CN103981110A - Construction method of gene engineered bacteria for arteannuic acid production - Google Patents
Construction method of gene engineered bacteria for arteannuic acid production Download PDFInfo
- Publication number
- CN103981110A CN103981110A CN201310048422.1A CN201310048422A CN103981110A CN 103981110 A CN103981110 A CN 103981110A CN 201310048422 A CN201310048422 A CN 201310048422A CN 103981110 A CN103981110 A CN 103981110A
- Authority
- CN
- China
- Prior art keywords
- gene
- arteannuinic acid
- relevant
- fragment
- terminator
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a construction method of gene engineered bacteria for arteannuic acid production, and the construction method comprises the following steps: A) construction of a gene expression module including an artemisinic acid production related gene, an upstream promoter of the artemisinic acid production related gene, and a downstream terminator of the artemisinic acid production related gene; and B) co-transformation of the gene expression module into saccharomyces cerevisiae. The method has the advantages of being simple and fast and efficient, avoids multiple cloning, and does not rely on enzyme digestion sites, many fragments can be together transformed, the homologous recombination efficiency is high, and the method can significantly shorten the construction time of the gene engineered bacteria.
Description
Technical field
The invention belongs to microorganism fermentation field, specifically, is the construction process that produces arteannuinic acid genetic engineering bacterium about a kind of.
Background technology
Artemisinin (artemisinin) is that Chinese Scientists is in 20 century 70s from Chinese traditional herbs sweet wormwood or claim the antimalarial effective monomer of separating-purifying Herba Artemisiae annuae (Artemisia annuaL.), its chemical nature is to contain " peroxide bridge " structure (1,2,4-trioxane ring) sesquiterpene lactones.Taking Artemisinin as parent nucleus through the Artemisinin succinate (Artesunate) of artificial semi-synthetic acquisition, Artemtherin (Artemether), the artemisinin-based drugs such as Artemisinin ether (arteether) and dihydroarteannuin, in blood, solvability is good, bioavailability is high, chloroquine resistance malaria and mortality cerebral malaria are had to special efficacy, become " based on the conjoint therapy of Artemisinin " (artemisinin-based combination therapies that the World Health Organization (WHO) advocates, ACTs) first-selected new antimalarial agent, wherein Artesunate, Artemether and Artemether compound have been put into WHO " basic medicine catalogue ".
The source of Artemisinin mainly contains 4 kinds of modes at present:
The first is directly from sweet wormwood, to extract, and this is the main source of current Artemisinin.But the distributional region of sweet wormwood is narrow, the content of Artemisinin in Artemisia annuna is very low, and therefore the Artemisinin of natural origin can not meet growing needs.
The second way is that this mode cost is high by the complete synthesis Artemisinin of producing of chemistry, and difficulty is large, can't put into production at present, substantially under test.
The third mode is to produce Artemisinin by cell cultures, research report, and the content that the sweet wormwood of laboratory culture is sent out Artemisinin in shape root can reach 550mg/L, also has a lot of work to do but will commercially produce.
The 4th kind of mode is to produce Artemisinin by metabolic engineering, builds arteannuinic acid biosynthetic pathway pattern bacterium, utilizes the pattern bacterium of restructuring to produce arteannuinic acid, then taking arteannuinic acid as raw material chemosynthesis Artemisinin.
Through research for many years, the biosynthesizing of Artemisinin is substantially illustrated, and mainly comprises three phases: the first step is acetyl-CoA forming method Thessaloniki tetra-sodium (FPP); Second step is to synthesize sesquiterpene AD by FPP; Final step is by the synthetic arteannuinic acid of AD or dihydroartemisinic acid.And then formation Artemisinin (Fig. 1).
2003, Martin etc. import 9 genes in intestinal bacteria, these 9 genes comprise synthetic MVA(mevalonic acid) encoding gene and ADS(false indigo-4, 11-diene synthase) gene, by rebuild MVA approach and false indigo-4 in intestinal bacteria, 11-diene route of synthesis, make it produce artemisinine key intermediate species false indigo-4, 11-diene (Martin VJ, Pitera DJ, Withers ST, et al.Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Nat Biotechnol, 2003, 21:796-803).
2006, Lindahl etc. in yeast saccharomyces cerevisiae, import containing the plasmid of ADS gene or by ADS gene integration to yeast chromosomal, obtain respectively 600 and 100 μ g/L ADs.This is in eukaryote, to produce for the first time artemisinine body, for utilizing from now on yeast production artemisinine precursor that possibility (Lindahl Ann-Louise is provided, Olsson M E., Merche P, et al, Production of the artemisinin preceursor amorpha-4,11-diene by engineered Saccharomyces cerevisiae, Biotechnology Letter, 2006,28:571-580).
The same year, Ro DK etc., taking yeast saccharomyces cerevisiae as Host Strains, import ADS, CYP71AV1 gene, have rebuild the pathways metabolism of blue or green punt-pole acid, and optimize by culture condition, obtain 115mg/L arteannuinic acid (Ro DK, Paradise EM., Ouellet M et al, Production of the antimalarial drug precursor artemisinic acid in engineerd yeast, Nature, 2006,440:940-943).
2007, patent application CN200710187922.8 disclosed the method structure arteannuinic acid production bacterium that imports HMG-CoA, ADS synthase and FPP synthase and other genes.
According to above-mentioned document, although can construct arteannuinic acid genetic engineering bacterium, while structure, need to successively arteannuinic acid be expressed to relevant gene integration on yeast karyomit(e), relate to multistage clone, and depend on suitable restriction enzyme site and select, complex operation, the cycle is long.
Summary of the invention
In order to overcome deficiency of the prior art, the present invention aims to provide a kind of construction process that produces arteannuinic acid genetic engineering bacterium.
The construction process of product arteannuinic acid genetic engineering bacterium of the present invention, comprises the following steps:
A) build genetic expression module, described genetic expression module comprises that arteannuinic acid produces relevant gene, described arteannuinic acid and produce promotor and the described arteannuinic acid of relevant upstream region of gene and produce the terminator in relevant gene downstream;
B) by described genetic expression module cotransformation to yeast saccharomyces cerevisiae.
According to the present invention, described steps A) described in genetic expression module also comprise the promotor of resistant gene, described resistant gene upstream and the terminator in described resistant gene downstream.
According to the present invention, described steps A) described in build genetic expression module and comprise the following steps:
A1) obtaining described arteannuinic acid by pcr amplification produces relevant gene, described resistant gene fragment, described arteannuinic acid and produces the promoter fragment of relevant upstream region of gene, the promoter fragment of described resistant gene upstream, described arteannuinic acid and produce the terminator fragment in relevant gene downstream and the terminator fragment in described resistant gene downstream;
A2) produce relevant gene, described resistant gene fragment, described arteannuinic acid taking described arteannuinic acid and produce the promoter fragment of relevant upstream region of gene, the promoter fragment of described resistant gene upstream, described arteannuinic acid and produce the terminator fragment in relevant gene downstream and the terminator fragment in described resistant gene downstream as template, obtain genetic expression module by overlapping pcr amplification.
According to a preferred embodiment of the invention, described steps A) described in arteannuinic acid to produce relevant gene be ads, amo and cpr.
According to a preferred embodiment of the invention, described ads, amo and cpr derive from sweet wormwood.
According to a preferred embodiment of the invention, described steps A) described in promotor be selected from ADH1p, TEF1p, TPI1p and PGK1p.
According to a preferred embodiment of the invention, described promotor derives from yeast saccharomyces cerevisiae.
According to a preferred embodiment of the invention, described steps A) described in terminator be selected from TKL1t, ADH1t, ENO2t and TDH2t.
According to a preferred embodiment of the invention, described terminator derives from yeast saccharomyces cerevisiae.
According to a preferred embodiment of the invention, described resistant gene is ble gene.
According to a preferred embodiment of the invention, described resistant gene derives from pGAPZ α A.
Beneficial effect of the present invention: exogenous genetic fragment is proceeded in yeast cell simultaneously, utilize yeast self born of the same parents endogenous substance system one step that each fragment is assembled, and further Homologous integration arrives the chromosome position of design, there is simple and quick efficient advantage, avoided multistage clone, also do not need to rely on restriction enzyme site, multi-disc section transforms jointly, homologous recombination efficiency is high, and 1-2 week can obtain engineering strain, and this good Yeast gene engineering method can obviously shorten engineering bacteria and build the time.The fermentation yield of the arteannuinic acid of the genetic engineering bacterium obtaining according to method of the present invention can reach about 1-1.5g/L, has good prospects for commercial application.
Brief description of the drawings
Fig. 1 is the Artemisinin route of synthesis schematic diagram of sweet wormwood (Artemisiae annuae), and wherein, ADS represents AD synthase, and AMO represents AD P450 monooxygenase, and CPR represents cytochrome P450 reductase.
Fig. 2 is PCR program 1.
Fig. 3 is PCR program 2.
Fig. 4 is Overlap PCR program.
embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting the scope of the invention.
embodiment 1, experimental program and design of primers
The experimental program design of the present embodiment is as follows:
Purchased from EUROSCARF, number Y10000, bacterial strain information: MAT α at yeast saccharomyces cerevisiae BY4742(; His3 △ 1; Leu2 △ 0; Lys2 △ 0; Ura3 △ 0) in express 3 the gene ads(GenBank:DQ241826 of Artemisinin route of synthesis that come from sweet wormwood), amo(GenBank:DQ872632) and cpr(GenBank:DQ984181), through yeast biased codons optimization, and express ble gene (Zeocin resistant gene) as selection markers simultaneously, select yeast chromosomal δ site as integration site, the integration assembling sequence of each gene module on karyomit(e) as shown in Figure 1.Each promotor and gene are numbered respectively, as shown in table 1.
Table 1, each promotor and gene numbering
| Promotor numbering | Promotor title | Gene numbering | Gene title |
| A | TEF1p | 1 | ads |
[0038]?
| B | TPI1p | 2 | amo |
| C | PGK1p | 3 | cpr |
Utilize overlapping PCR(overlap PCR) method build following genetic expression module: delta53 '-ADH1p-ble-TKL1t, TEF1p-ads-ADH1t, TPI1p-amo-ENO2t and PGK1p-cpr-TDH2t-delta55 ', the i.e. combination of A1B2C3.
Wherein, approach builds needs the information of little DNA fragmentation of pcr amplification as shown in table 2.
Table 2, little DNA fragmentation
According to the technical requirements of overlapping PCR, use the Vector NTI10.0 design PCR primer sequence above each small segment that is used for increasing, primer sequence is as shown in table 3.
Table 3, primer sequence
embodiment 2, genetic engineering bacterium structure
2.1, the structure of plasmid pLYADS, pLYAMO and pLYCPR
Reference literature (Yang RY; Feng LL; Yang XQ; Yin LL; Xu XL; Zeng QP.Quantitative transcript profiling reveals down-regulation of A sterol pathway relevant gene and overexpression of artemisinin biogenetic genes in transgenic Artemisia annua plants.Planta medica, 2008Oct; 74 (12): 1510-6.Epub2008Sep24.) obtain ads, amo and cpr gene, through yeast biased codons optimization synthetic (SEQ ID No:29-31), be inserted into respectively pMD19-T Simple Vector(purchased from Takara company) in, obtain plasmid pLYADS, pLYAMO and pLYCPR, be transformed into E.coli bacterial strain DH5 α and preserve.
2.2, the extraction of template DNA
The extracting method of yeast saccharomyces cerevisiae BY4742 chromosomal DNA is as follows: the full single bacterium colony of picking is connected to 3mlYPD liquid nutrient medium test tube (20ml shaking flask), 30 DEG C, 220rpm/min, the about 18h of incubated overnight left and right, collect bacterium liquid, use genome extraction agent box (purchased from Axygen company) to extract genome.
The extracting method of plasmid is as follows: the DH5 α bacterium that inoculation contains corresponding plasmid is in 3ml LB liquid nutrient medium (20ml shaking flask), wherein containing penbritin final concentration is 100 μ g/ml, 37 DEG C, 220rpm/min, the about 15h of incubated overnight left and right, is used plasmid to bring up again test kit (purchased from Axygen company) extracting plasmid pGAPZ α A(purchased from Invitrogen company), pLYADS, pLYAMO and pLYCPR.
2.3, the pcr amplification of little DNA fragmentation
Each little DNA fragmentation amplification is all used high-fidelity KOD-Neo-Plus enzyme (spinning company purchased from TOYOBO Japan), according to 50 μ l KOD-PCR system preparation pcr amplification reaction liquid (table 4).
The pcr amplification reaction liquid preparation system of table 4, little DNA fragmentation
PCR program 1 as shown in Figure 2.
Above-mentioned PCR program 1 is for the δ 53 ' that increases, δ 55 ', TEF1p, TPI1p, PGK1p, TKL1t, ADH1t, ENO2t, TDH2t, ble fragment.
PCR program 2 as shown in Figure 3.
Above-mentioned PCR program 2 is for amplifying ADH 1p, ads, amo, cpr fragment.
Use the agarose gel electrophoresis of 1% concentration to separate PCR product, rubber tapping, is used gel to reclaim test kit (purchased from Axygen company) purifying and reclaims fragment, and makes the heavy molten concentration of each fragment keep quite, saving backup.
The structure of 2.4, genetic expression module
Taking the each little DNA fragmentation that obtains in 2.3 as template, adopt Overlap PCR to build each genetic expression module delta53 '-ADH1p-ble-TKL1t, TEF1p-ads-ADH1t, TPI1p-amo-ENO2t, PGK1p-cpr-TDH2t-delta55 '.Use high-fidelity KOD-Neo-Plus enzyme (spinning company purchased from TOYOBO Japan), according to 50 μ l KOD-PCR system preparation pcr amplification reaction liquid (table 5).
The pcr amplification reaction liquid preparation system of table 5, genetic expression module
Overlap PCR program as shown in Figure 4.
Overlap PCR product is used to the agarose gel electrophoresis of 1% concentration, rubber tapping, is used gel to reclaim test kit (purchased from Axygen company) purifying and reclaims fragment, saves backup and transforms.
2.5, genetic expression module cotransformation yeast saccharomyces cerevisiae BY4742
Adopt lithium acetate conversion method by genetic expression module delta53 '-ADH1p-ble-TKL1t, TEF1p-ads-ADH1t, TPI1p-amo-ENO2t, PGK1p-cpr-TDH2t-delta55 ' cotransformation yeast saccharomyces cerevisiae BY4742.
Working method is as follows: picking list bacterium colony connects YPD test tube, in 30 DEG C, and 230rpm/min overnight incubation, the fresh 25ml YPD substratum (250ml shaking flask) of transferring, 30 DEG C, 230rpm/min shaking culture, treats bacterium liquid OD
600reach at 0.8～1.0 o'clock, 4 DEG C, 4000rpm, the centrifugal collection thalline of 5min, outwells supernatant, washs respectively thalline with ice bath sterilized water and 100mM LiAc, then uses the resuspended thalline of 100mM LiAc to cell concn approximately 2 × 10
9individual/mL, makes transformed competence colibacillus cell.Get 50 μ l competent cells, 4 DEG C, 13000rpm, 30s is centrifugal, and collection thalline is abandoned supernatant, adds successively the PEG6000 of 240 μ l50%, the LiAc of 36 μ l1M, 50 μ l strand milt DNA(are by the milt DNA boiling water boiling 5min depositing in advance, single stranded), the aseptic ddH of 24 μ l
2o and the plasmid DNA that is concentrated to 10 μ l left and right, vortex mixes, then 30 DEG C of water-bath 30min, 42 DEG C of water-bath 25min, 13000rpm, the centrifugal collection thalline of 30s, with rifle head sucking-off supernatant, add 1mlYPD, resuspended thalline, in 30 DEG C, more than 230rpm recovery 6h, 13000rpm after recovery, 30s is centrifugal, the resuspended thalline of 100 μ l sterilized water, it is that the microbiotic Zeocin(of 200 μ g/ml is purchased from Invitrogen company that coating contains final concentration) YPD solid medium, cultivate 3～4 days in 30 DEG C.
2.6, qualification
Selected clone is inoculated in 3ml YPD liquid nutrient medium test tube, 30 DEG C, 220rpm/min, the about 18h of incubated overnight left and right, gets bacterium liquid, uses genome extraction agent box (purchased from Axygen company) to extract genome, verify by PCR, obtain genotype positive colony.Above-mentioned construction process can repeat, and this is apparent for the person of ordinary skill of the art.
embodiment 3, the fermentation of genetic engineering bacterium and the detection of arteannuinic acid
3.1, fermentation
Seed culture method is as follows:
Single bacterium colony of the positive colony that screening obtains from culture plate picking embodiment 2, access 25ml YPD substratum (250ml shaking flask), 30 DEG C, 220rpm/min, cultivates 48h.
Fermentation culture method is as follows:
By seed liquor, according in 4% inoculative proportion access 25ml YPD substratum (250ml shaking flask), 30 DEG C, 220rpm/min, cultivates 120h.
3.2, the extraction of arteannuinic acid
The extracting method of arteannuinic acid is as follows:
Get appropriate fermented liquid, adjust pH to 8.5-9.0, be diluted to the multiple (as 50 times) needing with methyl alcohol, mixed solution is proceeded to centrifuge tube, 4000r/min, centrifugal 30min, collects supernatant liquor.
3.3, the detection of arteannuinic acid
Reference literature method (Zhang Dong etc., HPLC-UV-ELSD method is measured the content of Artemisinin in Artemisia annuna, Qinghaosu II and arteannuinic acid simultaneously. Acta Pharmaceutica Sinica, 2007,42 (9): 978-981) and, arteannuinic acid is carried out to HPLC detection.With arteannuinic acid standard substance in contrast, detected result shows that the genetic engineering bacterium that builds by above step can fermentative production arteannuinic acid, and through 10 batches of fermentations, the output of arteannuinic acid on average can reach 1.2g/L.
The detection of structure, fermentation and the arteannuinic acid of the genetic engineering bacterium of embodiment 4～8, five kind of different promoters and the assortment of genes
Ensure that the combination of resistant gene module is constant, other gene orders are constant, and ensure the promotor difference of each gene, build other five kinds of promotors and gene according to the genetic engineering bacterium of different order permutation and combination according to the method described in embodiment 1～2, wherein, five kinds of promotors and the assortment of genes put in order as follows: A1C2B3, B1A2C3, B1C2A3, C1A2B3, C1B2A3.
The output of the genetic engineering bacterium of above-mentioned five kinds of combinations being fermented, extracts and measure arteannuinic acid according to the method for embodiment 3, result is as shown in table 6.
The output (5 batches of mean value) of the arteannuinic acid of the genetic engineering bacterium that table 6, A1C2B3, B1A2C3, B1C2A3, C1A2B3, five kinds of combinations of C1B2A3 build
| Embodiment | Combination | The output (g/L) of arteannuinic acid |
| 4 | A1C2B3 | 1.23 |
| 5 | B1A2C3 | 1.16 |
| 6 | B1C2A3 | 1.42 |
| 7 | C1A2B3 | 1.03 |
| 8 | C1B2A2 | 1.52 |
Claims (11)
1. a construction process that produces arteannuinic acid genetic engineering bacterium, is characterized in that, comprises the following steps:
A) build genetic expression module, described genetic expression module comprises that arteannuinic acid produces relevant gene, described arteannuinic acid and produce promotor and the described arteannuinic acid of relevant upstream region of gene and produce the terminator in relevant gene downstream;
B) by described genetic expression module cotransformation to yeast saccharomyces cerevisiae.
2. the method for claim 1, is characterized in that, described steps A) described in genetic expression module also comprise the promotor of resistant gene, described resistant gene upstream and the terminator in described resistant gene downstream.
3. method as claimed in claim 1 or 2, is characterized in that, described steps A) described in build genetic expression module and comprise the following steps:
A1) obtaining described arteannuinic acid by pcr amplification produces relevant gene, described resistant gene fragment, described arteannuinic acid and produces the promoter fragment of relevant upstream region of gene, the promoter fragment of described resistant gene upstream, described arteannuinic acid and produce the terminator fragment in relevant gene downstream and the terminator fragment in described resistant gene downstream;
A2) produce relevant gene, described resistant gene fragment, described arteannuinic acid taking described arteannuinic acid and produce the promoter fragment of relevant upstream region of gene, the promoter fragment of described resistant gene upstream, described arteannuinic acid and produce the terminator fragment in relevant gene downstream and the terminator fragment in described resistant gene downstream as template, obtain genetic expression module by overlapping pcr amplification.
4. the method as described in claim 1 or 3, is characterized in that, described steps A) described in arteannuinic acid to produce relevant gene be ads, amo and cpr.
5. method as claimed in claim 4, is characterized in that, described ads, amo and cpr derive from sweet wormwood.
6. the method as described in claim 1 or 2 or 3, is characterized in that described steps A) described in promotor be selected from ADH1p, TEF1p, TPI1p and PGK1p.
7. method as claimed in claim 6, is characterized in that, described promotor derives from yeast saccharomyces cerevisiae.
8. the method as described in claim 1 or 2 or 3, is characterized in that described steps A) described in terminator be selected from TKL1t, ADH1t, ENO2t and TDH2t.
9. method as claimed in claim 8, is characterized in that, described terminator derives from yeast saccharomyces cerevisiae.
10. method as claimed in claim 2 or claim 3, is characterized in that, described resistant gene is ble gene.
11. methods as claimed in claim 10, is characterized in that, described resistant gene derives from pGAPZ α A.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310048422.1A CN103981110A (en) | 2013-02-07 | 2013-02-07 | Construction method of gene engineered bacteria for arteannuic acid production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310048422.1A CN103981110A (en) | 2013-02-07 | 2013-02-07 | Construction method of gene engineered bacteria for arteannuic acid production |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103981110A true CN103981110A (en) | 2014-08-13 |
Family
ID=51273280
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310048422.1A Pending CN103981110A (en) | 2013-02-07 | 2013-02-07 | Construction method of gene engineered bacteria for arteannuic acid production |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103981110A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316372A (en) * | 2014-08-04 | 2016-02-10 | 重庆乾泰生物医药有限公司 | Method for producing artemisinic acid through fermentation |
| CN106367361A (en) * | 2016-10-08 | 2017-02-01 | 天津大学 | Saccharomyces cerevisiae engineering strain as well as construction method and application thereof |
-
2013
- 2013-02-07 CN CN201310048422.1A patent/CN103981110A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| DAE-KYUN RO，ET AL: "Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid", 《BMC BIOTECHNOLOGY》 * |
| JACOB R. LENIHAN,ET AL: "Developing an Industrial Artemisinic Acid Fermentation Process to Support the Cost-Effective Production of Antimalarial Artemisinin-Based Combination Therapies", 《BIOTECHNOL. PROG.》 * |
| PATRICK J. WESTFALLA,ET AL: "Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin", 《PNAS》 * |
| RENE´ VERWAAL，ET AL: "High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316372A (en) * | 2014-08-04 | 2016-02-10 | 重庆乾泰生物医药有限公司 | Method for producing artemisinic acid through fermentation |
| CN105316372B (en) * | 2014-08-04 | 2019-02-01 | 重庆乾泰生物医药有限公司 | A kind of method of fermenting and producing Arteannuic acid |
| CN106367361A (en) * | 2016-10-08 | 2017-02-01 | 天津大学 | Saccharomyces cerevisiae engineering strain as well as construction method and application thereof |
| CN106367361B (en) * | 2016-10-08 | 2019-08-06 | 天津大学 | A kind of saccharomyces cerevisiae engineered yeast strain and its construction method, application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103509726B (en) | Method for producing beta-amyrin with saccharomyces cerevisiae engineering bacterium | |
| CN103416223B (en) | Method for improving cordycepin output in cordyceps militaris fermentation broth | |
| CN102199556B (en) | Saccharomyces cerevisiae genetic engineering bacteria with high ester yield and construction method thereof | |
| CN106635853A (en) | Recombinant saccharomyces cerevisiae capable of producing glycyrrhetinic acid, and construction method and applications thereof | |
| CN108395997A (en) | A kind of Yeast engineering bacteria of high yield Diterpenoids from bulbus | |
| CN104419701A (en) | Rapid assembling method of multi-fragment DNA yeast | |
| CN101173308A (en) | Method for ferment for producing adenomethionine with genetic engineering bacterium | |
| CN107746815A (en) | Produce recombinant Saccharomyces cerevisiae bacterium and its construction method of 13R manoyl oxides | |
| CN104017738A (en) | High-yield engineering strain kmust-SQS for ganoderic acid | |
| CN106318966B (en) | A method of 3-O- glucosyl group oleanolic acid and cellobiose oleanolic acid are synthesized using saccharomyces cerevisiae | |
| CN103865817A (en) | Construction method of genetically engineered bacterium for producing beta-carotene | |
| CN105368732A (en) | Industrial saccharomyces cerevisiae strain capable of producing xylitol and construction method of industrial saccharomyces cerevisiae strain | |
| CN108546722A (en) | A method of utilizing Radix Salviae Miltiorrhizae functional gene from the beginning biosynthesis Rosmarinic acid | |
| CN105567578B (en) | A kind of ganoderic acid high-yielding engineering bacterial strain kmust-SE | |
| CN103981110A (en) | Construction method of gene engineered bacteria for arteannuic acid production | |
| CN105296367B (en) | A kind of ganoderic acid high-yielding engineering bacterial strain kmust-VGB-1 | |
| CN109929828A (en) | A kind of Celastrus angulatus sesquiterpene synthase CaTPS1 and its gene order | |
| CN108823113A (en) | The industrial strain and method of efficient xylose metabolism producing and ethanol | |
| CN103865814B (en) | A kind of ganoderan high-yielding engineering bacterial strain Rmust and construction process thereof | |
| CN106399218A (en) | Bacillus subtilis engineering bacteria and application thereof | |
| CN105296368A (en) | Recombinant mortierella alpina strain for heterologous expression of MpFADS6 gene, construction method of recombinant mortierella alpina strain and application of recombinant mortierella alpina strain in production of EPA | |
| CN107523582A (en) | A kind of engineering bacteria for producing coniferyl alcohol, construction method and the purposes for producing coniferyl alcohol | |
| CN102154137B (en) | Temperature tolerance Saccharomyces cerevisiae and application thereof | |
| CN103146614B (en) | Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof | |
| CN106893726B (en) | Promoter and recombinant yeast strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140813 |
|
| RJ01 | Rejection of invention patent application after publication |