Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for producing sclareol

一种香紫苏醇、微生物的技术，应用在生物化学设备和方法、化学仪器和方法、酵素等方向，能够解决未公开香紫苏醇方法等问题

Active Publication Date: 2011-01-05
FIRMENICH SA
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] None of the prior art documents discloses a method for the biosynthetic production of sclareol from the acyclic diterpene precursor GGPP

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for producing sclareol
  • Method for producing sclareol
  • Method for producing sclareol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0188] Isolation of LPP synthase encoding cDNA from Clary sage by PCR method

[0189] A. Plant Material and RNA Extraction

[0190] Clary sage with flower buds (1.5-2 cm long, 1-2 days old) were collected from the field in Bassins, Switzerland, and frozen directly in liquid nitrogen. Concert from Invitrogen (Carlsbad, CA) TM Total RNA was extracted with Plant RNA Reagent, according to the manufacturer's manual, with 2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, CA) was used to purify mRNA by oligod T-cellulose affinity chromatography. Use Marathon TM cDNA Amplification Kit (Clontech, Mountain View, CA) was used to construct cDNA library.

[0191] B. Polymerase chain reaction for amplification of diterpene synthase cDNA

[0192] The amino acid sequences of class I and class II diterpene synthases from different plants were aligned and conserved motifs were selected. Degenerate oligonucleotide sequences were deduced from these conserved amino acid motifs. Designed usi...

Embodiment 2

[0198] Heterologous Expression of LPP Synthase from Clary Sage in Escherichia coli

[0199] pETDuet-1 (Novagen, Madison, WI), designed for expression under the control of the T7 promoter, was used for expression in E. coli cells. For construction of expression plasmids, forward and reverse primers SaTps-Nde (SEQ ID NO: 22) and SaTps designed to introduce an NdeI site immediately before the start codon and a KpnI site after the stop codon were used - Kpn (SEQ ID NO: 23), the open reading frame of SaTps1 (SEQ ID NO: 20) was amplified by PCR from a cDNA library. Since the reading frame contains an NdeI site at position 1614 of the reading frame, this amplification was performed in two steps by overlap extension PCR (Gene 77, 61-68, 1989 of Horton et al.), wherein primer SaTps-Nde( SEQ ID NO: 22) and SaTps-Kpn (SEQ ID NO: 23), together with primers Satps-mutlf (SEQ ID NO: 24) and Satps-mutlr (SEQ ID NO: 25), which were designed to remove the NdeI site and The amino acid sequen...

Embodiment 3

[0206] Purification and Enzyme Activity of LPP Synthase from Clary Sage

[0207] To further identify the recombinant diterpene synthase, we proceeded to purify SsLPPs3 and SsLPPs9 enzymes (SEQ ID NO: 1 and SEQ ID NO: 2).

[0208] The PCR2.1-Topo plasmid containing the SsLPPs3 cDNA and SsLPPs9 cDNA (SEQ ID NO: 4 and SEQ ID NO: 5) (Example 2) was digested with NdeI and SacI, and the insert was ligated into the pET28a(+) plasmid (Novagen) middle. The resulting expression plasmids (pET28-SsLPPs3 and pET28-SsLPPs9) contained cDNAs whose 5' end modifications (SEQ ID NO: 26 and SEQ ID NO: 27) were designed to express proteins with an N-terminal six-histidine tag . Purified under natural conditions using ProBond TM The purification system (Invitrogen) was performed according to the manufacturer's protocol except that, for elution, L-histidine was used instead of imidazole to minimize enzyme inhibition. Using this method, SsLPPs3 and SsLPPs9 recombinases can be purified to appare...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a method of producing sclareol, said method comprising a) contacting a particular polypeptide having a LPP synthase activity with GGPP and b) contacting a particular polypeptide having a sclareol synthase activity with labdenediol diphosphate (LPP) produced in step a). In particular, said method may be carried out in vitro or in vivo to produce sclareol, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of the polypeptides used in the method. Nucleic acids derived from Salvia sclarea and encoding the polypeptides of the invention, expression vectors containing said nucleic acids, as well as a non-human host organism or a cell transformed to harbor the same nucleic acids, are also part of the present invention.

Description

technical field [0001] The present invention provides a method of producing sclareol comprising contacting at least one polypeptide with geranylgeranyl pyrophosphate (GGPP). Specifically, the method can be performed in vitro or in vivo to produce sclareol, a compound that is very useful in the field of fragrances and seasonings. The invention also provides amino acid sequences of polypeptides for use in the methods of the invention. Nucleic acids encoding polypeptides of the invention and expression vectors containing said nucleic acids are also part of the invention. A non-human host organism or cell transformed for use in a method of producing sclareol is also an object of the present invention. Background technique [0002] Terpenes are found in most living organisms (microorganisms, animals and plants). These compounds are built from five-carbon units called isoprenes and are divided by the number of these units present in their structure. Thus, monoterpenes, sesquit...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/62C07D307/92
CPCC07K2319/00C12N9/88C12P7/18C07D311/92C07D307/92C12N9/16C12Y301/07004C12Y401/01033
Inventor M·沙尔克
Owner FIRMENICH SA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com