Production of manool

Patent metadata

Field	Value
Publication number	US-2019352673-A1
Application number	US-201716472120-A
Country	US
Kind code	A1
Filing date	Dec 18, 2017
Priority date	Dec 22, 2016
Publication date	Nov 21, 2019
Grant date	—

Abstract

Described herein are methods of producing (+)-manool, the methods including: contacting geranylgeranyl diphosphate with a copalyl diphosphate (CPP) synthase to form a (9S, 10S)-copalyl diphosphate and contacting the CPP with a sclareol synthase enzyme to form (+)-manool and derivatives thereof. Also described herein are nucleic acids encoding CPP synthases and sclareol synthases for use in the methods. Further described herein are expression vectors and non-human host organisms and cells including nucleic acids encoding a CPP synthase and a sclareol synthase as described herein.

First claim

1 . A method of producing (+)-manool, the method comprising:
a) contacting geranylgeranyl diphosphate (GGPP) with a copalyl diphosphate (CPP) synthase to form a copalyl diphosphate, wherein the CPP synthase comprises
a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or
b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or
c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and
b) contacting the CPP with a sclareol synthase to form the (+)-manool; and c) optionally isolating the (+)-manool.

2 . The method of claim 1 , wherein the CPP synthase comprises
a) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or b) a polypeptide comprising an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or c) a polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21.

3 . The method of claim 1 , wherein step a) further comprises culturing a non-human host organism or cell capable of producing GGPP and transformed to express at least one polypeptide comprising
a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and having a CPP synthase activity, under conditions conducive to a production of CPP.

4 . The method of claim 1 , wherein the method further comprises, prior to step a), transforming a non-human host organism or cell capable of producing GGPP with
a) at least one nucleic acid encoding a polypeptide comprising
a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or
b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 17 and SEQ ID NO: 18; or
c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and
having a CPP synthase activity, so that said organism or cell expresses said polypeptide having a CPP synthase activity; and
b) at least one nucleic acid encoding a polypeptide having a sclareol synthase activity, so that said organism or cell expresses said polypeptide having a sclareol synthase activity.

5 . The method as recited in claim 4 , wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25.

6 . The method as recited in claim 1 , further comprising processing the (+)-manool to a (+)-manool derivative using a chemical or biochemical synthesis or a combination of both.

7 . The method as recited in claim 6 , wherein the derivative is an alcohol, acetal, aldehyde, acid, ether, ketone, lactone, acetate or an ester.

8 . The method as recited in claim 6 , wherein the derivative is selected from the group consisting of copalol, copalal, manooloxy, Z-11, gamma-ambrol and ambrox.

9 . A method for transforming a host cell or non-human organism, the method comprising transforming a host cell or non-human organism with a nucleic acid encoding a polypeptide having a copalyl diphosphate synthase activity and a nucleic acid encoding a polypeptide having a sclareol synthase activity,
wherein the polypeptide having copalyl diphosphate activity comprises
a) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or
b) an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or
c) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 and SEQ ID NO: 21; and
wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25.

10 . The method as recited in claim 4 , wherein the host cell or non-human organism is a plant, a prokaryote, or a fungus.

11 . The method as recited in claim 4 , wherein the non-human host organism or cell is E. coli or Saccharomyces cerevisiae.

12 . An expression vector comprising
a) a nucleic acid encoding a polypeptide having a CPP synthase activity comprising
a) an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or
b) an amino acid sequence having at least 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or
c) an amino acid sequence having at 98%, 99% or 100% sequence identity SEQ ID NO: 20 or SEQ ID NO: 21; or
b) a nucleic acid encoding a polypeptide having a CPP synthase activity comprising a nucleotide sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to a nucleic acid sequence as set forth in SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

13 . The expression vector of claim 12 further comprising
a) a nucleic acid encoding a polypeptide having a sclareol synthase activity, wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25; or
b) a nucleic acid encoding a polypeptide having a sclareol synthase activity comprising a nucleotide sequence having at least 90%, 95%, 98%,99% or 100% sequence identity to SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 33, or SEQ ID NO: 34.

14 . A non-human host organism or cell comprising
a) the expression vector as recited in claim 12 ; or b) a nucleic acid encoding a polypeptide having a copalyl diphosphate synthase activity and a nucleic acid encoding a polypeptide having a sclareol synthase activity,
wherein the polypeptide having copalyl diphosphate activity comprises
i. an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 15; or
ii. an amino acid sequence having at least 71%, 72%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18; or
iii. an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 21; and

wherein the polypeptide having sclareol synthase activity comprises an amino acid sequence having at least 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 23, or SEQ ID NO: 25; and
wherein at least one

Assignees

• Firmenich & Cie

Inventors

• Schalk Michel
• Daviet Laurent
• ROCCI Letizia
• Solis Escalante Daniel

Classifications

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C12P7/02 Primary

  containing a hydroxy group · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12N15/81

  for yeasts · CPC title