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System and microorganism for producing taxadiene, and application of system or microorganism

A technology of taxadiene and microorganisms, applied in the field of synthetic biology, to achieve the effect of broad application prospects

Active Publication Date: 2014-02-12
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, some other researchers are working to elucidate the synthetic pathway of paclitaxel, but there are still at least five functional genes that have not been cloned. Among these enzymes, researchers agree that catalyzing geranyl-geranyl Taxadiene synthase (TS), which phosphorylates taxadiene, is the first decisive step in the synthesis of taxadiene

Method used

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  • System and microorganism for producing taxadiene, and application of system or microorganism
  • System and microorganism for producing taxadiene, and application of system or microorganism
  • System and microorganism for producing taxadiene, and application of system or microorganism

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Experimental program
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Embodiment 1

[0047] Embodiment 1 Mevalonate pathway plasmid construction

[0048] Escherichia coli BL21(DE3) genomic DNA and Saccharomyces cerevisiae INVSC1 genomic DNA were purified with Qiagen's Blood and Cell Culture DNA Mini Kit.

[0049] Plasmid pMH1 contains the first three genes of the mevalonate pathway: atoB gene (acetoacetyl-CoA thioesterase) from Escherichia coli BL21 (DE3), erg13 (HMG-CoA synthase, 3- hydroxymethyl-glutaryl-CoA synthase) and thmg1 (HMG-CoA reductase, 3-hydroxymethyl-glutaryl-CoA reductase, deletes the transmembrane region of HMG1). Plasmid pFZ81 contains the last four genes of the mevalonate pathway: erg12 (mevalonate kinase), erg8 (mevalonate-5-phosphate kinase) and mvd1 (mevalonate-5-phosphate kinase) from Saccharomyces cerevisiae INVSC1 pyrophosphate kinase), derived from the idi (isopentenyl pyrophosphate isomerase) gene of Escherichia coli BL21 (DE3). All genes were amplified by PCR, and the primers used are listed in Table 1.

[0050] The specific cons...

Embodiment 2

[0056] Example 2 Taxadiene Production Metabolic Pathway Plasmid Construction

[0057] The codon-optimized geranylgeranyl pyrophosphate synthase ggpps gene derived from Taxus canadensis (sequence shown in SEQ ID NO.1) and the codon-optimized taxadiene derived from Taxus brevifolia were synthesized Enzyme ts gene for gene synthesis (sequence shown in SEQ ID NO.2), followed by primers GS-duet-f, GS-duet-r and TS-duet-f, TS-duet-r amplification, while the vector pETduet-1 was amplified with duet-TG-f, duet-TG-r, and the three fragments were connected together by Gibson method to obtain plasmid pFZ131 ( Figure 5 ). The replicon of plasmid pFZ131 was replaced with pSC01 replicon by simple cloning method, the specific operation is as follows: Plasmid pFZ131 was amplified with primers duet-pSC101-f, duet-pSC101-r, and pSC101-f, pSC101-r were used as primers for pSC101 plasmid The template amplifies the pSC101 replicon, and then connects the two fragments to obtain the plasmid pXC02...

Embodiment 3

[0058] Example 3 Construction of Taxadiene Production Strains

[0059] The two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transformed into Escherichia coli BL21(DE3) to obtain BL21(DE3) / pMH1 / pFZ81, and then pXC02 was transformed into BL21(DE3) / pMH1 / pFZ81 to obtain purple The taxadiene producing strain T2. Transform pFZ131 into BL21(DE3) / pMH1 / pFZ81 to obtain taxadiene producing strain T4.

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Abstract

The invention discloses a system and microorganism for producing taxadiene, and application of the system or the microorganism, and belongs to the field of synthetic biology. The system for producing the taxadiene comprises mevalonate pathway related genes or functional equivalents thereof, and taxadiene synthetized related genes or functional equivalents thereof. The mevalonate pathway related genes are selected from atoB and idi of escherichia coli BL21, and erg13, thmg1, erg12, erg8, and mvd1 of brewer's yeast INVSC1; gene nucleotide sequences of the taxadiene synthetized related genes ggpps are shown by SEQID No.1 and SEQID No.2. The microorganism for producing the taxadiene is the microorganism containing the system for producing the taxadiene, by applying the system into the microorganism, the microorganism for producing the taxadiene is obtained. The microorganism produced in the invention can produce culture for the taxadiene stably, lays a foundation for further using the microorganism to produce paclitaxel, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of synthetic biology and relates to a system for producing taxadiene, microorganisms and applications 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 phase II-III clinical studies, it was found that paclitaxel is mainly suitable for ovarian cancer and breast cancer, and has certain curative effects on lung cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma, and brain tumors. It is currently the most promising drug. And the commercial application of the most mature anticancer drug. There are three main ways to obtain paclitaxel today: (1) the most traditional extraction from natural plant bark, (2) extraction of intermediate products such as baccatin III from plant leaves and other tissues, followed by chemical synthesis t...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N1/15C12N1/19C12N1/21C12P5/00C12R1/07C12R1/19C12R1/465
Inventor 刘天罡朱发银曹小迎张宇琛卞光凯邓子新
Owner WUHAN UNIV
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