen from Table 1, the volume variance between each group is not obvious, and illustrating to carry out treatment to conserved sequence can be real The raising of existing terpene substances yield, and non-conserved sequences do not make significant difference to this.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

SEQUENCE LISTING

<110>University Of Tianjin

<120>A kind of promoter and restructuring yeast strains

<130> MP1701567

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1479

<212> DNA

<213>Artificial sequence

<400> 1

accaagttcc cgctcgattt gattcatgaa gaaggacaaa agaactattt aatgttcatg 60

aagatgattg aggaagaaaa ggaaaaaatt agaatacagc aggagcaaat gggcgggcaa 120

acatttacgc tgaaagacta tgttgaaggt aacttgcctt cgccagaaga acaaatgaaa 180

atacaattgg agaagcagaa ggaggtagac gccttatttg aagaggaaaa gaaaaagaag 240

aagattgctg aatccaaata attttcatgt aaaccctctt ctcatgtatc tacgtatcta 300

tgtgtgtatg taaatgtacc tgtacactcc ccacaccctc attttgttac tgtcatgtga 360

ataaaactta tgtatattgc taacttacta ccactgcacc tcctaacatc accatactac 420

gtacaaacac gcctatttat tttttctatg ttaaatttta acgatgtaga cacacctaat 480

gatctgatgc gctttgcata tctcatattc cttcactagc ataaaaatcc aaaaaaaaag 540

aatatttagg ccgaatggaa ttattcgtaa cgtcatacga aaaaagtttc aattcgtaca 600

atgcctggca tgttcattcg aatataaggc cgccgccttc cagtcagggt agccaaaagt 660

ataatcccgg gtggaaacta aactaaaaac cgtactcaca actttccgcg gacgctaaca 720

gacaaataga cacactatca ggtcaggaac tgccgtcaca tacgacactg cccctcacgt 780

aagggcatga tagaattgga ttatgtaaaa ggtgaagata ccattgtaga agcaaccagc 840

acgtcgccgt ggctgatgag gtctcctctt gcccgggccg cagaaaagag gggcagtggc 900

ctgtttttcg acataaatga ggggcatggc cagcaccgag acgtcattgt tgcatatggc 960

gtatccaagc cgaaacggcg ctcgcctcat ccccacggga ataaggcagc cgacaaaaga 1020

aaaacgaccg aaaaggaacc agaaagaaaa aagagggtgg gcgcgccgcg gacgtgtaaa 1080

aagatatgca tccagcttct atatcgcttt aactttaccg ttttgggcat cgggaacgta 1140

tgtaacattg atctcctctt gggaacggtg agtgcaacga atgcgatata gcaccgacca 1200

tgtgggcaaa ttcgtaataa attcggggtg agggggattc aagacaagca accttgttag 1260

tcagctcaaa cagcgattta acggttgagt aacacatcaa aacaccgttc gaggtcaagc 1320

ctggcgtgtt taacaagttc ttgatatcat atataaatgt aataagaagt ttggtaatat 1380

tcaattcgaa gtgttcagtc ttttacttct cttgttttat agaagaaaaa acatcaagaa 1440

acatctttaa catacacaaa cacatactat cagaataca 1479

<210> 2

<211> 1146

<212> DNA

<213>Artificial sequence

<400> 2

atggcttact ctgctatggc aactatgggt tataatggta tggctgcatc ttgtcatact 60

ttgcatccaa catcaccatt aaaaccattt catggtgcat ctacatcatt ggaagctttt 120

aatggtgaac atatgggttt gttgagaggt tactctaaga gaaagttgtc ttcatacaag 180

aacccagctt caagatcttc aaatgctact gttgcacaat tgttgaatcc accacaaaag 240

ggtaaaaagg cagttgaatt cgatttcaat aagtacatgg attctaaagc tatgacagtt 300

aatgaagcat tgaataaggc tattccatta agatatccac aaaagatata tgaatctatg 360

agatactcat tgttagctgg tggtaaaaga gttagaccag ttttgtgtat tgctgcatgt 420

gaattagttg gtggtactga agaattggct attccaacag cttgtgcaat cgaaatgatc 480

catactatgt ctttgatgca tgatgatttg ccatgtatcg ataacgatga tttgagaaga 540

ggtaaaccaa caaaccataa gatcttcggt gaagatactg ctgttacagc tggtaatgct 600

ttgcattcat acgcattcga acatatcgct gtttctactt caaaaacagt tggtgcagat 660

agaatcttga gaatggtttc tgaattaggt agagctactg gttcagaagg tgttatgggt 720

ggtcaaatgg ttgatattgc atctgaaggt gacccatcaa tcgatttgca aactttggaa 780

tggatccata tccataagac agctatgttg ttggaatgtt ctgttgtttg tggtgcaatt 840

attggtggtg cttcagaaat cgttatcgaa agagcaagaa gatacgctag atgtgttggt 900

ttgttgttcc aagttgttga tgatatctta gatgttacta agtcttcaga tgaattgggt 960

aaaacagctg gtaaagattt gatctctgat aaggctacat acccaaagtt gatgggttta 1020

gaaaaggcaa aggaattttc tgatgaattg ttgaacagag ctaagggtga attgtcatgt 1080

tttgatccag ttaaagctgc accattgtta ggtttggcag attacgttgc ttttagacaa 1140

aattaa 1146

<210> 3

<211> 1641

<212> DNA

<213>Artificial sequence

<400> 3

atgtctttga ctgaagaaaa gccaatcaga ccaatcgcta acttcccacc atctatctgg 60

ggtgaccaat tcttgatcta cgaaaagcaa gttgaacaag gtgttgaaca aatcgttaac 120

gacttgaaga aggaagttag acaattgttg aaggaagctt tggacatccc aatgaagcac 180

gctaacttgt tgaagttgat cgacgaaatc caaagattgg gtatcccata ccacttcgaa 240

agagaaatcg accacgcttt gcaatgtatc tacgaaactt acggtgacaa ctggaacggt 300

gacagatctt ctttgtggtt cagattgatg agaaagcaag gttactacgt tacttgtgac 360

gttttcaaca actacaagga caagaacggt gctttcaagc aatctttggc taacgacgtt 420

gaaggtttgt tggaattgta cgaagctact tctatgagag ttccaggtga aatcatcttg 480

gaagacgctt tgggtttcac tagatctaga ttgtctatca tgactaagga cgctttctct 540

actaacccag ctttgttcac tgaaatccaa agagctttga agcaaccatt gtggaagaga 600

ttgccaagaa tcgaagctgc tcaatacatc ccattctacc aacaacaaga ctctcacaac 660

aagactttgt tgaagttggc taagttggaa ttcaacttgt tgcaatcttt gcacaaggaa 720

gaattgtctc acgtttgtaa gtggtggaag gctttcgaca tcaagaagaa cgctccatgt 780

ttgagagaca gaatcgttga atgttacttc tggggtttgg gttctggtta cgaaccacaa 840

tactctagag ctagagtttt cttcactaag gctgttgctg ttatcacttt gatcgacgac 900

acttacgacg cttacggtac ttacgaagaa ttgaagatct tcactgaagc tgttgaaaga 960

tggtctatca cttgtttgga cactttgcca gaatacatga agccaatcta caagttgttc 1020

atggacactt acactgaaat ggaagaattc ttggctaagg aaggtagaac tgacttgttc 1080

aactgtggta aggaattcgt taaggaattc gttagaaact tgatggttga agctaagtgg 1140

gctaacgaag gtcacatccc aactactgaa gaacacgacc cagttgttat catcactggt 1200

ggtgctaact tgttgactac tacttgttac ttgggtatgt ctgacatctt cactaaggaa 1260

tctgttgaat gggctgtttc tgctccacca ttgttcagat actctggtat cttgggtaga 1320

agattgaacg acttgatgac tcacaaggct gaacaagaaa gaaagcactc ttcttcttct 1380

ttggaatctt acatgaagga atacaacgtt aacgaagaat acgctcaaac tttgatctac 1440

aaggaagttg aagacgtttg gaaggacatc aacagagaat acttgactac taagaacatc 1500

ccaagaccat tgttgatggc tgttatctac ttgtgtcaat tcttggaagt tcaatacgct 1560

ggtaaggaca acttcactag aatgggtgac gaatacaagc acttgatcaa gtctttgttg 1620

gtttacccaa tgtctatcta a 1641

<210> 4

<211> 1182

<212> DNA

<213>Artificial sequence

<400> 4

atggcttata ccgcaatggc agcaggaact cagtcattgc agttgaggac agtcgcctct 60

taccaggagt gcaactcaat gaggtcttgc ttcaagttga ccccattcaa gtcattccac 120

ggtgtcaact tcaacgttcc ttctttaggt gccgccaact gcgaaatcat gggtcacttg 180

aaattgggtt ctttgccata caaacagtgt tcagtatcat ctaagtcaac taagactatg 240

gcccagttgg tagatttggc agagaccgag aaagccgagg gaaaggatat cgagttcgat 300

tttaacgagt atatgaagtc taaggctgtc gctgttgatg cagccttgga taaggccatc 360

cctttggagt atccagagaa gatccatgag tctatgaggt actcattgtt ggccggagga 420

aaaagggtca gacctgcatt atgcatcgct gcttgcgagt tagtaggtgg ttctcaggac 480

ttggccatgc caaccgcatg tgccatggaa atgattcata ccatgtcatt gattcacgat 540

gatttgcctt gcatggacaa cgacgacttc agaaggggaa agcctaccaa tcacaaggtt 600

ttcggagagg acactgctgt tttagccggt gacgcattgt tatctttcgc ttttgaacac 660

atcgccgttg ccacatcaaa aactgtccca tctgacagga ccttgagagt catttctgag 720

ttgggtaaaa ccatcggttc acagggattg gtcggaggtc aggtagtcga catcacttct 780

gagggagacg ccaacgtcga cttaaagaca ttggagtgga ttcacattca caagactgcc 840

gtcttgttgg aatgctctgt tgtttctgga ggaatcttgg gtggagctac cgaggatgag 900

attgctagaa taagaagata cgccaggtgc gtcggtttgt tgttccaggt tgtcgacgac 960

attttggatg tcaccaagtc ttcagaggaa ttgggaaaga ccgccggtaa agacttattg 1020

accgacaagg ctacctaccc taagttgatg ggtttggaga aggccaaaga gtttgcagca 1080

gaattagcta ccagggcaaa ggaagagttg tcatcattcg accagatcaa ggcagcccct 1140

ttgttaggat tggccgatta catcgctttc aggcaaaact aa 1182

<210> 5

<211> 924

<212> DNA

<213>Artificial sequence

<400> 5

atggtttctg gttctaaggc tggtgtctca ccacacaggg agattgaggt catgaggcag 60

tctattgacg atcacttggc tggtttgttg cctgagactg actctcagga cattgtctca 120

ttggcaatga gggagggtgt catggctcca ggtaagagga taaggccttt gttgatgttg 180

ttggcagcta gggacttgag gtaccagggt tctatgccta ctttgttgga cttggcttgc 240

gctgtcgaat tgactcacac tgcatcattg atgttggacg acatgccttg catggacaac 300

gcagaattaa ggaggggtca gccaacaaca cacaagaagt tcggtgagtc agtcgcaatt 360

ttggcatcag ttggtttgtt atcaaaggct ttcggattga ttgctgcaac tggtgactta 420

ccaggtgaga ggagggcaca ggctgtcaac gagttgtcta ctgctgtcgg agtccaggga 480

ttggtcttgg gtcagttcag ggacttgaac gacgcagctt tggacaggac tccagacgct 540

atattgtcta caaaccactt aaagacagga attttgttct ctgctatgtt gcagatagtc 600

gctattgcat ctgcttcttc tccatctaca agggagactt tgcacgcttt cgcattggac 660

ttcggtcagg ctttccagtt gttggacgac ttgagggacg atcacccaga gacaggaaag 720

gacaggaaca aggatgcagg taaatcaact ttggtcaaca ggttaggtgc agacgctgct 780

aggcagaagt taagggagca cattgactct gctgacaagc acttgacttt cgcttgccca 840

cagggaggtg ctattaggca gttcatgcac ttgtggttcg gtcaccactt agctgactgg 900

tcacctgtca tgaagatagc ttaa 924

Claims

1. a kind of promoter, it is characterised in that on the basis of yeast strain ALD6 promoter full length sequences, carry out such as the next item down or Multinomial treatment：

(1) conserved sequence in one or more snippets ALD6 promoter is knocked out；

(3) conserved sequence in one or more snippets ALD6 promoter is mutated；

(4) conserved sequence in one or more snippets ALD6 promoter is replaced；

2. promoter according to claim 1, it is characterised in that on the basis of yeast strain ALD6 promoter full length sequences, To carry out the promoter after one of following item treatment：

(2) conserved sequence in one section of ALD6 promoter is knocked out；

(3) conserved sequence in one section of ALD6 promoter is mutated；

(4) sequence comprising conserved sequence in one or more snippets ALD6 promoter is knocked out, to the guarantor in one section of ALD6 promoter Sequence is kept to be mutated；

3. promoter according to claim 1 or claim 2, it is characterised in that the conserved sequence is selected from ALD6 promoter total length sequences 1-377bp in row, 630-654bp, 701-787bp, 846-900bp, 916-925bp, 941-1116bp, 1184-1205bp, Nine sections of conserved sequences of 1227-1313bp, 1430-1469bp.

4. promoter according to claim 1 or claim 2, it is characterised in that including in described one or more snippets ALD6 promoter The sequence of conserved sequence is selected from 1-629bp sequences, 630-700bp sequences, 701-845bp sequences, 846-915bp sequences, 916- 940bp sequences, 941-1183bp sequences, 1184-1226bp sequences, 1227-1429bp sequences, 1430-1479bp sequences, 378- 654bp sequences, 655-787bp sequences, 787-900bp sequences, 788-900bp sequences, 901-925bp sequences, 926-1116bp Sequence, 1117-1205bp sequences, 1117-1191bp sequences, 1206-1313bp sequences, 1314-1469bp sequences.

5. promoter according to claim 1 or claim 2, it is characterised in that described to sport base substitution mutation.

6. promoter according to claim 2, it is characterised in that the resistance label is KanMX resistance labels.

7. promoter described in claim 1-6 any one is building terpenoid production with yeast strain and in production terpene Application in class compound.

8. apply according to claim 7, it is characterised in that the terpenoid is geraniol, sweet wormwood diene, geranyl One or more in geraniol, cryptosterol, lycopene.

9. the production terpenoid yeast strain of a kind of restructuring, it is characterised in that using described in claim 1-6 any one Promoter replaces original ALD6 promoters.

10. yeast strain according to claim 9, it is characterised in that the yeast strain is Wine brewing yeast strain.