CN103146729A - Microzyme for producing taxadiene and construction method thereof - Google Patents

Abstract

The invention discloses a microzyme for producing taxadiene and a construction method thereof. The method comprises the following steps: (1) constructing a carrier SyBE_001175; (2) constructing a carrier SyBE_001187; and (3) introducing the carriers SyBE_001175 and SyBE_001187 into Saccharomyces cerevisiae W303-1A or Saccharomyces cerevisiae BY4742 to obtain the microzyme for producing taxadiene. Compared with the prior art, the microzyme for producing taxadiene disclosed in the invention has the advantages of environmental protection and lower cost, and provides a feasible method for producing taxadiene.

Description

Produce yeast and the construction process of Japanese yew diene

Technical field

The present invention relates to a kind of yeast and construction process of producing the Japanese yew diene.

Background technology

Taxol is a kind of effective cancer therapy drug, and traditional preparation method is included as Ramulus et folium taxi cuspidatae is carried out plant extract, but in Ramulus et folium taxi cuspidatae, not only content is extremely low, and to cut down in a large number the Ramulus et folium taxi cuspidatae trees be the environment unfriendly act; Although the method for culture plant cell has solved the problem of extensive felling Ramulus et folium taxi cuspidatae, because of the plant cell growth cycle longer, therefore it is not high to obtain the efficient of taxol yet; Chemical synthesis not only synthesis step is complicated, and each step yield is not high, carry out scale operation and certainly will produce a large amount of polluting wastes if use this method, and cost is expensive.

The fast development that the great demand in the fields such as human health, the energy, environment is also being drawn synthetic biology.Gene element (promotor, transcriptional control zone, ribosome bind site, open reading frame, terminator etc.) is needed according to the through engineering approaches target, and organic reconstruct and coupling together has just formed the functional gene module.By existing bio-networks is used, introduce simultaneously new functional gene module, give expression to the product that n cell can not synthesize or content is extremely low.

A large amount of, fruitful work has been done in U.S. Croteau R professor's laboratory in the biosynthetic research of taxol, through the effort of more than ten years, the most of biosynthetic pathway of taxol is clear and definite.The Japanese yew diene is as the key precursor in the pacilitaxel approach, and the lifting of its output has very great meaning.

Professor Croteau of Washington State University is engaged in the parsing of pacilitaxel approach and Cloning of Genes Related work the earliest, has in succession cloned a plurality of genes of pacilitaxel since the nineties in last century, for the microorganism taxol biosynthesis provides basic substance.Calendar year 2001, Texas peasants and workers' Scott etc. has realized the biosynthesizing of Japanese yew diene first in intestinal bacteria, and output is 1.3mg/l.The Boronat of Barcelona, ESP university in 2004 etc. has realized the synthetic of Japanese yew diene in Arabidopis thaliana, output is 600ng/g DW.Jennewein of German Darmstadt technology university in 2005 etc. has synthesized the Japanese yew diene in yeast, output reaches 8.7mg/L.2006, professor Croteau etc. realized the synthetic of Japanese yew diene-5 α-alcohol at yeast, are about 25 μ g/L.2010, the Stephanopoulos seminar of Massachusetts Institute of Technology (MIT) successfully realized the synthetic of Japanese yew diene in intestinal bacteria, and output is up to 1020 ± 80mg/L.Although the output of Japanese yew diene has reached higher level in intestinal bacteria, but when this seminar carries out follow-up Japanese yew diene-5 α-alcohol synthetic, the seldom product of amount but only detected, this may be difficult to effectively bring into play due to the P450 enzyme its catalysis under colibacillary background.That therefore, carries out Japanese yew diene and follow-up precursor in yeast syntheticly has original advantage and an important meaning.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, a kind of Japanese yew diene synthase gene is provided.

Second purpose of the present invention is to provide a kind of protein of Japanese yew diene synthase gene coding.

The 3rd purpose of the present invention is to provide a kind of saccharomycetic construction process of producing the Japanese yew diene.

The 4th purpose of the present invention is to provide a kind of yeast of producing the Japanese yew diene.

Technical scheme of the present invention is summarized as follows:

A kind of Japanese yew diene synthase gene is the described nucleotide sequence of sequence table SEQ ID NO:11.

The protein of above-mentioned Japanese yew diene synthase gene coding is the described aminoacid sequence of sequence table SEQ ID NO:12.

A kind of saccharomycetic construction process of producing the Japanese yew diene is to comprise the steps:

(1) structure of carrier S yBE_001175:

1. adopt the OE-PCR method to be stitched together promotor, GGPP synthase gene (crtE), terminator, must arrive the fragment that two ends comprise Hind III and Apa I site, connect into episomal vector pRS425;

2. adopt the OE-PCR method to be stitched together the Japanese yew diene synthase gene shown in promotor, sequence table SEQ ID NO:11, terminator, must arrive the fragment that two ends comprise Sac I and Hind III site, connect into the carrier that 1. step obtains, obtain carrier S yBE_001175;

(2) structure of carrier S yBE_001187:

1. yeast complex type promotor, tHMGR gene, terminator are used the OE-PCR method and be stitched together, must arrive the fragment that two ends comprise Xho I and Apa I site, be connected to integrating vector pRS403;

2. yeast complex type promotor, ERG20 gene, terminator are used the OE-PCR method and be stitched together, must arrive the fragment that two ends comprise Sac I and Xho I two sites, be connected to the carrier that 1. step obtains, obtain carrier S yBE_001187;

Described GGPP synthase gene is the described nucleotide sequence of sequence table SEQ ID NO:9; Described tHMGR gene is the described nucleotide sequence of sequence table SEQ ID NO:3; Described ERG20 gene is the described nucleotide sequence of sequence table SEQ ID NO:7;

(3) described carrier S yBE_001175, SyBE_001187 are imported in yeast saccharomyces cerevisiae W303-1A or yeast saccharomyces cerevisiae BY4742, obtain producing the yeast of Japanese yew diene.

The yeast that above-mentioned a kind of saccharomycetic construction process of producing the Japanese yew diene builds.

The yeast of production Japanese yew diene of the present invention is with respect to more environmental protection of prior art, with low cost, for the production of Japanese yew diene provides a kind of feasible method.

Description of drawings

Fig. 1 carrier S yBE_001175(pRS425-TS-crtE) build collection of illustrative plates.

Fig. 2 carrier S yBE_001187 (pRS403-tHMGR-ERG20) builds collection of illustrative plates.

Japanese yew diene route of synthesis in Fig. 3 yeast.

Fig. 4 tunning is measured gas chromatogram.

Japanese yew diene mass spectrum in Fig. 5 tunning.

Fig. 6 Japanese yew diene GC-MS typical curve.

Embodiment

Below by synthetic Japanese yew diene preferred embodiment and illustrate by reference to the accompanying drawings all respects of the present invention and feature.It should be appreciated by those skilled in the art, these embodiment just are used for explanation, and do not limit the scope of the invention.Under the condition that does not deviate from claims scope, those skilled in the art can carry out various modifications and improvement to various aspects of the present invention, and these modifications and improvement also belong to protection scope of the present invention.For example, promotor and the expression vector that uses in embodiment being replaced with other promotors and expression vector commonly used in this area, is that those of ordinary skill in the art can understand and realize.

In addition, unless it should be noted that and specialize, below in embodiment various materials and reagent used be all material and reagent commonly used in this area, can obtain by conventional commercial sources; Method therefor is and well known to a person skilled in the art ordinary method.

Embodiment 1: the acquisition of Japanese yew diene route of synthesis genes involved in yeast

The acquisition of A, tHMGR gene (yeast brachymemma HMG-CoA reductase gene)

According to yeast HMG-CoA reductase gene primers, SEQ ID NO:1tHM-F:5 '-ATGGTTTTAACCAATAAAACAGTCATTTCT-3 ' and SEQ ID NO:2tHM-R:5 '-TTAGGATTTAATGCAGGTGACG-3 ', take W303-1A strain gene group as masterplate, use the pfu enzyme to carry out PCR(95 ℃, 3min; 95 ℃, 30s, 57 ℃, 35s, 72 ℃, 2min, 32cycles; 72 ℃, 5min; 4 ℃ ,+∞) amplification obtains the 1509bp fragment.Be cloned into the pMD18-T carrier, check order, confirm not undergo mutation.Nucleotides sequence is classified as shown in SEQ ID NO:3, aminoacid sequence SEQ ID NO:4.

The acquisition of B, ERG20 gene (yeast FPP and GPP synthase gene)

According to yeast ERG20 gene order design primer, SEQ ID NO:5E20-F:5 '-ATGGCTTCAGAAAAAGAAAT-3 ' and SEQ ID NO:6E20-R:5 '-CTATTTGCTTCTCTTGTAAACTT-3 ', take W303-1A strain gene group as masterplate, use the pfu enzyme to carry out PCR(95 ℃, 3min; 95 ℃, 30s, 50 ℃, 35s, 72 ℃, 75s, 32cycles; 72 ℃, 5min; 4 ℃ ,+∞) amplification obtains the 1059bp fragment, is cloned into the pMD18-T carrier, checks order, and confirms not undergo mutation.Nucleotide sequence SEQ ID NO:7.Aminoacid sequence SEQ ID NO:8.

Acquisition and the optimization of C, GGPP synthase gene (the living Erwinia crtE gene of grass)

By codon optimized, make the codon of the living Erwinia GGPP synthase gene of grass have the yeast Preference, and suitably evade restriction enzyme site commonly used.The majorizing sequence that produces is: SEQ ID NO:9, corresponding aminoacid sequence is SEQ ID NO:10.

The acquisition of D, Taxadiene Synthase gene and optimization

By codon optimized, make the codon of Japanese yew diene synthase gene have the yeast Preference, and suitably evade restriction enzyme site commonly used.The majorizing sequence that produces is: SEQ ID NO:11, corresponding aminoacid sequence is SEQ ID NO:12.

Embodiment 2: Vector construction

The HMG-CoA synthase gene of A, carrier S yBE_001187(brachymemma and the expression vector of ERG20 gene) structure

1. yeast complex type promotor TDH3p, tHMGR gene, terminator are used the OE-PCR method and be stitched together, must arrive the fragment that two ends comprise Xho I and Apa I site, be connected to integrating vector pRS403;

2. yeast complex type promotor TDH3p, ERG20 gene, terminator are used the OE-PCR method and be stitched together, must arrive the fragment that two ends comprise Sac I and Xho I two sites, be connected to the carrier that 1. step obtains, obtain carrier S yBE_001187(and see Fig. 2);

B, carrier S yBE_001175(contain optimize after the livings Erwinia GGPP synthase gene of grass and optimize rear Japanese yew diene Synthase Gene Expression carrier) structure:

1. promotor TDH3p(also can be selected GAL1p etc.), GGPP synthase gene crtE, terminator adopt the OE-PCR method to be stitched together, and must arrive the fragment that two ends comprise Hind III and Apa I site, connects into episomal vector pRS425;

2. promotor TDH3p(also can be selected GAL1p etc.), the Japanese yew diene synthase gene shown in sequence table SEQ ID NO:11, terminator adopt the OE-PCR method to be stitched together, must arrive the fragment that two ends comprise Sac I and Hind III site, connect into the carrier that 1. step obtains, obtain carrier S yBE_001175(and see Fig. 1);

The evaluation of D, expression vector

The above-mentioned expression vector that builds is transformed into respectively in intestinal bacteria DH-5 α, and the upgrading grain carries out the evaluation that single, double enzyme is cut and checked order, and connect into the plasmid corresponding position to guarantee the purpose fragment, and base sequence is not undergone mutation.

Embodiment 3: the saccharomycetic acquisition of producing the Japanese yew diene

Adopt the Lithium Acetate method to carry out the yeast conversion of carrier.Wherein integrative plasmid carries out linearizing in advance, and to be integrated into yeast saccharomyces cerevisiae BY4742 genome corresponding site, the sequestered plasmid directly is transformed in yeast saccharomyces cerevisiae BY4742.After transforming, yeast adopts SD-drop solid medium (yeast nitrogen that deaminizes, 6.7g/l; Glucose, 20g/l; Dropout mix, 0.2%; Solid is added 2% agar powder) screen, the transformant that obtains is transferred to and cultivates 36h in liquid nutrient medium, extracts yeast plasmid or genome as template, carries out the PCR checking, to get rid of false-positive interference.Confirm correct positive strain, plate streaking or glycerol stock are preserved.

The MVA approach that utilizes yeast self to exist, key gene HMG-CoA reductase gene (HMGR) and FPP synthase gene (ERG20) that this approach is related to raise, introduce simultaneously GGPP synthase gene crtE and Japanese yew diene synthase gene, obtain the Wine brewing yeast strain (route map is seen Fig. 3) that possesses production Japanese yew two olefinic functionalities.

Embodiment 4: yeast fermentation is cultivated

Verify correct positive transformant, connect bacterium and enter in 3ml SD-drop liquid nutrient medium to cultivate 30h, switching enters 50ml YPD(1% yeast and soaks powder; 2% peptone; 2% glucose).Making initial OD 600 values is 0.05,30 ℃, and 200rpm cultivates 70h.

Also switchable to the SD-Drop(yeast nitrogen that deaminizes, 6.7g/l; Glucose, 20g/l; Dropout mix, 0.2%) ferment in substratum.

Embodiment 5: the qualitative and quantitative of yeast fermentation product

A, product qualitative

Get the 400ul fermented liquid to the 1.5ml centrifuge tube, add the equal-volume normal hexane, ultrasonication 20min mixes vortex 20min.4 ℃ of centrifugal 5min.Get upper strata normal hexane layer, carry out GC-MS and detect.Obtain spectrogram and see Fig. 4 and Fig. 5.

B, tunning quantitatively

With the mark product configuration concentration gradient that separation and purification obtains, use GC drawing standard curve, determine that linearity range is 10mg/L-100mg/L (Fig. 6).The sample that after the function stem fermentation, extraction obtains is measured, and calculates output by the matched curve formula, obtains function stem output 9.7mg/L.

The bacterial strain that obtains carried out repeatedly fermenting experiment, and due to reasons such as bacterial strain self plasmid stabilities, Japanese yew diene output is between 8.6-13.3mg/L.

Carrier S yBE_001175, SyBE_001187 are imported in yeast saccharomyces cerevisiae W303-1A, obtain producing the yeast of Japanese yew diene.Experimental results show that: the Japanese yew diene output of producing is between 6.5-10.7mg/L.

Claims (4)

1. a Japanese yew diene synthase gene, is characterized in that it is the described nucleotide sequence of sequence table SEQ ID NO:11.

2. the protein of a kind of Japanese yew diene synthase gene coding of claim 1, is characterized in that it is the described aminoacid sequence of sequence table SEQ ID NO:12.

3. a saccharomycetic construction process of producing the Japanese yew diene, is characterized in that comprising the steps:

(1) structure of carrier S yBE_001175:

1. adopt the OE-PCR method to be stitched together promotor, GGPP synthase gene, terminator, must arrive the fragment that two ends comprise Hind III and Apa I site, connect into episomal vector pRS425;

2. adopt the OE-PCR method to be stitched together the Japanese yew diene synthase gene shown in promotor, sequence table SEQ ID NO:11, terminator, must arrive the fragment that two ends comprise Sac I and Hind III site, connect into the carrier that 1. step obtains, obtain carrier S yBE_001175;

(2) structure of carrier S yBE_001187:

1. yeast complex type promotor, tHMGR gene, terminator are used the OE-PCR method and be stitched together, must arrive the fragment that two ends comprise Xho I and Apa I site, be connected to integrating vector pRS403;

2. yeast complex type promotor, ERG20 gene, terminator are used the OE-PCR method and be stitched together, must arrive the fragment that two ends comprise Sac I and Xho I two sites, be connected to the carrier that 1. step obtains, obtain carrier S yBE_001187;

Described GGPP synthase gene is the described nucleotide sequence of sequence table SEQ ID NO:9; Described tHMGR gene is the described nucleotide sequence of sequence table SEQ ID NO:3; Described ERG20 gene is the described nucleotide sequence of sequence table SEQ ID NO:7;

(3) described carrier S yBE_001175, SyBE_001187 are imported in yeast saccharomyces cerevisiae W303-1A or yeast saccharomyces cerevisiae BY4742, obtain producing the yeast of Japanese yew diene.

4. claim 3 a kind of produces the yeast that the saccharomycetic construction process of Japanese yew diene builds.

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