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Taxadiene overexpression vector and application thereof

A technology of over-expression vector and taxadiene, applied in the direction of vector, nucleic acid vector, introduction of foreign genetic material using vector, etc., can solve problems such as need for further research, difficult commercial production, unstable metabolism of detection means, etc., and achieve screening. High efficiency, stable and effective production, high efficiency of transformant screening

Inactive Publication Date: 2018-02-09
WUHAN J1 BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the defects of detection methods and unstable metabolism, it is difficult to be used in commercial production
[0004] Therefore, the acquisition of taxol-producing bacteria with stable metabolism and high yield still needs further study

Method used

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  • Taxadiene overexpression vector and application thereof
  • Taxadiene overexpression vector and application thereof
  • Taxadiene overexpression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Application of different exogenous promoters of embodiment 1 promoter in Alternaria alternaria

[0052] The present invention selects green fluorescent protein (GFP) as a screening marker for promoter strength, and puts it under the control of promoters from different sources to study the ability of promoters from different sources to promote gene expression in TPF6 in vivo. With primers trpC-F-EcoRI, trpC-R-SacI; gpdA-F-EcoRI, gpdA-R-SacI; alcA-F-EcoRI, alcA-R-SacI; oliC-F-EcoRI, oliC-R-SacI from The promoters trpC (SEQ ID NO.1), gpdA (SEQ ID NO.2), alcA (SEQ ID NO.3) and oliC (SEQ ID NO.4) were amplified by PCR in Aspergillus nidulans. The promoter agdA( SEQ ID NO.5) and glaA (SEQ ID NO.6). Then the primers GFP-F-SacI, GFP-R-BstEII; GFP-F-XbaI, GFP-R-BstEII were amplified respectively to obtain gfp fragments with different restriction sites (SEQ ID NO.7). Finally, the promoters trpC, gpdA, alcA, oliC, glaA and agdA were connected with the digested gfp and the plasm...

Embodiment 2

[0055] Example 2. Construction of Taxadiene Overexpression Plasmid in Fungus TPF6

[0056] In order to realize the stable production of taxadiene in TPF6 and provide sufficient precursors for the synthesis of paclitaxel and other intermediate metabolites, the taxadiene synthase TS gene derived from Taxus brevifolia was optimized according to the codon bias of TPF6 Afterwards, gene synthesis (SEQ ID NO.8) was carried out, followed by PCR amplification with primers TS-Aa-F-SacI and TS-Aa-R-BstEII respectively to obtain ts fragments, which were digested by SacI-BstEII and connected to the same Plasmid pGB123 was obtained from plasmid pGB94. In order to obtain the trpC-IDI-Aa-niaD expression element, select primers trpC-F-EcoRI, trpC-IDI-Aa-R; niaD-IDI-Aa-F, niaD-R-SpeI from Aspergillus nidulans FGSC ) were amplified by PCR to obtain the trpC promoter and the niaD (SEQ ID NO.9) terminator. Primers IDI-Aa-trpC-F, IDI-Aa-niaD-R were amplified to obtain the idi gene (SEQ ID NO.10) ...

Embodiment 3

[0057]Example 3. Acquisition of recombinant Alternaria—Agrobacterium-mediated genetic manipulation method of Alternaria

[0058] The plasmid pGB127 constructed in Example 2 for transforming Alternaria was transformed into Agrobacterium tumefaciens EHA105 competent cells by using the method of calcium transfer. Then the transformants were picked and transferred to 8 mL of LB medium containing 50 mg / L kanamycin, and cultured overnight at 28° C. (12-15 h), so that the OD reached 0.8-1.2. Subsequently 5000rpm, 5min collects thalline, and with IM (0.5g / L NH 4 NO 3 , 0.3g / L NaCl, 0.01g / L CaCl 2 2H 2 O, 0.6g / L MgSO 4 ·7H 2 O, 0.136g / L KH 2 PO 4 , 0.5mg / L CuSO 4 ·5H 2 O, 0.5mg / L ZnSO 4 ·7H 2 O, 0.5 mg / L H 3 BO 3 , 0.5mg / L Na 2 MoO 4 2H 2 O, 1.3mg / L Na 2 -EDTA·2H 2 O, 1mg / L FeSO 4 ·7H 2 0, 6.3g / L glycerol, 8.5g / L MES, 2g / L glucose, 0.2mmol / L acetosyringone) culture medium to dilute Agrobacterium to OD600 is 0.5, then cultivate 6h under the same conditions to obtain ...

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Abstract

The invention discloses a taxadiene overexpression vector and an application thereof. The vector comprises at least one genes selected from taxadiene synthase TS gene, idi gene and tHMG1 gene. The overexpression vector provided by the invention, when transformed into a fungus capable of producing taxol, can achieve overexpression of a key gene for the synthesis of GGPP and taxadiene in the fungus,so that the accumulation of the precursor substances GGPP and taxadiene can be achieved in the fungus capable of producing the taxol, and more precursor substances can be provided for the accumulation of subsequent metabolic intermediates to the greatest extent; therefore, the taxol yield of recombinant microorganism containing the overexpression vector of the invention can be effectively improved, and the metabolic stability of fermentation production of the taxol can be effectively improved.

Description

technical field [0001] The invention relates to taxadiene overexpression vector and application thereof. Background technique [0002] In 1963, American scientists first isolated the crude extract of paclitaxel from a kind of Pacific fir bark and wood, and found that it had high activity on mouse tumor cells. Later, through II-III clinical studies, paclitaxel was widely used in the treatment of ovarian cancer, breast cancer and other cancers, and it is now an anticancer drug with the most potential and the most mature commercial application. However, with the year-by-year logging and slow growth, the available yew has become less and less, making the imbalance between supply and demand of yew increasingly serious, making it difficult to achieve sustainable commercial production. Nowadays, paclitaxel is mainly produced by chemical semi-synthesis: by extracting intermediate products such as baccatin III, 10-deacetyl baccatin III, etc. from yew leaves and other tissues, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15C12P17/02C12R1/645
CPCC12N9/0006C12N9/88C12N9/90C12N15/80C12N2800/22C12N2800/60C12P17/02C12Y101/01034C12Y402/03017C12Y503/03002
Inventor 刘天罡卞光凯傅帅苑玉杰
Owner WUHAN J1 BIOTECH
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