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Fusion protein for producing 5[alpha]-hydroxytaxadiene and application of fusion protein

A technology of taxadiene and fusion protein, applied in the field of synthetic biology and medicine

Active Publication Date: 2021-03-30
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report in the field of successful heterologous synthesis of 5α-hydroxytaxadiene in plant cells

Method used

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  • Fusion protein for producing 5[alpha]-hydroxytaxadiene and application of fusion protein
  • Fusion protein for producing 5[alpha]-hydroxytaxadiene and application of fusion protein
  • Fusion protein for producing 5[alpha]-hydroxytaxadiene and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Embodiment 1: Fusion protein tp(TS) / trT5H / trCPR sequence design

[0118]The taxadiene synthase (TS) sequence length from Taxus brevifolia is 862aa (Genbank access no.U48796.1), and the specific sequence is as follows (SEQ ID NO:3), where the underline is the signal fragment :

[0119] MAQLSFNAALKMNALGNKAIHDPTNCRAKSEGQMMWVCSKSGRTRVKMSRGSGGPGPVVM MSSSTGTSKVVSETSSTIVDDIPRLSANYHGDLWHHNVIQTLETPFRESSTYQERADELVVKIKDMFNALGDGDISPSAYDTAWVARVATISSDGSEKPRFPQALNWVLNNQLQDGSWGIESHFSLCDRLLNTINSVIALSVWKTGHSQVEQGTEFIAENLRLLNEEDELSPDFEIIFPALLQKAKSLGINLPYDLPFIKYLSTTREARLTDVSAAADNIPANMLNALEGLEEVIDWKKIMRFQSKDGSFLSSPASTACVLMNTGDEKCFTFLNNLLDKFGGCVPCMYSIDLLERLSLVDNIEHLGIGRHFKQEIKVALDYVYRHWSERGIGWGRDSLVPDLNTTALGLRTLRTHGYDVSSDVLNNFKDENGRFFSSAGQTHVELRSVVNLFRASDLAFPDEGAMDDARKFAEPYLRDALATKISTNTKLFKEIEYVVEYPWHMSIPRLEARSYIDSYDDDYVWQRKTLYRMPSLSNSKCLELAKLDFNIVQSLHQEELKLLTRWWKESGMADINFTRHRVAEVYFSSATFEPEYSATRIAFTKIGCLQVLFDDMADIFATLDELKSFTEGVKRWDTSLLHEIPECMQTCFKVWFKLMEEVNNDVVKVQGRDMLAHIRKPWELYFNCYVQEREWLD...

Embodiment 2

[0132] Embodiment 2: Construction of the recombinant plasmid of TS, T5H, CPR, tp(TS) / trT5H / trCPR

[0133]The total RNA of the needle leaves of Taxus brevifolia was extracted, and the cDNA of Taxus was obtained by Takara reverse transcription kit. Using cDNA as a template and using pEAQ-TS-F / R, pEAQ-T5H-F / R, pEAQ-CPR-F / R as primer pairs respectively, TS, T5H and CPR sequences were obtained by PCR cloning and blunt-ended Cloning method Clone into pEASY-Blunt vector, construct pEASY-TS, pEASY-T5H, pEASY-CPR respectively. The PCR amplification system is 50 μL (PrimeSTAR Max Premix, 25 μL; the final concentration of double primers is 0.2-0.3 μM; 1.0 μL from DNA; the remaining volume is made up with sterilized distilled water); PCR reaction conditions: pre-denaturation at 98°C for 2 minutes, then denaturation at 98°C 10s, annealing at 55°C for 15s, extension at 72°C for 20s, 35 cycles, detected by agarose electrophoresis, fragments of about 2.5kb, 1.5kb, and 2.1kb were obtained aft...

Embodiment 3

[0141] Example 3: Localization of TS, T5H and tp(TS) / trT5H / trCPR in plant cells

[0142] With tp(TS), TS and GFP construct fusion protein vector pEAQ-TS / GFP and pEAQ-tp(TS) / GFP; tp(T5H), T5H and GFP construct fusion protein vector pEAQ-T5H / GFP and pEAQ-tp( T5H) / GFP; tp(TS) / trT5H / trCPR and CFP were used to construct fusion protein vector pEAQ-tp(TS) / trT5H / trCPR / CFP. The specific construction process is as follows:

[0143] Using pEAQ-TS as a template, pEAQ-TS as a forward primer, and tp(TS)-GFP-R as a reverse primer and perform PCR amplification to obtain the N-terminal chloroplast localization peptide base sequence tp(TS) of TS, The size is about 180bp. Using pEASY-GFP as a template, tp(TS)-GFP-F as a forward primer, and pEAQ-GFP as a reverse primer, PCR amplification was performed to obtain a DNA fragment GFP expressing green fluorescent protein, with a size of about 750 bp. Further, the DNA fragments tp(TS) and GFP obtained by the above two amplifications were used as tem...

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Abstract

The invention relates to a preparation method of a paclitaxel key intermediate 5[alpha]-hydroxytaxadiene. According to the present invention, the gene in the paclitaxel partial synthesis pathway is introduced into the chloroplast of the plant tobacco through the genetic engineering method so as to carry out engineering modification, such that the tobacco can produce the 5[alpha]-hydroxytaxadiene.

Description

technical field [0001] The invention relates to the technical fields of synthetic biology and medicine, in particular, the invention relates to a protein for producing 5α-hydroxytaxadiene and its application. Background technique [0002] Paclitaxel is a diterpene alkaloid compound derived from the yew plant. It is the best natural anticancer drug discovered so far. It has been widely used clinically in the treatment of breast cancer, ovarian cancer, some head and neck cancers, and lung cancer. Like most plant-derived natural products, paclitaxel is only distributed in Taxus plants, and its content is extremely low, so its source is greatly limited. The synthesis of paclitaxel has been successfully realized through the method of total chemical synthesis, but many reaction steps and low conversion rate make this method unfeasible. Although it is feasible to obtain paclitaxel by taxus endophyte and taxus plant cell culture, the yield is extremely low. At present, the importa...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N9/88C07K19/00C12N15/62C12P7/02C12P17/02
CPCC12N9/88C12N9/0083C12N9/0042C12P7/02C12P17/02C12Y106/02004C12Y402/03017C12Y114/99037C07K2319/02
Inventor 王勇李建华王凯博
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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