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Sesquiterpene synthases and methods of use

A sesquiterpene and synthase technology, applied in the fields of sesquiterpene synthase and its preparation and use, can solve the problems of unacceptable chemical synthesis cost and the like

Active Publication Date: 2005-11-30
FIRMENICH SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity of the structure of some sesquiterpenes, the cost of their chemical synthesis is often unacceptable

Method used

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  • Sesquiterpene synthases and methods of use
  • Sesquiterpene synthases and methods of use
  • Sesquiterpene synthases and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Isolation of sesquiterpene synthase cDNA by RT-PCR.

[0089] The deduced amino acid sequences of plant sesquiterpene synthases were aligned, conserved regions were identified and plant sesquiterpene synthase-specific oligonucleotides were designed. In order to obtain high sequence homology, the sequences were divided into two groups (Fig. 4). The first group included the sequences of the following enzymes: gimaene C synthase from tomato (Lycopersicon esculentum) cv.VFNT cherry (Colby et al, 1998), from Mentha x piperita (Crock et al, 1997 ), (E)-β-farnesene synthase from North American fir (Steele et al, 1998), and sesquiterpene synthesis from orange (GenBank accession number AF288465) Enzyme, 5-epi-aristolocene synthase from tobacco (Facchini and Chappell, 1992) and pepper (Back et al, 1998), vetispiradiene synthase from potato and Hyoscyamus muticus (Back and Chappel, 1995) . The second group included the sequences of the following enzymes: (+)-delta-jun...

Embodiment 2

[0095] Example 2: Isolation of sesquiterpene synthase cDNA by 5' / 3'-RACE

[0096] To isolate the full-length sequence of the sesquiterpene synthase, first use the 5′ / 3′-RACE method with Marathon TM cDNA Amplification Kit (Clontech), starting from 1 μg of mRNA purified from total RNA prepared by Hot Borate technology. The quality of the synthetic cDNA is poor, basically all are small cDNA (average size 0.5Kb). However, with this cDNA, the 3' ends of GFTpsB and GFTpsC were obtained by gene-specific primers, GFTpsBRF1 and GFTpsBRF2 were used for GFTpsB, and GFTpsCRF1 and GFTpsCRF2 were used for GFTpsC (see Table 1). The mRNA prepared by the guanidinium thiocyanate / phenol extraction method obtained higher quality cDNA with an average size of 2Kb, so that the 5'-end sequence of GFTpsC could be isolated with gene-specific primers GFTpsCRR1 and GFTpsRR2 (see Table 1), thereby Complete the full-length sequence of this clone. The 5'-terminal of GFTpsB and the 5'-terminal and 3'-ter...

Embodiment 3

[0103] Example 3: cDNA library screening and EST sequencing

[0104] In order to obtain the partial cloned full-length cDNA obtained by PCR method, a cDNA library prepared from mRNA of pomelo peel was constructed.

[0105] Using Uni-ZAP(R) XR Library Construction Kit (Stratagene), cDNA synthesis and library construction were performed starting from 7.5 μg of grapefruit peel mRNA (prepared by guanidine thiocyanate / phenol method) according to the manufacturer's recommended method. The initial titer of the library was 2 × 10 7 PFU (plaque forming unit), the average length of the insert is 1.1Kb.

[0106] The sesquiterpene synthase encoding cDNA was isolated from the cDNA library described above using two methods: EST sequencing and screening with DNA probes. For EST (Expressed Sequence Tag) sequencing, a portion of the library was used to excise the pBluescript phagemid in the Uni-ZAP XR vector using Stratagene's massexcision protocol. The resulting transformed colonies (576) ...

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Abstract

The invention relates to sesquiterpene synthases and methods of their production and use. In one embodiment, the invention provides nucleic acids comprising a nucleotide sequence as described herein that encodes for at least one sesquiterpene synthases. In a further embodiment, the invention also provides for sesquiterpene synthases and methods of making and using these enzymes. For example, sesquiterpene synthases of the invention may be used to convert famesyl-pyrophosphate to various oxygenated and aliphatic sesquiterpenes including valencene, bicyclo-germacrene, cubebol and delta- cadinene.

Description

[0001] This application claims priority to US Provisional Application No. 60 / 415,765, filed October 4, 2002, the contents of which are incorporated herein by reference. This application also claims priority from International Application No. PCT / IB02 / 05070, filed December 2, 2002, the contents of which are incorporated herein by reference. [0002] The present application relates to sesquiterpene synthases and methods of making and using the same. In one embodiment, the invention provides a nucleic acid comprising a nucleotide sequence described herein encoding at least one sesquiterpene synthase. In another embodiment, the present invention also provides sesquiterpene synthase and methods for its preparation and use. For example, the sesquiterpene synthases of the invention can be used to convert farnesyl pyrophosphate to a variety of oxidized and aliphatic sesquiterpenes, including valencene, bicyclo-gemaene, piperene, Solanol and delta-cadinene. Background of the inventio...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N9/88C12N15/00C12N15/52
CPCC12N9/88
Inventor 米歇尔·沙尔克安东尼·克拉克
Owner FIRMENICH SA
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