EP4281570A1 - In-vitro synthetic platform for the generation of isoprenoids - Google Patents

In-vitro synthetic platform for the generation of isoprenoids

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Publication number
EP4281570A1
EP4281570A1 EP22700507.1A EP22700507A EP4281570A1 EP 4281570 A1 EP4281570 A1 EP 4281570A1 EP 22700507 A EP22700507 A EP 22700507A EP 4281570 A1 EP4281570 A1 EP 4281570A1
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EP
European Patent Office
Prior art keywords
kinase
diphosphate
process according
seq
hemiterpene
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EP22700507.1A
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German (de)
French (fr)
Inventor
Michael DIRKMANN
Andreas Vogel
Hanna DUDEK
Sebastian Bartsch
Lena SCHILLING
Rico Czaja
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C Lecta GmbH
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C Lecta GmbH
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Publication of EP4281570A1 publication Critical patent/EP4281570A1/en
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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa

Definitions

  • the invention relates to a process for isoprenoid production by an in-vitro synthetic platform involving at least the steps of phosphorylating a first hemiterpene alcohol with a first phosphate donor under catalysis of a first kinase thereby obtaining a first hemiterpene monophosphate and phosphorylating the latter with a second phosphate donor under catalysis of a second kinase thereby obtaining at least first hemiterpene diphosphate.
  • the invention further relates to a composition comprising at least first hemiterpene diphosphate obtainable by the process according to the invention.
  • isoprenoids including cannabinoids, terpenes and terpenoids, have gained great importance in a variety of industrially relevant applications for the production of pharmaceuticals, cosmetics, perfumes, flavors, animal feed supplements and nutraceuticals.
  • terpenes and terpenoids may be obtained from natural sources i.e. by extraction.
  • natural sources i.e. by extraction.
  • a fundamental problem associated thereto regards the low availability, above all, due to the low amenability of such natural sources to large-scale production.
  • terpenoids are largely derived from two major diphosphorylated building blocks: isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Following their generation, such diphosphorylated building blocks may be subjected to sequential condensation under catalysis of specific prenyltransferases thereby resulting in geranyl pyrophosphate (GPP), famesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) which then may be converted to the more complex terpenoids under catalysis of appropriate terpene synthases. The resulting terpenoids may be then further functionalized either chemically or under enzymatic catalysis.
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • TMP terpene mini -path
  • AP promiscuous acid phosphatase
  • IPK isopentenyl phosphate kinase
  • WO 2011/009132 A2 relates to methods and compositions pertaining to the synthesis of isoprenoid diphosphates by using selected enzymes (i.e. isopentyl monophosphate kinase (IMK)) and methods to identify amino acid substitutions within the IMKs which increase isoprenoid production rate.
  • IMK isopentyl monophosphate kinase
  • this method requires expensive starting materials such as isoprenoid diphosphate which direct impacts on the economic feasibility of the whole process.
  • WO 2019/023192 Al relates to herbicide-detoxifying enzymes, compositions containing one or more of the enzymes, and plant seeds treated with the enzymes.
  • WO 2019/232025 A2 relates to cells and cell-free systems for the conversion of isopentenols to isoprenoid precursors which are reacted with enzymes (choline kinase from S. cerevisiae) that phosphorylate short chain alcohol to produce the corresponding monophosphate, followed by phosphorylation of the latter to afford diphosphates upon reaction with a suitable enzyme (e.g. isopentenyl phosphate kinase (IPK)).
  • IPK isopentenyl phosphate kinase
  • IKI isopentenyl pyrophosphate isomerase
  • IDI is also disclosed which is instrumental for the conversion of one or more synthetic precursors into the respective constitutional isomer.
  • WO 2020/150340 Al discloses methods for preparing isoprenoid subunits, as well as methods of employing these isoprenoid subunits for the synthesis of isoprenoids.
  • a primary alcohol is reacted with an enzyme performing a first phosphorylation such as a phosphatase that exhibits bidirectional activity, a single enzyme or a system involving two independent kinases (i.e. a first and second kinase), in the presence of ATP to generate the corresponding diphosphates.
  • a first phosphorylation such as a phosphatase that exhibits bidirectional activity, a single enzyme or a system involving two independent kinases (i.e. a first and second kinase)
  • PhoN a non-specific phosphatase from S. flexneri, shows a superior activity in the phosphorylation of DMAA/ISO.
  • the processes of the prior art are not satisfactory in every respect and there is a demand for improved
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • a first aspect of the invention relates to a process for the preparation of a first hemiterpene diphosphate comprising the steps of:
  • step (a2) phosphorylating the first hemiterpene monophosphate obtained in step (ai) with a second phosphate donor under catalysis of a second kinase thereby obtaining a first hemiterpene diphosphate and a second phosphate acceptor; wherein "terpene” encompasses terpenes as well as terpenoids according to IUPAC.
  • terpene is preferably not limited to “terpenes” according to IUPAC, but is to be interpreted in a broader meaning to additionally also encompass “terpenoids” according to IUPAC.
  • terpenoids are natural products and related compounds formally derived from isoprene units.
  • terpenes contain oxygen in various functional groups. This class is subdivided according to the number of carbon atoms in the same manner as are terpenes.
  • the skeleton of terpenoids may differ from strict additivity of isoprene units by the loss or shift of a fragment, generally a methyl group (see https://goldbook.iupac.org/terms/view/T06279).
  • terpenes are pure hydrocarbons, whereas “terpenoids” contain oxygen in various functional groups.
  • terpene in the nomenclature of compounds containing oxygen in various functional groups, such as “terpene alcohol”, which in accordance with IUPAC nomenclature, due to the presence of the alcohol as oxygen functional group, is not a terpene but a terpenoid according to IUPAC.
  • terpene refers to both, the pure hydrocarbons (terpenes according to IUPAC) and the derivatives contain oxygen in various functional groups (terpenoids according to IUPAC).
  • terpene preferably refers to a derivative containing oxygen in at least one of various functional groups.
  • hemiterpene diphosphate is preferably to be interpreted as a terpene or a terpenoid according to IUPAC, more specifically as a hemiterpene or hemiterpenoid comprising up to five carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage.
  • a hemiterpene diphosphate may for instance be obtained by reaction of pyrophosphoric acid (H4P2O7) with a hemiterpene alcohol, or analogously by reacting phosphoric acid (H3PO4) with a hemiterpene monophosphate.
  • H4P2O7 pyrophosphoric acid
  • H3PO4 phosphoric acid
  • Step (ai) of the process according to the invention involves phosphorylation of a hemiterpene alcohol.
  • a "hemiterpene alcohol” is preferably to be interpreted as a terpene or a terpenoid according to IUPAC, more specifically as a hemiterpene or hemiterpenoid comprising up to five carbons atoms as well as at least one alcoholic group within the hydrocarbon skeleton.
  • Step (a2) of the process according to the invention involves phosphorylation of a hemiterpene monophosphate.
  • a "hemiterpene monophosphate” is preferably to be interpreted as a terpene or a terpenoid according to IUPAC, more specifically as a hemiterpene or hemiterpenoid comprising up to five carbons atoms as well as one phosphate group.
  • a hemiterpene monophosphate may for instance be obtained by reacting phosphoric acid (H3PO4) with a hemiterpene alcohol.
  • phosphorylation refers to the attachment of a phosphate group to an organic molecule via a suitable mechanism for example by condensation, substitution or addition.
  • Steps (ai) and (a2) of the process according to the invention involve phosphorylation under catalysis of a kinase.
  • a "kinase” is preferably to be interpreted as an enzyme that catalyzes the transfer of phosphate groups from a phosphate donor to specific substrates.
  • Steps (ai) and (a2) of the process according to the invention involve phosphorylation with a phosphate donor.
  • a "phosphate donor” is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction.
  • Steps (ai) and (a2) of the process according to the invention involve phosphorylation with a phosphate donor obtaining a phosphate acceptor.
  • a "phosphate acceptor" is preferably to be interpreted as a chemical species deriving from a dephosphorylation reaction of the corresponding phosphate donor, i.e. presenting at least one phosphate unit less than the corresponding phosphate donor.
  • the phosphate acceptor is preferably characterized by a molecular structure amenable to undergo addition of a phosphate unit thereby re- pristinating the corresponding phosphate donor.
  • (deoxy) shall preferably be understood as being entirely optional, i.e. covering both deoxy and non-deoxy variants of the same chemical substance.
  • conversion is preferably to be interpreted as the ratios between the molar amount of consumed reactant and the molar amount of reactant initially employed in the reaction.
  • first kinase is preferably to be interpreted as an enzyme capable of catalyzing the monophosphorylation of an alcohol, said alcohol being preferably a hemiterpene alcohol, and even more preferably isoprenol and/or prenol.
  • the first kinase is an enzyme capable of catalyzing the transfer of a phosphate group selected from the group consisting of:
  • - phosphotransferase with alcohol group as acceptor preferably selected from the group consisting of ethanolamine kinase (EC 2.7.1.82), amide-alcohol kinase, ceramide kinase (EC 2.7.1.138), pantoate kinase (EC 2.7.1.169), undecaprenol kinase (EC 2.7.1.66), choline kinase (EC 2.7.1.32), glycerol kinase (EC 2.7.1.30), glycerone kinase (EC 2.7.1.29), mevalonate kinase (EC 2.7.1.30), and hydrox- yethylthiazole kinase (EC 2.7.1.50);
  • phosphotransferases with a phosphate group as an acceptor EC 2.7.4; preferably selected from the group consisting of polyphosphate kinase, phosphomevalonate kinase, adenylate kinase, nucleosidephosphate kinase, deoxycytidylate kinase, nucleoside-diphosphate kinase, phosphomethylpyrimidine kinase, guanylate kinase, dTMP kinase, nucleoside-triphosphate-adenylate kinase, (deoxy) adenylate kinase, T2-induced deoxynucleotide kinase, (deoxy) nucleoside-phosphate kinase, UMP/CMP kinase, thiamine-diphosphate kinase, thiamine-phosphate kinase, 3-phosphoglyceroyl--phosphat
  • - phosphotransferases with a carboxy group as acceptor EC 2.7.2: acetate kinase (EC 2.7.2.1), acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10), glutamate 5-ki- nase (EC 2.7.2.11), glutamate 1-kinase (EC 2.7.2.13), branched-chain-fatty-acid kinase (EC 2.7.2.14), propionate kinase (EC 2.7.2.15), 2-phosphoglycerate kinase (EC 2.7.2.
  • [amino group carrier protein] -L-2 -aminoadipate 6-kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase( EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8).
  • the first kinase is a hydroxyethylthiazole kinase from Escherichia coli (EC 2.7. 1.50).
  • the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • the first kinase is a choline kinase from Plasmodium falciparum (PfCK) (EC 2.7.1.32).
  • the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • the first kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.
  • the first kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the first kinase according to the invention may essentially consist of said amino acid sequence.
  • said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the first kinase is to be immobilized on a solid support, e.g. for purification purposes.
  • step (ai) is performed in a liquid reaction medium and the first kinase is employed in a concentration of at least 0.0001 mg/ml.
  • step (ai) is performed in a liquid reaction medium and the first kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, more preferably at most 10 mg/ml.
  • the first "hemiterpene alcohol” is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to five carbons atoms as well as at least one alcoholic group within the hydrocarbon skeleton.
  • the first hemiterpene alcohol is preferably selected from the group consisting of isoprenol, 3-methyl-3-buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Methyl-2-propen-l-ol, 3 -methylene- 1 -pentanol, 2-hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-methyl-4-penten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-bu- ten-l-ol, 3-methyl-2-penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl-(2£)-penten-l-ol, (2E)-butene-2-methyl- 1,4-dio
  • the first hemiterpene alcohol is isoprenol or prenol, and even more preferably isoprenol.
  • the first hemiterpene alcohol is employed in a concentration of at least 0.1 mM; preferably of at least 0.5 mM, more preferably of at least 1.0 mM.
  • step (ai) is performed in a liquid reaction medium and the first hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, more preferably at most 1000 mM.
  • the "first phosphate donor” is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction.
  • the first phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably the first phosphate donor is a nucleoside mono- or polyphosphate preferably selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glu- cose-6-phosphate, fructose- 1,6-bisphosphate, or 3 -phosphoglycerate.
  • the first phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil and even more preferably of adenosine.
  • the first phosphate donor is adenosine triphosphate (ATP).
  • ATP adenosine triphosphate
  • step (ai) is preferably performed in a liquid reaction medium and the first phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, more preferably at least 0.01 mM.
  • step (ai) is performed in a liquid reaction medium and the first phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, more preferably at most 10 mM.
  • step (a2) of the process according to the invention the first hemiterpene monophosphate obtained from step (ai) is reacted with a second phosphate donor under catalysis of a second kinase thereby obtaining a first hemiterpene diphosphate and a second phosphate acceptor.
  • the "second kinase” preferably refers to an enzyme capable of catalyzing the phosphorylation of a mono- or polyphosphorylated alcohol, preferably of a mono- or polyphosphorylated hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isopentenyl monophosphate and/or dimethylallyl monophosphate.
  • the second kinase in an enzyme capable of catalyzing the transfer of a phosphate group, such as phosphotransferase with a phosphate group as acceptor.
  • the second kinase in an enzyme selected from the group consisting of polyphosphate kinase (EC 2.7.4.
  • phosphomevalonate kinase (EC 2.7.4.2), adenylate kinase (EC 2.7.4.3), nucleoside-phosphate kinase (EC 2.7.4.4), thiamine-diphosphate kinase (EC 2.7.4.15), thi- amone-phosphate kinase (EC 2.7.4.16), famesyl-diphosphate kinase (EC 2.7.4.18), isopentenyl phosphate kinase (EC 2.7.4.26), and famesyl phosphate kinase (EC 2.7.4.32), phosphotransferases with a carboxy group as acceptor (EC 2.7.2): acetate kinase (EC 2.7.2.1), acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10), glutamate 5-kinase (EC 2.7.2.
  • [amino group carrier protein] -L-2 -aminoadipate 6- kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase (EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8), preferably a isopentenyl phosphate kinase (EC 2.7.4.26).
  • said isopentenyl phosphate kinase is selected from an organism selected from the group consisting of Arabidopsis thaliana, Branchiostoma floridae, Meth- anolobus tindarius,Methanococcus vannielii, aus Roseiflexus castenholzii, Thermoplasma acidophilum, Methanothermobacter thermoautotropicus , Methanocaldococcus jannaschii, Haloferax volcanii, Sul- folobus solfataricus, Trichoplax adhaerens, or Methanosalsum zhilinae.
  • the second kinase is a isopentenyl phosphate kinase from Roseiflexus castenholzii.
  • the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • the second kinase is a isopentenyl phosphate kinase from Methanol- obus tindarius.
  • the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61 %, or at least 62%, or at least 63 %, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in
  • the second kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of the first kinase, SEQ ID NO:3 or SEQ ID NO:4.
  • the second kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the second kinase according to the invention may essentially consist of said amino acid sequence.
  • said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-ter- minus and/or at the C-terminus of said subsequence.
  • Such extension may be advantageous, for example, when the second kinase is to be immobilized on a solid support, e.g. for purification purposes.
  • step (ai) is performed in a liquid reaction medium and the second kinase is employed in a concentration of at least 0.0001 mg/ml.
  • step (ai) is performed in a liquid reaction medium and the second kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, most preferably at most 10 mg/ml.
  • the "second phosphate donor” is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction.
  • the second phosphate donor preferably belongs to the same chemical category as the first phosphate donor, i.e. organic or inorganic monophosphate or polyphosphate, and is capable of donating a phosphate unit during the course of a reaction.
  • the second phosphate donor may not substantially differ from the first phosphate donor and preferably be a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3 -phosphoglycerate.
  • the second phosphate donor is the same as the first phosphate donor.
  • the second phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil and preferably of adenosine.
  • this second phosphate donor is adenosine triphosphate.
  • step (a2) is preferably performed in a liquid reaction medium and the second phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, most preferably at least 0.1 mM.
  • the second phosphate donor substantially differs from the first phosphate donor i.e. it has different molecular weights.
  • the second phosphate donor and the first phosphate donor might differ by at one methine group.
  • the second phosphate donor and the first phosphate donor differ by at most one methine group.
  • step (a2) is performed in a liquid reaction medium and the second phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, most preferably at most 10 mM.
  • the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least
  • the first hemiterpene alcohol is isoprenol
  • the first and second phosphate donor is adenosine triphosphate
  • the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%,
  • Another aspect of the invention relates to a process for the preparation of a monoterpene diphosphate comprising the process for the preparation of a first hemiterpene diphosphate according to the invention as described above.
  • the process additionally comprises the steps of:
  • step (b) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a monoterpene diphosphate synthase thereby obtaining the monoterpene diphosphate.
  • Step (b) of the process according to the invention involves linking under catalysis of a monoterpene diphosphate synthase.
  • the "monoterpene diphosphate” synthase is preferably an enzyme capable of catalyzing the condensation of one unit of the first hemiterpene diphosphate obtained in step (a2) with one unit of the second hemiterpene diphosphate provided in step (as).
  • the monoterpene diphosphate synthase is a geranyl diphosphate synthase (GPPS) (EC 2.5. 1.1) and/or the monoterpene diphosphate is a geranyl diphosphate (GPP).
  • GPPS geranyl diphosphate synthase
  • GPP geranyl diphosphate
  • the second hemiterpene diphosphate is obtained in step (a2) and/or in step (ai).
  • the process may preferably entail performing step (as) as sub-step of steps (ai) and/or (a2) in an overall setting in which the second hemiterpene diphosphate is obtained concomitantly with the first hemiterpene monophosphate or diphosphate during the phosphorylation of the corresponding first hemiterpene alcohol or first hemiterpene monophosphate respectively, either in the same or in separate reaction environments.
  • the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
  • the monoterpene diphosphate is geranyl pyrophosphate (GPP); and/or
  • the monoterpene diphosphate synthase is a geranyl diphosphate synthase (GPPS) belonging to EC 2.5. 1.1.
  • GPPS geranyl diphosphate synthase
  • Another aspect of the invention relates to a process for the preparation of a sesquiterpene diphosphate comprising
  • the process additionally comprises the steps of:
  • step (c) linking the first hemiterpene diphosphate obtained in step (a2) with the second hemiterpene diphosphate provided in step (as) under catalysis of a sesquiterpene diphosphate synthase thereby obtaining the sesquiterpene diphosphate.
  • a "sesquiterpene diphosphate" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to fifteen carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage.
  • Step (c) of the process according to the invention involves linking under catalysis of a sesquiterpene diphosphate synthase.
  • the "sesquiterpene diphosphate synthase” is preferably an enzyme capable of catalyzing the sequential condensation of one or more units of the first hemiterpene diphosphate obtained in step (a2) with one unit of the second hemiterpene diphosphate provided in step (as).
  • the second hemiterpene diphosphate may be obtained in step (as) and/or in step (ai).
  • the process may preferably entail performing step (as) as sub-step of steps (ai) and/or (as) in an overall setting in which the second hemiterpene diphosphate is obtained concomitantly with the first hemiterpene monophosphate or diphosphate during the phosphorylation of the corresponding first hemiterpene alcohol or first hemiterpene monophosphate respectively, either in the same or in separate reaction environments.
  • the sesquiterpene diphosphate synthase is a famesyl diphosphate synthase (FPPS) (EC 2.5.1.10).
  • FPPS famesyl diphosphate synthase
  • the sesquiterpene diphosphate synthase is preferably an FPPS from an organismus selected from the group consisting of Geobacillus stearothermophilus, Bos Taurus, Gallus gallus, Pseudomonas aeruginosa, Rhizosolenia setigera, Ricinus communis, Lupinus albus, Homo sapiens, Artemisia spiciformi or Abies grandis.
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and
  • the sesquiterpene diphosphate synthase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.
  • SEQ ID NO:5 amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.
  • said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence.
  • extension may be advantageous, for example, when the sesquiterpene diphosphate synthase is to be immobilized on a solid support, e.g. for purification purposes.
  • the sesquiterpene diphosphate is famesyl diphosphate (FPP).
  • the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
  • the sesquiterpene diphosphate is famesyl pyrophosphate (FPP); and/or
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • Another aspect of the invention relates to a process for the preparation of a diterpene diphosphate comprising
  • the process additionally comprises the steps of:
  • step (d) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a diterpene diphosphate synthase thereby obtaining the diterpene diphosphate.
  • a “diterpene diphosphate” is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to twenty carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage.
  • Step (d) of the process according to the invention involves linking under catalysis of a diterpene diphosphate synthase.
  • the "sesquiterpene diphosphate synthase” is preferably to be interpreted as an enzyme capable of catalyzing the sequential condensation of one or more units of the first hemiterpene diphosphate obtained in step (a2) with one unit of the second hemiterpene diphosphate provided in step (as).
  • the second hemiterpene diphosphate may be obtained in step (a2) and/or in step (ai).
  • the process may preferably entail performing step (as) as sub-step of steps (ai) and/or (as) in an overall setting in which the second hemiterpene diphosphate is obtained concomitantly with the first hemiterpene monophosphate or diphosphate during the phosphorylation of the corresponding first hemiterpene alcohol or first hemiterpene monophosphate respectively, either in the same or in separate reaction environments.
  • the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) (EC 2.5.1.29).
  • GPPS geranylgeranyl diphosphate synthase
  • the diterpene diphosphate is geranylgeranyl diphosphate (GGPP).
  • the process of the invention is characterized in that:
  • the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
  • the diterpene diphosphate is geranylgeranyl pyrophosphate (GGPP); and/or
  • the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) belonging to EC 2.5.1.29.
  • GPPS geranylgeranyl diphosphate synthase
  • Another aspect of the invention relates to a process for the preparation of a tetraterpene comprising
  • the process additionally comprises the step of:
  • tetraterpene is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to forty carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage.
  • Step (e) of the process according to the invention involves linking under catalysis of a tetraterpene synthase.
  • the "tetraterpene synthase” is preferably an enzyme capable of catalyzing the sequential condensation of two or more units of the diterpene diphosphate obtained in step (d).
  • the tetraterpene synthase is a phytoene synthase (EC 2.5.1.32).
  • the tetraterpene is phytoene.
  • the process is characterized in that:
  • the tetraterpene is phytoene
  • the tetraterpene synthase is a phytoene synthase belonging to EC 2.5.1.32.
  • (B) a monoterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (b));
  • (C) a sesquiterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (c));
  • step (as) are described, that analogously apply to any of the above processes (B) through (E).
  • step (as) comprises the sub-steps of:
  • step (as.2) phosphorylating the second hemiterpene monophosphate obtained in step (as.i) with a fourth phosphate donor under catalysis of a fourth kinase thereby obtaining the second hemiterpene diphosphate and a fourth phosphate acceptor.
  • first phosphate acceptor preferably to be interpreted as a chemical species deriving from a dephosphorylation reaction of the corresponding phosphate donor, i.e. presenting at least one phosphate unit less than it.
  • the first, second, third or fourth phosphate acceptor is preferably characterized by a molecular structure amenable to undergo addition of a phosphate unit thereby divulgtinating the corresponding phosphate donor.
  • the "second hemiterpene alcohol” is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to five carbons atoms as well as at least one alcoholic group within the hydrocarbon skeleton.
  • the second hemiterpene alcohol is a constitutional isomer of the first hemiterpene alcohol.
  • substitutional isomer has the same meaning as it would be understood by a skilled artesian in the art i.e. a molecule presenting the same molecular formula of a parent molecule, however in a different special arrangement.
  • the second hemiterpene alcohol is selected from the group consisting of isoprenol, 3-methyl-3-buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Me- thyl-2-propen-l-ol, 3 -methylene- 1 -pentanol, 2-hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-me- thyl-4-penten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-buten-l-ol, 3- methyl-2-penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl-(2£)-penten-l-ol, (2E)-butene-2 -methyl-
  • the second hemiterpene alcohol is isoprenol or prenol, and most preferably prenol.
  • the second hemiterpene alcohol is employed in a concentration of at least 0. 1 mM; preferably of at least 0.5 mM, most preferably of at least 1.0 mM.
  • the second hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, most preferably at most 1000 mM.
  • the "third kinase” is preferably to be interpreted as an enzyme capable of catalyzing the phosphorylation of an alcohol, said alcohol being preferably a hemiterpene alcohol, and even more preferably isoprenol and/or prenol.
  • the third kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • the third kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of the first kinase, second kinase, or SEQ ID NO: 1.
  • the third kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the third kinase according to the invention may essentially consist of said amino acid sequence.
  • said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the third kinase is to be immobilized on a solid support, e.g. for purification purposes.
  • the "fourth kinase” preferably refers to an enzyme capable of catalyzing the phosphorylation of a mono- or polyphosphorylated alcohol, preferably of a mono- or polyphosphorylated hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isopentenyl monophosphate and/or dimethylallyl monophosphate.
  • the fourth kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • the fourth kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of the first kinase, second kinase, third kinase, or SEQ ID NO:3.
  • the fourth kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the fourth kinase according to the invention may essentially consist of said amino acid sequence.
  • said overall amino acid sequence may be extended, i.e.
  • Such extension may be advantageous, for example, when the fourth kinase is to be immobilized on a solid support, e.g. for purification purposes.
  • the "third phosphate donor” is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction.
  • the third phosphate donor preferably belongs to the same chemical category as the first or second phosphate donor, i.e. organic or inorganic monophosphate or polyphosphate, and is capable of donating a phosphate unit during the course of a reaction.
  • the third phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the third phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phos- phate, fructose- 1,6-bisphosphate, and 3 -phosphoglycerate.
  • the third phosphate donor has the same molecular structure as the first and /or second phosphate donor.
  • the "fourth phosphate donor” is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction.
  • the fourth phosphate donor preferably belongs to the same chemical category as the first, second or third phosphate donor, i.e. organic or inorganic monophosphate or polyphosphate, and is capable of donating a phosphate unit during the course of a reaction.
  • the fourth phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phos- phate, fructose- 1,6-bisphosphate, 3 -phosphoglycerate.
  • the fourth phosphate donor has the same molecular structure as the first and second and/or third phosphate donor.
  • the molar ratio of the first hemiterpene alcohol relative to second hemiterpene alcohol is at least 1: 10, or at least 1:2, or preferably at least 1: 1, or most preferably at least 2: 1 or 3: 1.
  • the molar ratio of the first hemiterpene alcohol relative second hemiterpene alcohol is at most 100: 1; preferably at most 10: 1 or at most 5: 1, or preferably at most 3: 1.
  • steps (ai), (a2), (as.i), (a-, 2) is repeated at least once;
  • steps (ai), (a2), (as.i), (as.2) are performed in a single reactor;
  • step (as) comprises the substep of
  • an "isomerase” is preferably to be interpreted as an enzyme capable of altering the isomeric structure of a given initial isomeric substrate to afford a different isomeric substrate presenting the same molecular formula as compared with the initial substrate and different structural arrangement.
  • the isomerase is selected from the group consisting of steroid delta isomerase (EC 5.3.3.1), vinylacetyl-CoA delta isomerase (EC 5.3.3.3), muconolactone delta-isomerase (EC 5.3.3.4), 5- carboxymethyl-2-ydroxymuconate delta isomerase (EC 5.3.3.10), isopiperitenone delta-isomerase (EC 5.3.3.11), polyenoic fatty acid isomerase (EC 5.3.3.13), trans-2,3-dihydro-3-hydroxyanthranilate isomerase (EC 5.3.3.17), lutein isomerase (5.3.3.22) and isopentenyl diphosphate isomerase (EC 5.3.3.2) and even more preferably the isomerase is a isopentenyl diphosphate isomerases (EC 5.3.3.2).
  • steroid delta isomerase EC 3.1
  • vinylacetyl-CoA delta isomerase EC 5.3.3.3
  • muconolactone delta-isomerase
  • the isomerase is a isopentenyl diphosphate isomerase from Schizosaccharomyces pombe, Claviceps purpurea, Phaffia rhodozyma, Homo sapiens, Sus scrofa, Gallus gallus, Solanum lycopersicum, Haematococcus lacustris, Gossypium barbadense, Escherichia coli, or Saccharomyces cerevisiae.
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 90%, or at least 9
  • the Isomerase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24 SEQ ID NO:25.
  • SEQ ID NO: 15 SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24 SEQ ID NO:25.
  • the Isomerase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the Isomerase according to the invention may essentially consist of said amino acid sequence.
  • said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence.
  • extension may be advantageous, for example, when the Isomerase is to be immobilized on a solid support, e.g. for purification purposes.
  • the second hemiterpene diphosphate is selected from the group consisting of isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), 3-methyl-3-buten- 2-ol diphosphate, tiglic alcohol diphosphate, angelicic alcohol diphosphate, senecioic alcohol diphosphate and isovaleric alcohol diphosphate.
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • 3-methyl-3-buten- 2-ol diphosphate 3-methyl-3-buten- 2-ol diphosphate
  • tiglic alcohol diphosphate tiglic alcohol diphosphate
  • angelicic alcohol diphosphate senecioic alcohol diphosphate
  • isovaleric alcohol diphosphate isovaleric alcohol diphosphate.
  • the second hemiterpene diphosphate is a structural isomer; preferably a constitutional isomer of the first hemiterpene diphosphate.
  • the second hemiterpene diphosphate is isopentenyl pyrophosphate (IPP) or dimethylallyl pyrophosphate (DMAPP).
  • the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP).
  • the processes (A) through (E) according to the invention are characterized in that: - at least steps (ai) and (a2) are performed simultaneously; and/or
  • steps (ai), (a2), (as.i), (as.2), (as s) is repeated at least once;
  • steps (ai), (a2), (as.i), (as.2), (as s) are performed in a single reactor;
  • steps (ai) and (a ,). are performed simultaneously;
  • the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%,
  • the first hemiterpene alcohol is isoprenol
  • the first, second, third and fourth phosphate donor is adenosine triphosphate
  • the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%,
  • step (as) comprises the sub-steps of:
  • the isomerase is a isopentenyl diphosphate isomerases an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in
  • the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP); and/or
  • the third phosphate donor and fourth phosphate donor are adenosine triphosphate.
  • any of the processes (A) to ( E) and all their preferred embodiments as described above take place at a temperature of 2.0 °C to 99.5 °C, or 3.0°C to 95.0°C, or 4.0°C to 90.0°C, or 5.0°C to 85.0°C, or 15.0°C to 80.0°C, or 25.0°C to 75.0°C, or 28.0°C to 75.0°C, or 30.0°C to 60.0°C, or 33.0°C to 50.0°C, or 35.0°C to 45.0°C.
  • any of the processes (A) to ( E) and all their preferred embodiments as described above take place in a liquid reaction medium, wherein said liquid reaction medium is characterized by a pH in the range of 5.0 to 8.5, or 5.3 to 8.5, or 5.5 to 8.5, or 5.8 to 8.5, or 6.0 to 8.5, or 6.4 to 8.5, or 6.6 to 8.5, or 6.9 to 8.5, 7.5 to 8.5, or 8.0 to 8.5.
  • the process additionally comprises the step of:
  • step (f) reacting a monoterpene diphosphate obtained in step (b) under catalysis of a monoterpene synthase thereby obtaining the monoterpene.
  • the "monoterpene synthase” is preferably to be interpreted as an enzyme capable of catalyzing the rearrangement and/or cyclization of GPP into a monoterpene i.e. an organic compound comprising up to 10 carbon atoms.
  • the monoterpene synthase is a an selected from the group consisting of squalensyn- thase (EC 2.5.1.21), 1,8-cineol synthase, (EC 4.2.3.108), (-)-sabinene synthase (EC 4.2.3.109), (+)-sab- inene synthase (EC 4.2.3.110), 5-carene-synthase (EC 4.2.3.107) and (R)-limonene synthase (EC 4.2.3.20), preferably wherein the monoterpene synthase is (-)-sabinene synthase (EC 4.2.3.109), (+)- sabinene synthase (EC 4.2.3.110), 5-carene-synthase (EC 4.2.3.107).
  • squalensyn- thase EC 2.5.1.21
  • 1,8-cineol synthase EC 4.2.3.108
  • the monoterpene is selected from the group consisting of squalen 1,8-cineol, (-)- sabinene, (+)-sabinene, 5-carene and (R)-limonene, preferably wherein the monoterpene is squalen, (-)- sabinene, (+)-sabinene or 5-3-carene.
  • Another aspect of the invention relates to a process for the preparation of carvone comprising the process for the preparation of a monoterpene, particularly for the preparation of (R)-limonene, according to the invention as described above.
  • step (gi) reacting (R)-limonene obtained in step (f) under catalysis of a Limonene-6-hydroxylase (EC 1.14.13.48) thereby obtaining carvenol;
  • the process according to the invention is characterized in that:
  • steps (gi) or (g 2 ) are repeated at least once; and/or at least one of steps (gi) or (g 2 ) is performed in a single reactor; and/or steps (gi) or (g 2 ) are performed simultaneously.
  • Another aspect of the invention relates to a process for the preparation of a cannanbinoide comprising the process for the preparation of a monoterpene diphosphate, particularly for the preparation of GPP according to the invention as described above.
  • the process additionally comprises the step of:
  • step (hi) reacting a monoterpene diphosphate, particularly GPP, obtained in step (b) with an olivetolic acid under catalysis of an aromatic prenyltransferase thereby obtaining cannabigerolic acid;
  • CBDA cannabidiol
  • THC tetrahydrocannabinol
  • the aromatic prenyltransferase is a an selected from the group consisting of aspul- vinone dimethylallyltransferase (EC 2.5.1.35), trihydroxypterocarpan dimethylallyltransferase (EC 2.5.1.36), 4-hydroxybenzoic acid oligoprenyltransferase (EC 2.5.1.39), naringenin 8-dimethylallyltrans- ferase (EC 2.5.1.70), 4-hydroxybenzoate geranyltransferase (EC 2.5.1.93), fumigaclavine A dimethylallyltransferase (EC 2.5.1.100), geranylpyrophosphate :olivetolate geranyltransferase (EC 2.5.1.102), 4- hydroxyphenylpyruvate: dimethylallyl transferase (EC 2.5.1.111), homogentisate geranylgeranyltransferase (EC 2.5.1.116), flaviolin linalyltransfera
  • the process according to the invention is characterized in that:
  • steps (hi) (h 2 ) or (h ) is repeated at least once;
  • steps (hi) (h 2 ) or (ha) is performed in a single reactor;
  • Another aspect of the invention relates to a process for the preparation of a sesquiterpene comprising the process for the preparation of a sesquiterpene diphosphate according to the invention as described above. [0182] In this context, it is preferred that, besides steps (as) and (c) leading to the sesquiterpene diphosphate according to the invention as described above, the process additionally comprises the step of:
  • step (i) reacting a sesquiterpene diphosphate obtained in step (c) under catalysis of a sesquiterpene synthase thereby obtaining the sesquiterpene.
  • the sesquiterpene synthase is an enzyme capable of catalyzing the rearrangement and/or cyclization of FPP into a sesquiterpene i.e. an organic compound comprising up to 15 carbon atoms, preferably wherein the sesquiterpene synthase is a an enzyme selected from the group consisting of valencene synthase (EC 4.2.3.73), a-bisabolol-synthase (EC 4.2.3.138), (S)-beta-bisabolene synthase (EC 4.2.3.55), patchoulol synthase (EC 4.2.3.70), [3-elemene synthase (EC 4.2.3.x), 5-cadinene synthase (EC 4.2.3.13), (+)-endo-beta-bergamotene synthase (EC 4.2.3.53), (-)-endo-alpha-bergamo- tene synthase (EC 4.2.
  • the sesquiterpene is selected from the group consisting of valencene, a-bisabolol, [3- bisabolene, patchoulol, [3-elemene, 5-cadinene, a-trans-bergamotene, [3-cis-bergamotene, [3-trans-berga- motene, zingiberene, longifolene, a-santalene, [3-santalene.
  • Another aspect of the invention relates to a process for the preparation of sesquiterpenoid comprising the process for the preparation of a sesquiterpene according to the invention as described above.
  • step (i) leading to the sesquiterpene the process additionally comprises the step of:
  • step (j) reacting the sesquiterpene obtained in step (i) under catalysis of a monooxygenase (EC 1. 14.x.x) thereby obtaining sesquiterpenoid.
  • the sesquiterpenoid is selected from the group consisting of nootkatone, hemandul- cin, a-santalol, [3-santalol.
  • Another aspect of the invention relates to a process for the preparation of famesol comprising the process for the preparation of a sesquiterpene diphosphate, particularly for the preparation of FPP, according to the invention as described above.
  • the process additionally comprises the step of: (k) reacting a sesquiterpene diphosphate, particularly FPP, obtained in step (c) under catalysis of a FPP-diphosphatase thereby obtaining farnesol.
  • the FPP-diphosphatase is a phosphatase belonging to EC 3.6.1.x and/or EC 3.1.3.x.
  • Another aspect of the invention relates to a process for the preparation of a sclareol comprising the process for the preparation of a diterpene diphosphate, particularly for the preparation of GGPP, according to the invention as described above.
  • the process additionally comprises the step of:
  • step (h) reacting a diterpene diphosphate, particularly GGPP obtained in step (d) under catalysis of a lab- denediol synthase (EC 4.2.1.133) thereby obtaining the labdenediol diphosphate (LDPP); and
  • step (h) reacting labdenediol diphosphate obtained in step (h) under catalysis of a sclareol synthase (EC 4.2.3. 141) thereby obtaining the sclareol.
  • steps (h) or (I2) is repeated at least once;
  • steps (h) or (I2) is performed in a single reactor;
  • Another aspect of the invention relates to a process for the preparation of a tetraterpenoid, comprising the process for the preparation of a tetraterpene, particularly comprising the process for the preparation of phytoene according to the invention as described above.
  • the process additionally comprises the step of:
  • step (m) reacting phytoene obtained in step (e) under catalysis of one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ⁇ -carotene desaturase (EC 1.3.99.26), lycopene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpenoid.
  • one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ⁇ -carotene desaturase (EC 1.3.99.26), lycopene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpenoid.
  • the tetraterpenoid is selected from the group consisting of phytofluene, 8-carotene, lycopene, neurosporene and [3-carotene.
  • Another aspect of the invention relates to a process for the preparation of ionone, comprising the process for the preparation of a tetraterpenoid, particularly comprising the process for the preparation of [3-carotene according to the invention as described above.
  • the process additionally comprises the step of:
  • step (n) reacting the tetraterpenoid, particularly p-carotene, obtained in step (m) under catalysis of a carotenoid dioxygenase (EC 1.13.11.71 thereby obtaining ionone).
  • a carotenoid dioxygenase EC 1.13.11.71
  • P-carotene constitutes a particularly preferred choice
  • other tetraterpenoids such as of phytoene, phytofluene, 8-carotene, lycopene, neurosporene represent suitable starting material from which ionone can be derived.
  • (B) a monoterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (b));
  • (C) a sesquiterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (c));
  • (G) carvone (preferably encompassing steps (ai), (as), (as), (b), (f), (gi) and (gs));
  • (H) a cannabinoide (preferably encompassing steps (ai), (as), (as), (b), (hi), (hs) and (hs));
  • (L) sclareol (preferably encompassing steps (ai), (as), (as), (d), (h) and (E);
  • any of the processes (A) to ( N) and all their preferred embodiments as described above take place at a temperature of 2.0 °C to 99.5 °C, or 3.0°C to 95.0°C, or 4.0°C to 90.0°C, or 5.0°C to 85.0°C, or 15.0°C to 80.0°C, or 25.0°C to 75.0°C, or 28.0°C to 75.0°C, or 30.0°C to 60.0°C, or 33.0°C to 50.0°C, or 35.0°C to 45.0°C.
  • any of the processes (A) to ( N) and all their preferred embodiments as described above preferably take place in a liquid reaction medium, wherein said liquid reaction medium is characterized by a pH in the range of 5.0 to 8.5, or 5.3 to 8.5, or 5.5 to 8.5, or 5.8 to 8.5, or 6.0 to 8.5, or 6.4 to 8.5, or 6.6 to 8.5, or 6.9 to 8.5, 7.5 to 8.5, or 8.0 to 8.5.
  • the processes (A) through (N) according to the invention comprise a phosphate regeneration system enabling the phosphorylation of the first, second, third, and/or fourth phosphate acceptor thereby regenerating the corresponding phosphate acceptor.
  • any phosphate regeneration system known in the art can be employed.
  • suitable phosphate regeneration systems may be polyphosphate kinase ATP regeneration systems, creatine kinase ATP regeneration systems, acetate kinase ATP regeneration systems, pyruvate kinase ATP regeneration systems, glucose-6-phosphate kinase ATP regeneration systems, fructose- 1,6- bisphosphate kinase ATP regeneration systems, 3 -phosphoglycerate kinase ATP regeneration systems.
  • the process according to the invention may comprise the additional step of
  • step (o) phosphorylating the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2), the third phosphate acceptor optionally obtained in step (as.i) and/or the fourth phosphate acceptor optionally obtained in step (a3.2) with a phosphorylating agent, optionally under catalysis of a fifth kinase, thereby obtaining first phosphate donor, second phosphate donor, third phosphate donor, and/or fourth phosphate donor, respectively.
  • a "phosphorylating agent” is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit to the first, second, third or fourth phosphate acceptor during the course of a reaction.
  • the phosphorylating agent is polyphosphate, phosphocreatine, acetyl phosphate, phosphopyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3 -phosphoglycerate; more preferably acetyl phosphate
  • the "fifth kinase” is preferably an enzyme capable of catalyzing the phosphorylation of the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2), the third phosphate acceptor optionally obtained in step (as.i), and/or fourth phosphate acceptor optionally obtained in step (a3.2).
  • the fifth kinase is an acetate kinase belonging to EC 2.7.2.1 or EC 2.7.4.1.
  • the fifth kinase is an acetate kinase from Geobacillus kaustophilus, Escherichia coli, Rhodococcus erythropolis, Acinetobacter baylyi, Butyrivibrio fibrisolvens, Geobacillus caldoxylosilyti- cus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus shackletonii or Sinorhizobium meliloti.
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%,
  • the fifth kinase according to the invention preferably comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, SEQ ID NO:35 SEQ ID NO:36 or SEQ ID NO:37.
  • the fifth kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the fifth kinase according to the invention may essentially consist of said amino acid sequence.
  • the Fifth kinase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence
  • said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence.
  • Such extension may be advantageous, for example, when the fifth kinase is to be immobilized on a solid support, e.g. for purification purposes.
  • the phosphorylating agent is acetyl phosphate
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least
  • compositions comprising (i) first hemiterpene diphosphate obtainable by the process according to the invention as described above in combination with (ii) a first kinase, (iii) a first phosphate donor, (iv) a second kinase, and (v) a second phosphate donor or any combination of the foregoing.
  • the composition further comprises (vi) a phosphorylating agent, and/or (vii) a fifth kinase, preferably wherein the phosphorylating agent is acetyl phosphate and/or wherein the fifth kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at
  • the composition according to the invention further comprises a (i) monoterpene obtainable by the process according to the invention as described above in combination with a (ii) monoterpene synthase.
  • the monoterpene is preferably selected from the group consisting of squalene 1,8-cineol, (-)-sabinene, (+)-sabinene, 5-carene and (R)-limonene, preferably wherein the monoterpene is squalene, (-)-sabinene, (+)-sabinene or 5-3-carene.
  • the composition further comprises a (i) sesquiterpene diphosphate obtainable by the process according to the invention as described above in combination with a (ii) sesquiterpene diphosphate synthase.
  • the composition further comprises a (i) sesquiterpene obtainable by the process according to the invention as described above in combination with a (ii) sesquiterpene synthase.
  • the sesquiterpene is preferably selected from the group consisting of valencene, a-bisabolol, [3-bisabolene, patchoulol, [3-elemene, 5-cadinene, a-trans-bergamotene, [3-cis-bergamotene, P-trans-bergamotene, zingiberene, longifolene, a-santalene, -santalene.
  • the composition further comprises a (i) sesquiterpenoid obtainable by the process according to the invention as described above in combination with a (ii) monooxygenase (EC 1.14.x.x).
  • a (i) sesquiterpenoid obtainable by the process according to the invention as described above in combination with a (ii) monooxygenase (EC 1.14.x.x).
  • the sesquiterpenoid is preferably selected from the group consisting of noot- katone, hemandulcin, a-santalol, -santalol.
  • the composition further comprises a (i) diterpene diphosphate obtainable by the process according to the invention as described above in combination with a (ii) diterpene diphosphate synthase.
  • the composition further comprises (i) sclareol obtainable by the process according to the invention as described above in combination with a (ii) labdenediol diphosphate and a sclareol synthase (EC 4.2.3.141).
  • the composition further comprises a (i) tetraterpene obtainable by the process according to the invention as described above in combination with a (ii) tetraterpene synthase.
  • the tetraterpene is preferably phytoene.
  • the composition further comprises a (i) tetraterpenoid obtainable by the process according to the invention as described above in combination with (ii) one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ⁇ -carotene desaturase (EC 1.3.99.26), lyco- pene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpene
  • a tetraterpenoid obtainable by the process according to the invention as described above in combination with (ii) one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ⁇ -carotene desaturase (EC 1.3.99.26), lyco- pene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the t
  • the tetraterpenoid is preferably selected from the group consisting of phyto- fluene, 8-carotene, lycopene, neurosporene and [3-carotene.
  • the composition further comprises (i) ionone obtainable by the process according to the invention as described above in combination with a (ii) carotenoid dioxygenase (EC 1.13.11.71).
  • Clause 1 A process for the preparation of a first hemiterpene diphosphate comprising the steps of: (ai) phosphorylating a first hemiterpene alcohol with a first phosphate donor under catalysis of a first kinase thereby obtaining a first hemiterpene monophosphate and a first phosphate acceptor; and (a2) phosphorylating the first hemiterpene monophosphate obtained in step (ai) with a second phosphate donor under catalysis of a second kinase thereby obtaining a first hemiterpene diphosphate and a second phosphate acceptor; wherein "terpene” encompasses terpenes as well as terpenoids according to IUPAC.
  • Clause 2 The process according to clause 1, wherein the first kinase is an enzyme capable of catalyzing the phosphorylation of an alcohol; preferably of a hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isoprenol and/or prenol.
  • the first kinase is an enzyme capable of catalyzing the phosphorylation of an alcohol; preferably of a hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isoprenol and/or prenol.
  • Clause 3 The process of clauses 1 or 2, wherein the first kinase in an enzyme capable of catalyzing the transfer of a phosphate group; preferably wherein said first kinase is selected from the group consisting of:
  • - phosphotransferase with alcohol group as acceptor preferably selected from the group consisting of ethanolamine kinase (EC 2.7.1.82), amide-alcohol kinase, ceramide kinase (EC 2.7.1.138), pantoate kinase (EC 2.7.1.169), undecaprenol kinase (EC 2.7.1.66), choline kinase (EC 2.7.1.32), glycerol kinase (EC 2.7.1.30), glycerone kinase (EC 2.7.1.29), mevalonate kinase (EC 2.7.1.30), and hydrox- yethylthiazole kinase (EC 2.7.1.50); and/or
  • - phosphotransferases with a phosphate group as an acceptor EC 2.7.4; preferably selected from the group consisting of polyphosphate kinase, phosphomevalonate kinase, adenylate kinase, nucleoside- phosphate kinase, deoxycytidylate kinase, nucleoside-diphosphate kinase, phosphomethylpyrimidine kinase, guanylate kinase, dTMP kinase, nucleoside-triphosphate-adenylate kinase, (deoxy) adenylate kinase, T2-induced deoxynucleotide kinase, (deoxy)nucleoside-phosphate kinase, UMP/CMP kinase, thiamine-diphosphate kinase, thiamine-phosphate kinase, 3-phosphoglyce
  • acetate kinase EC 2.7.2. 1
  • acetate kinase acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10)
  • glutamate 5-ki- nase EC 2.7.2.11
  • glutamate 1-kinase EC 2.7.2.13
  • branched-chain-fatty-acid kinase EC 2.7.2.14
  • propionate kinase EC 2.7.2.15
  • 2-phosphoglycerate kinase EC 2.7.2.
  • [amino group carrier protein] -L-2 -aminoadipate 6-kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase( EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8).
  • Clause 4 The process according to any of the preceding clauses, wherein the first kinase is a hydroxyethylthiazole kinase from Escherichia coli (EC 2.7.1.50).
  • Clause 5 The process according to any of the preceding clauses, wherein the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least
  • Clause 6 The process according to any of the preceding clauses, preferably according to any of clauses 1 to 3, wherein the first kinase is a choline kinase from Plasmodium falciparum (PfCK) (EC 2.7.1.32).
  • PfCK Plasmodium falciparum
  • Clause 7 The process according to any of the preceding clauses, preferably according to any of clauses 1 to 3 or 6 wherein the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 8 The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first kinase is employed in a concentration of at least 0.0001 mg/ml.
  • step (ai) is performed in a liquid reaction medium and wherein the first kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, more preferably at most 10 mg/ml.
  • Clause 10 The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is selected from the group consisting of isoprenol, 3-methyl-3-buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Methyl-2-propen-l-ol, 3 -methylene- 1 -pentanol, 2- hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-methyl-4-penten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-buten-l-ol, 3-methyl-2-penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl- (2E)-penten-l-ol, (2E)-butene-2 -methyl
  • Clause 11 The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is isoprenol or prenol,
  • Clause 12 The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is isoprenol.
  • Clause 13 The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is employed in a concentration of at least 0.1 mM; preferably of at least 0.5 mM, more preferably of at least 1.0 mM.
  • Clause 14 The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, more preferably at most 1000 mM.
  • the first phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate preferably selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3-phospho- gly cerate.
  • Clause 16 The process according to any of the preceding clauses, wherein the first phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil.
  • Clause 17 The process according to any of the preceding clauses, wherein the first phosphate donor is adenosine triphosphate.
  • Clause 18 The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, more preferably at least 0.01 mM.
  • step (ai) is performed in a liquid reaction medium and wherein the first phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, more preferably at most 10 mM.
  • Clause 20 The process according to any of the preceding clauses, wherein the second kinase is an enzyme capable of catalyzing the phosphorylation of a phosphorylated alcohol; preferably of a phosphorylated hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isopentenyl monophosphate and/or dimethylallyl monophosphate.
  • the second kinase is an enzyme capable of catalyzing the phosphorylation of a phosphorylated alcohol; preferably of a phosphorylated hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isopentenyl monophosphate and/or dimethylallyl monophosphate.
  • Clause 21 The process according to any of the preceding clauses, wherein the second kinase in an enzyme capable of catalyzing the transfer of a phosphate group; preferably wherein said second kinase is a phosphotransferase with a phosphate group as acceptor; preferably selected from the group consisting of polyphosphate kinase (EC 2.7.4.
  • phosphomevalonate kinase (EC 2.7.4.2), adenylate kinase (EC 2.7.4.3), nucleoside-phosphate kinase (EC 2.7.4.4), thiamine-diphosphate kinase (EC 2.7.4.15), thi- amone-phosphate kinase (EC 2.7.4.16), famesyl-diphosphate kinase (EC 2.7.4.18), isopentenyl phosphate kinase (EC 2.7.4.26), and famesyl phosphate kinase (EC 2.7.4.32), phosphotransferases with a carboxy group as acceptor (EC 2.7.2): acetate kinase (EC 2.7.2.1), acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10), glutamate 5-kinase (EC 2.7.2.
  • [amino group carrier protein] -L-2 -aminoadipate 6- kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase( EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8).
  • Clause 22 The process according to any of the preceding clauses, wherein the second kinase is a isopentenyl phosphate kinase (EC 2.7.4.26).
  • Clause 23 The process according to any of the preceding clauses, wherein the second kinase is a isopentenyl phosphate kinase from Arabidopsis thaliana, Branchiostoma floridae, Methanolobus tindarius, Methanococcus vannielii, aus Roseiflexus castenholzii, Thermoplasma acidophilum, Methanothermo- bacterthermoautotropicus, Methanocaldococcus jannaschii, Haloferax volcanii, Sulfolobus solfataricus, Trichoplax adhaerens, or Methanosalsum zhilinae.
  • the second kinase is a isopentenyl phosphate kinase from Arabidopsis thaliana, Branchiostoma floridae, Methanolobus tindarius, Methanococcus vannielii, aus Roseiflexus castenholzii,
  • Clause 24 The process according to any of the preceding clauses, wherein the second kinase is a isopentenyl phosphate kinase from Roseiflexus castenholzii.
  • Clause 25 The process according to any of the preceding clauses, wherein the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least
  • Clause 26 The process according to any of the preceding clauses, preferably according to any of clauses 1 to 21 or 23, wherein the second kinase is a isopentenyl phosphate kinase from Methanolobus tindarius.
  • Clause 27 The process according to any of the preceding clauses, preferably according to any of clauses 1 to 21 or 23 or 26, wherein the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%,
  • Clause 28 The process according to any of the preceding clauses, wherein the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least
  • Clause 29 The process according to any of the preceding clauses, wherein step (a2) is performed in a liquid reaction medium and wherein the second kinase is employed in a concentration of at least 0.0001 mg/ml.
  • Clause 30 The process according to any of the preceding clauses, wherein step (a2) is performed in a liquid reaction medium and wherein the second kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, most preferably at most 10 mg/ml.
  • Clause 31 The process according to any of the preceding clauses, wherein the second phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3-phospho- glycerate.
  • the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3-phospho- glycerate.
  • Clause 32 The process according to any of the preceding clauses, wherein the second phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil.
  • Clause 33 The process according to any of the preceding clauses, wherein the second phosphate donor is adenosine triphosphate.
  • Clause 34 The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the second phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, most preferably at least 0.1 mM.
  • Clause 35 The process according to any of the preceding clauses, wherein the second phosphate donor and the first phosphate donor have different molecular weights.
  • Clause 36 The process according to any of the preceding clauses, wherein the second phosphate donor and the first phosphate donor differ by at least one methine group.
  • Clause 37 The process according to any of the preceding clauses, wherein the second phosphate donor and the first phosphate donor differ by at most one methine group.
  • Clause 38 The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the second phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, most preferably at most 10 mM.
  • Clause 39 The process according to any of the preceding clauses, wherein the second phosphate donor is the same as the first phosphate donor.
  • the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%,
  • the first hemiterpene alcohol is isoprenol
  • the first and second phosphate donor is adenosine triphosphate
  • the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or
  • Clause 41 A process for the preparation of a monoterpene diphosphate comprising the process according to any of the preceding clauses.
  • Clause 42 The process according to any of the preceding clauses, preferably according to clause 41 additionally comprising the steps of:
  • step (b) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a monoterpene diphosphate synthase thereby obtaining the monoterpene diphosphate.
  • Clause 43 The process according to any of the preceding clauses, preferably according to clause 42, wherein the second hemiterpene diphosphate is obtained in step (as) and/or in step (ai).
  • Clause 44 The process according to any of the preceding clauses, preferably according to any of clauses 42 to 43, wherein the monoterpene diphosphate synthase is an enzyme capable of catalyzing the condensation of one unit of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
  • Clause 45 The process according to any of the preceding clauses, preferably according to any of clauses 42 to 44, wherein the monoterpene diphosphate synthase is a geranyl diphosphate synthase (GPPS) (EC 2.5.1.1).
  • GPPS geranyl diphosphate synthase
  • Clause 46 The process according to any of the preceding clauses, preferably according to any of clauses 41 to 44, wherein the monoterpene diphosphate is a geranyl diphosphate (GPP).
  • GPP geranyl diphosphate
  • Clause 47 The process according to any of the preceding clauses, preferably according to any of clauses 41 to 45 wherein
  • the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
  • the monoterpene diphosphate is geranyl pyrophosphate (GPP); and/or
  • Clause 48 A process for the preparation of a sesquiterpene diphosphate comprising the process according to any of the preceding clauses, preferably according to any of clauses 1 to 40.
  • Clause 49 The process according to any of the preceding clauses, preferably according to clause 48 additionally comprising the steps of:
  • step (c) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a sesquiterpene diphosphate synthase thereby obtaining the sesquiterpene diphosphate.
  • Clause 50 The process according to any of the preceding clauses, preferably according to clause 49, wherein the second hemiterpene diphosphate is obtained in step (as) and/or in step (ai).
  • Clause 51 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 50, wherein the sesquiterpene diphosphate synthase is an enzyme capable of catalyzing the sequential condensation of one or more units of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
  • the sesquiterpene diphosphate synthase is an enzyme capable of catalyzing the sequential condensation of one or more units of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
  • Clause 52 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 51, wherein the sesquiterpene diphosphate synthase is a famesyl diphosphate synthase (FPPS) (EC 2.5.1.10).
  • FPPS famesyl diphosphate synthase
  • Clause 53 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 52, wherein the sesquiterpene diphosphate synthase is an FPPS from Geobacillus stea- rothermophilus, Bos Taurus, Gallus gallus, Pseudomonas aeruginosa, Rhizosolenia setigera, Ricinus communis, Lupinus albus, Homo sapiens, Artemisia spiciformi or Abies grandis.
  • the sesquiterpene diphosphate synthase is an FPPS from Geobacillus stea- rothermophilus, Bos Taurus, Gallus gallus, Pseudomonas aeruginosa, Rhizosolenia setigera, Ricinus communis, Lupinus albus, Homo sapiens, Artemisia spiciformi or Abies grandis.
  • Clause 54 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 9
  • Clause 55 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least
  • Clause 56 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least
  • Clause 57 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least
  • Clause 58 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least
  • Clause 59 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at
  • Clause 60 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least
  • Clause 61 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at
  • Clause 62 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at
  • Clause 63 The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at
  • Clause 64 The process according to any clauses 48 to 63, wherein the sesquiterpene diphosphate is famesyl diphosphate (FPP).
  • FPP famesyl diphosphate
  • Clause 65 The process according to any of the preceding clauses, preferably according to any of clauses 48 to 64 wherein
  • the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
  • the sesquiterpene diphosphate is famesyl pyrophosphate (FPP); and/or
  • the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%
  • Clause 66 A process for the preparation of a diterpene diphosphate comprising the process according to any of the preceding clauses, preferably according to any of clauses 1 to 40.
  • Clause 67 The process according to any of the preceding clauses, preferably according to clause 66 additionally comprising the steps of:
  • step (a2) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and (d) linking the first hemiterpene diphosphate obtained in step (a2) with the second hemiterpene diphosphate provided in step (as) under catalysis of a diterpene diphosphate synthase thereby obtaining the diterpene diphosphate.
  • Clause 68 The process according to any of the preceding clauses, preferably according to clause 67, wherein the second hemiterpene diphosphate is obtained in step (as) and/or in step (ai).
  • Clause 69 The process according to any of the preceding clauses, preferably according to any of clauses 67 to 68, wherein the diterpene diphosphate synthase is an enzyme capable of catalyzing the sequential condensation of one or more unit of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
  • the diterpene diphosphate synthase is an enzyme capable of catalyzing the sequential condensation of one or more unit of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
  • Clause 70 The process according to any of the preceding clauses, preferably according to any of clauses 67 to 69, wherein the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) (EC 2.5.1.29).
  • GPPS geranylgeranyl diphosphate synthase
  • Clause 71 The process according to any of the preceding clauses, preferably according to any of clauses 66 to 70, wherein the diterpene diphosphate is geranylgeranyl diphosphate (GGPP).
  • GGPP geranylgeranyl diphosphate
  • Clause 72 The process according to any of the preceding clauses, preferably according to any of clauses 66 to 71, wherein
  • the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
  • the diterpene diphosphate is geranylgeranyl pyrophosphate (GGPP); and/or
  • the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) belonging to EC 2.5.1.29.
  • GPPS geranylgeranyl diphosphate synthase
  • Clause 73 A process for the preparation of a tetraterpene comprising the process for the preparation of a diterpene diphosphate according to any of the preceding clauses, preferably according to any of clauses 66 to 72.
  • Clause 74 The process according to any of the preceding clauses, preferably according to clause 73 additionally comprising the step of:
  • step (e) linking at least two units of the diterpene diphosphate obtained in step (d) under catalysis of a tetraterpene synthase thereby obtaining the tetraterpene.
  • Clause 75 The process according to any of the preceding clauses, preferably according to clause 74, wherein the tetraterpene synthase is an enzyme capable of catalyzing the sequential condensation of two or more units of the diterpene diphosphate obtained in step (d).
  • Clause 76 The process according to any of the preceding clauses, preferably according to any of clauses 74 to 75, wherein the tetraterpene synthase is a phytoene synthase (EC 2.5.1.32).
  • Clause 77 The process according to any of the preceding clauses, preferably according to any of clauses 73 to 76, wherein the tetraterpene is phytoene.
  • Clause 78 The process according to any of the preceding clauses, preferably according to any of clauses 73 to 77, wherein - the tetraterpene is phytoene; and/or
  • the tetraterpene synthase is a phytoene synthase belonging to EC 2.5.1.32.
  • step (as) comprises the substeps of
  • step (as.2) phosphorylating the second hemiterpene monophosphate obtained in step (as.i) with a fourth phosphate donor under catalysis of a fourth kinase thereby obtaining the second hemiterpene diphosphate and a fourth phosphate acceptor.
  • Clause 80 The process according to any of the preceding clauses, preferably according to clause 79, wherein the second hemiterpene alcohol is selected from the group consisting of isoprenol, 3-methyl-3- buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Methyl-2 -propen- l-ol, 3 -methylene- 1 -pentanol, 2-hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-methyl-4-pen- ten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-buten-l-ol, 3 -methyl -2- penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl-(2E)-penten-l
  • Clause 81 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 80, wherein the second hemiterpene alcohol is preferably a constitutional isomer of the first hemiterpene alcohol.
  • Clause 82 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 81, wherein the second hemiterpene alcohol is isoprenol or prenol,
  • Clause 83 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 82, wherein the second hemiterpene alcohol is prenol.
  • Clause 84 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 83, wherein the second hemiterpene alcohol is employed in a concentration of at least 0.1 mM; preferably of at least 0.5 mM, most preferably of at least 1.0 mM.
  • Clause 85 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 84, wherein the second hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, most preferably at most 1000 mM.
  • Clause 86 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 85, wherein the third kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or
  • Clause 87 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 86, wherein the fourth kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%,
  • Clause 88 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 87, wherein the third phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the third phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6- phosphate, fructose- 1,6-bisphosphate, and 3 -phosphoglycerate.
  • the third phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the third phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6- phosphate, fructose- 1,6-bisphosphate, and 3 -phosphoglycer
  • Clause 89 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 88, wherein the third phosphate donor has the same molecular structure as the first and /or second phosphate donor.
  • Clause 90 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 89, wherein the fourth phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6- phosphate, fructose- 1,6-bisphosphate, 3 -phosphoglycerate.
  • the fourth phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6- phosphate, fructose- 1,6-bisphosphate, 3 -phosphoglycerate.
  • Clause 91 The process according to any of the preceding clauses, preferably according to any of clauses 78 to 90, wherein the fourth phosphate donor has the same molecular structure as the first and second and/or third phosphate donor.
  • Clause 92 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 91, wherein the molar ratio of the first hemiterpene alcohol relative to second hemiterpene alcohol is at least 1 : 10, or at least 1 : 2, or preferably at least 1 : 1 , or most preferably at least 2 : 1 or 3 : 1.
  • Clause 93 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 92, wherein the molar ratio of the first hemiterpene alcohol relative second hemiterpene alcohol is at most 100: 1; preferably at most 10 : 1 or at most 5 : 1 , or preferably at most 3: 1.
  • Clause 94 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 93, wherein
  • steps (ai), (a2), (as.i), (a-, 2) is repeated at least once;
  • steps (ai), (a2), (as.i), (a-, 2) are performed in a single reactor; and/or
  • step (as) comprises the substep of
  • Clause 96 The process according to any of the preceding clauses, preferably according to clause 95, wherein the isomerase is selected from the group consisting of steroid delta isomerase (EC 5.3.3. 1), vinylacetyl-CoA delta isomerase (EC 5.3.3.3), muconolactone delta-isomerase (EC 5.3.3.4), 5-carbox- ymethyl-2-ydroxymuconate delta isomerase (EC 5.3.3.10), isopiperitenone delta-isomerase (EC 5.3.3.11), polyenoic fatty acid isomerase (EC 5.3.3.13), trans-2,3-dihydro-3-hydroxyanthranilate isomerase (EC 5.3.3.17), lutein isomerase (5.3.3.22) and isopentenyl diphosphate isomerase (EC 5.3.3.2).
  • Clause 97 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 96, wherein the isomerase is a isopentenyl diphosphate
  • Clause 98 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 97, wherein the isomerase is a isopentenyl diphosphate isomerase from Schizosaccharo- myces pombe, Claviceps purpurea, Phaffia rhodozyma, Homo sapiens, Sus scrofa, Gallus gallus, Sola- num lycopersicum, Haematococcus lacustris, Gossypium barbadense, Escherichia coli, or Saccharomy- ces cerevisiae.
  • the isomerase is a isopentenyl diphosphate isomerase from Schizosaccharo- myces pombe, Claviceps purpurea, Phaffia rhodozyma, Homo sapiens, Sus scrofa, Gallus gallus, Sola- num lycopersicum, Haematococcus
  • Clause 99 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least
  • Clause 100 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%
  • Clause 101 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%
  • Clause 102 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 9
  • Clause 103 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
  • Clause 104 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 9
  • Clause 105 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 9
  • Clause 106 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 9
  • Clause 107 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 9
  • Clause 108 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 9
  • Clause 109 The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 9
  • Clause 110 The process according to any of the preceding clauses, wherein the second hemiterpene diphosphate is selected from the group consisting of isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), 3-methyl-3-buten-2-ol diphosphate, tiglic alcohol diphosphate, angelicic alcohol diphosphate, senecioic alcohol diphosphate and isovaleric alcohol diphosphate.
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • 3-methyl-3-buten-2-ol diphosphate 3-methyl-3-buten-2-ol diphosphate
  • tiglic alcohol diphosphate tiglic alcohol diphosphate
  • angelicic alcohol diphosphate senecioic alcohol diphosphate
  • isovaleric alcohol diphosphate isovaleric alcohol diphosphate.
  • Clause 111 The process according to any of the preceding clauses, wherein the second hemiterpene diphosphate is a structural isomer; preferably a constitutional isomer of the first hemiterpene diphosphate.
  • Clause 112 The process according to any of the preceding clauses, wherein the second hemiterpene diphosphate is isopentenyl pyrophosphate (IPP) or dimethylallyl pyrophosphate (DMAPP).
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • Clause 114 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 113, wherein
  • steps (ai), (a2), (as.i), (as.2), (as s) is repeated at least once;
  • steps (ai), (a2), (as.i), (as.2), (as s) are performed in a single reactor;
  • steps (a-, 2) and (as s) are performed simultaneously;
  • steps (a2) and (as s), are performed simultaneously.
  • Clause 115 The process according to any of the preceding clauses, preferably according to any of clauses 79 to 114, wherein
  • the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%,
  • the first hemiterpene alcohol is isoprenol
  • the first, second, third and fourth phosphate donor is adenosine triphosphate
  • the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%,
  • step (as) comprises the sub-steps of:
  • the isomerase is a isopentenyl diphosphate isomerases an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in
  • the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP); and/or
  • the third phosphate donor and fourth phosphate donor are adenosine triphosphate.
  • Clause 116 A process for the synthesis of a monoterpene comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a monoterpene diphosphate according to any clauses 41 to 47 or any of clauses 79 to 115.
  • Clause 117 The process according to any of the preceding clauses, preferably according to clause 116 additionally comprising the step of:
  • step (f) reacting a monoterpene diphosphate obtained in step (b) under catalysis of a monoterpene synthase thereby obtaining the monoterpene.
  • Clause 118 The process according to any of the preceding clauses, preferably according to clause 117, wherein the monoterpene synthase is an enzyme capable of catalyzing the rearrangement and/or cyclization of GPP into a monoterpene i.e.
  • the monoterpene synthase is a an selected from the group consisting of squalenesynthase (EC 2.5.1.21), 1,8-cineol synthase, (EC 4.2.3.108), (-)-sabinene synthase (EC 4.2.3.109), (+)-sabinene synthase (EC 4.2.3.110), 5-carene-synthase (EC 4.2.3.107) and (R)-limonene synthase (EC 4.2.3.20), preferably wherein the monoterpene synthase is (-)-sabinene synthase (EC 4.2.3.
  • Clause 120 A process for the synthesis of a carvone comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a monoterpene according to any clauses 116 to 119.
  • Clause 121 The process according to any of the preceding clauses, preferably according to clause 119 additionally comprising the steps of:
  • step (gi) reacting (R)-limonene obtained in step (f) under catalysis of a Limonene-6-hydroxylase (EC 1.14.13.48) thereby obtaining carvenol;
  • Clause 122 The process according to any of the preceding clauses, preferably according to clause 121, wherein
  • steps (gi) or (g 2 ) is repeated at least once;
  • steps (gi) or (g 2 ) is performed in a single reactor;
  • Clause 123 A process for the synthesis of a cannanbinoide comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a monoterpene diphosphate according to any clauses 41 to 47 or any of clauses 78 to 114.
  • Clause 124 The process according to any of the preceding clauses, preferably according to clause 123 additionally comprising the steps of:
  • step (hi) reacting a monoterpene, particularly GPP diphosphate obtained in step (b) with an olivetolic acid under catalysis of an aromatic prenyltransferase thereby obtaining cannabigerolic acid;
  • CBDA cannabidiol
  • THC tetrahydrocannabinol
  • Clause 125 The process according to any of the preceding clauses, preferably according to clause 124 wherein the aromatic prenyltransferase is a an selected from the group consisting of aspulvinone dimethylallyltransferase (EC 2.5.1.35), trihydroxypterocarpan dimethylallyltransferase (EC 2.5.1.36), 4- hydroxybenzoic acid oligoprenyltransferase (EC 2.5.1.39), naringenin 8 -dimethylallyltransferase (EC 2.5.1.70), 4-hydroxybenzoate geranyltransferase (EC 2.5.1.93), fumigaclavine A dimethylallyltransferase (EC 2.5.1.100), geranylpyrophosphate: olivetolate geranyltransferase (EC 2.5.1.102), 4-hydroxy- phenylpyruvate: dimethylallyl transferase (EC 2.5.1.111), homogentisate geranylgeranyltransferas
  • Clause 126 The process according to any of the preceding clauses, preferably according to clause 124, wherein
  • steps (hi) (I12) or (h ) is repeated at least once;
  • steps (hi) (I12) or (I13) is performed in a single reactor;
  • Clause 127 A process for the synthesis of a sesquiterpene comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a sesquiterpene diphosphate according to any clauses 48 to 65 or any of clauses 79 to 115.
  • Clause 128 The process according to any of the preceding clauses, preferably according to clause 127 additionally comprising the step of:
  • step (i) reacting a sesquiterpene diphosphate obtained in step (c) under catalysis of a sesquiterpene synthase thereby obtaining the sesquiterpene.
  • Clause 129 The process according to any of the preceding clauses, preferably according to clause 127 and/or 128, wherein the sesquiterpene synthase is an enzyme capable of catalyzing the rearrangement and/or cyclization of FPP into a sesquiterpene i.e.
  • the sesquiterpene synthase is a an enzyme selected from the group consisting of valencene synthase (EC 4.2.3.73), a-bisabolol-synthase (EC 4.2.3.138), (S)-beta-bisabolene synthase (EC 4.2.3.55), patchoulol synthase (EC 4.2.3.70), p-elemene synthase (EC 4.2.3.x), 5-cadinene synthase (EC 4.2.3.13), (+)-endo-beta-bergamotene synthase (EC 4.2.3.53), (-)-endo-alpha-bergamo- tene synthase (EC 4.2.3.54), exo-alpha-bergamotene synthase (EC 4.2.3.81), zingiberene synthase (EC 4.2.3.65), longifolene synthase (4.2.
  • Clause 130 The process according to any of the preceding clauses, preferably according to any of clauses 127 to 129, wherein the sesquiterpene is selected from the group consisting of valencene, a- bisabolol, P-bisabolene, patchoulol, P-elemene, 5-cadinene, a-trans-bergamotene, P-cis-bergamotene, P- trans-bergamotene, zingiberene, longifolene, a-santalene, P-santalene.
  • the sesquiterpene is selected from the group consisting of valencene, a- bisabolol, P-bisabolene, patchoulol, P-elemene, 5-cadinene, a-trans-bergamotene, P-cis-bergamotene, P- trans-bergamotene, zingiberene, longifolene, a-santalene, P-santalene
  • Clause 131 A process for the synthesis of a sesquiterpenoid comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a sesquiterpene according to any clauses 127 to 130.
  • Clause 132 The process according to any of the preceding clauses, preferably according to clause 131 additionally comprising the steps of:
  • Clause 134 A process for the synthesis of a famesol comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a sesquiterpene diphosphate according to any clauses 48 to 65 or any of clauses 79 to 115.
  • Clause 135 The process according to any of the preceding clauses, preferably according to clause 134 additionally comprising the steps of:
  • step (k) reacting a sesquiterpene diphosphate obtained in step (c) under catalysis of a FPP-diphosphatase thereby obtaining fame sol.
  • Clause 136 The process according to any of the preceding clauses, preferably according to clause 135 wherein the FPP-diphosphatase is a phosphatase belonging to EC 3.6.1.x and/or EC 3.1.3.x.
  • Clause 137 A process for the synthesis of a sclareol comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a diterpene diphosphate according to any clauses 66 to 72 or any of clauses 79 to 115.
  • Clause 138 The process according to any of the preceding clauses, preferably according to clause 137 additionally comprising the step of:
  • step (h) reacting a diterpene diphosphate, particularly GGPP, obtained in step (d) under catalysis of a labdenediol synthase (EC 4.2.1.133) thereby obtaining the labdenediol diphosphate (LDPP); and
  • step (h) reacting labdenediol diphosphate obtained in step (h) under catalysis of a sclareol synthase (EC 4.2.3. 141) thereby obtaining the sclareol.
  • Clause 139 The process according to any of the preceding clauses, preferably according to any of clauses 137 to 138, wherein
  • steps (h) or (I2) is repeated at least once;
  • steps (h) or (I2) is performed in a single reactor;
  • Clause 140 A process for the synthesis of a tetraterpenoid comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a tetraterpene according to any clauses 73 to 78 or any of clauses 79 to 115.
  • Clause 141 The process according to any of the preceding clauses, preferably according to clause 140 additionally comprising the step of:
  • step (m) reacting phytoene obtained in step (e) under catalysis of one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ⁇ -carotene desaturase (EC 1.3.99.26), lycopene-a-cyclase (EC 5.5.1.18), lycopene P-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpene.
  • Clause 142 The process according to any of the preceding clauses, preferably according to clause 141, wherein the tetraterpenoid is selected from the group consisting of phytofluene, 8-carotene, lycopene, neurosporene and P-carotene.
  • Clause 143 A process for the synthesis of ionone comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a tetraterpenoid according to any clauses 140 to 142.
  • Clause 144 The process according to any of the preceding clauses, preferably according to clause 143 wherein the process comprises the additional step of:
  • step (n) reacting the tetraterpenoid obtained in step (m) under catalysis of a carotenoid dioxygenase (EC 1.13.11.71 thereby obtaining ionone) .
  • Clause 145 The process according to any of the preceding clauses, preferably according to clause 144, wherein the tetraterpenoid is selected from the group consisting of phytoene, phytofluene, 8-carotene, lycopene, neurosporene and P-carotene.
  • step (o) phosphorylating the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2), the third phosphate acceptor optionally obtained in step (as.i) and/or the fourth phosphate acceptor optionally obtained in step (a3.2) with a phosphorylating agent, optionally under catalysis of a fifth kinase, thereby obtaining first phosphate donor, second phosphate donor, third phosphate donor, and/or fourth phosphate donor, respectively.
  • Clause 147 The process according to any of the preceding clauses, preferably according to clause 146, wherein the phosphorylating agent is acetyl phosphate, polyphosphate, phosphocreatine, acetyl phosphate, phosphopyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, 3 -phosphoglycerate, preferably wherein the phosphorylating agent is acetyl phosphate
  • Clause 148 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 147, wherein the fifth kinase is an enzyme capable of catalyzing the phosphorylation of the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2) and/or the third phosphate acceptor optionally obtained in step (as.i).
  • the fifth kinase is an enzyme capable of catalyzing the phosphorylation of the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2) and/or the third phosphate acceptor optionally obtained in step (as.i).
  • Clause 149 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 148, wherein the fifth kinase is an acetate kinase belonging to EC 2.7.2. 1 or EC 2.7.4.1.
  • Clause 150 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 149, wherein the fifth kinase is an acetate kinase from Geobacillus kaustophilus, Escherichia coli, Rhodococcus erythropolis, Acinetobacter baylyi, Butyrivibrio fibrisolvens, Geobacillus cald- oxylosilyticus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus pu- milus, Bacillus shackletonii or Sinorhizobium meliloti.
  • the fifth kinase is an acetate kinase from Geobacillus kaustophilus, Escherichia coli, Rhodococcus erythropolis, Acinetobacter baylyi, Butyrivibrio fibrisolvens, Geobacillus cald- oxylosilyticus, Bacillus am
  • Clause 151 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 152 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 153 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 154 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 155 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 156 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 157 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 158 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least
  • Clause 159 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 160 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least
  • Clause 161 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 162 The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at
  • Clause 163 The process according to any of the preceding clauses, preferably according to any of clauses to clauses 146 to 162, wherein
  • the phosphorylating agent is acetyl phosphate
  • the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or
  • Clause 164 The process according to any of the preceding clauses, wherein the process take place at a temperature of 2.0 °C to 99.5 °C, or 3.0°C to 95.0°C, or 4.0°C to 90.0°C, or 5.0°C to 85.0°C, or 15.0°C to 80.0°C, or 25.0°Cto 75.0°C, or 28.0°Cto 75.0°C, or 30.0°C to 60.0°C, or 33.0°C to 50.0°C, or 35.0°C to 45.0°C.
  • Clause 165 The process according to any of the preceding clauses, wherein the process take place in a liquid reaction medium, wherein said liquid reaction medium is characterized by a pH in the range of 5.0 to 8.5, or 5.3 to 8.5, or 5.5 to 8.5, or 5.8 to 8.5, or 6.0 to 8.5, or 6.4 to 8.5, or 6.6 to 8.5, or 6.9 to 8.5, 7.5 to 8.5, or 8.0 to 8.5.
  • Clause 166 A composition comprising a (i) first hemiterpene diphosphate obtainable by the process according to any of the proceeding clauses in combination with (ii) a first kinase, (iii) a first phosphate donor, (iv) a second kinase, and (v) a second phosphate donor or any combination of the foregoing.
  • Clause 167 The composition according to clause 166 further comprising a (i) monoterpene diphosphate obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 41 to 47 or any of clauses 79 to 115 in combination with a (ii) monoterpene diphosphate synthase.
  • Clause 168 The composition according to any of clauses 166 or 167 further comprising a (i) monoterpene obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 116 to 119 or any of clauses 79 to 115 in combination with a (ii) monoterpene synthase.
  • Clause 169 The process according to clause 167, wherein the monoterpene is selected from the group consisting of squalene 1,8-cineol, (-)-sabinene, (+)-sabinene, 5-carene and (R)-limonene, preferably wherein the monoterpene is squalene, (-)-sabinene, (+)-sabinene or 5-3-carene.
  • Clause 170 The composition according to any of clauses 166 to 169 further comprising a (i) sesquiterpene diphosphate obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 48 to 65 or any of clauses 79 to 115 in combination with a (ii) sesquiterpene diphosphate synthase.
  • Clause 171 The composition according to clause 170 further comprising a (i) sesquiterpene obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 127 to 130 or any of clauses 79 to 115 in combination with a (ii) sesquiterpene synthase.
  • Clause 172 The composition according to clause 171, wherein the sesquiterpene is selected from the group consisting of valencene, a-bisabolol, P-bisabolene, patchoulol, P-elemene, 5-cadinene, a-trans- bergamotene, P-cis-bergamotene, P-trans-bergamotene, zingiberene, longifolene, a-santalene, P-san- talene.
  • the sesquiterpene is selected from the group consisting of valencene, a-bisabolol, P-bisabolene, patchoulol, P-elemene, 5-cadinene, a-trans- bergamotene, P-cis-bergamotene, P-trans-bergamotene, zingiberene, longifolene, a-santalene, P-san- talene.
  • Clause 173 The composition according to any of clauses 166 to 171 further comprising a (i) sesquit- erpenoid obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 131 to 133 or any of clauses 79 to 115 in combination with a (ii) monooxygenase (EC 1.14.x.x).
  • Clause 174 The composition according to clause 173, wherein the sesquiterpenoid is selected from the group consisting of nootkatone, hemandulcin, a-santalol, P-santalol.
  • Clause 175 The composition according to any of clauses 166 to 174 further comprising a (i) diterpene diphosphate obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 66 to 72 or any of clauses 79 to 115 in combination with a (ii) diterpene diphosphate synthase.
  • Clause 176 The composition according to clause 175 further comprising a (i) sclareol obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 137 to 139 in combination with a (ii) labdenediol diphosphate and a sclareol synthase (EC 4.2.3.141).
  • Clause 177 The composition according to any of clauses 166 to 176 further comprising a (i) tetraterpene obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 73 to 78 or any of clauses 79 to 115 in combination with a (ii) tetraterpene synthase.
  • Clause 178 The composition according to any clause 177, wherein the tetraterpene is phytoene.
  • Clause 179 The composition according to any of clauses 166 to 178 further comprising a (i) tetraterpenoid obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 140 to 142 or any of clauses 79 to 115 in combination with (ii) one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ⁇ -carotene desaturase (EC 1.3.99.26), lycopene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpene Clause 180: The composition according to clause 179, wherein the tetraterpenoid is selected from the group consisting of phytofluene, 8-carotene, lycopene, neurosporene and [3-carotene.
  • Clause 181 The composition according to any of clauses 166 to 180 further comprising a (i) ionone obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 143 to 145 or any of clauses 79 to 115 in combination with a (ii) carotenoid dioxygenase (EC 1.13.11.71).
  • Cloning The genes were cloned into the expression vector pLElA18 (derivative of pRSF-lb, Novagen) containing an N-terminal Hise-tag for purification of the enzymes. The resulting plasmids were each used for transformation of E. coli BL21(DE3) cells.
  • the eluted solution was rebuffered into 100 mM Tris-HCl-buffer pH 7,5, 150 mM NaCl, 10% (v/v) glycerol using PD-10 desalting columns according to the manufacturer (GE Healthcare). Aliquots of the purified enzyme solution were shock frozen in liquid nitrogen and stored at -20 °C.
  • EcHTK For the determination of the standard activity of EcHTK (SEQ ID NO: 1), the synthesis of IP (isoprenol) from IOH (isoprenol) and ATP was assayed.
  • An EcHTK (SEQ ID NO: 1) unit (U) corresponds to the synthesis of 1 pmol IP per minute from 100 mM IOH and 1,25 mM ATP in 200 mM Tris-HCL buffer pH 8.5, 50 mM MgCl 2 , 150 mM PolyP, 0. 1 mg/ml SmPPK (SEQ ID NO:37) at 30 °C. Reaction progress was determined discontinuously by stopping reaction after a given reaction time through heating at 90 °C for 10 min and quantifying the amount of synthesized IP via HPLC calibrated with an external standard.
  • RcIMK SEQ ID NO:3
  • IPP IPP from IP and ATP
  • An RcIMK (SEQ ID NO:3) unit (U) corresponds to the synthesis of 1 pmol IPP per minute from 50 mM IP and 5 mM ATP in 200 mM Tris-HCL buffer pH 8.5, 20 mM MgC’T. 100 mM AcP, 5 U/ml GkACK (SEQ ID NO:26) at 30 °C. Reaction progress was determined discontinuously by stopping reaction after a given reaction time through heating at 90 °C for 10 min and quantifying the amount of synthesized IP via HPLC calibrated with an external standard.
  • GsFPPS SEQ ID NO:5
  • a GsFPPS unit (U) corresponds to the synthesis of 1 pmol FPP per minute from 1 mM IPP and 1 mM DMAPP in 50 mM Tris-HCL buffer pH 7.5, 0.40 mM MgC’T at 30 °C. Reaction progress was determined discontinuously by stopping reaction after a given reaction time by adding one volume of abs. MeOH and quantifying the amount of synthesized IP via HPLC calibrated with an external standard.
  • EXAMPLE 2 Screening of different kinase for the synthesis of the IP and DMAP
  • EXAMPLE 3 Synthesis of IPP and DMAPP under employment of a polyphosphate-based ATP- regeneration system.
  • EXAMPLE 3.2 Synthesis of DMAPP - under employment of an Isomerase
  • IPP isopentenyl diphosphate
  • DMAPP dimethylallyl diphosphate
  • a reaction containing 50 mM IPP, 5 mM MgCT and 0.2 mg/ml SpIDI (SEQ ID NO: 15) in 50 mM Tris-HCl, pH 7.5 was used.
  • the reaction was incubated at 30 °C for 5 h to give Dimethylallyl pyrophosphate DMAPP.
  • the isomerization from IPP to DMAPP can either be performed in a separate step, as described here. Or it can be coupled to the reaction from IOH to IPP described in example 3.1.
  • the DMAPP yield can be significantly increased as compared to the case in which a polyphosphate-based ATP-regeneration system with SmPPK (SEQ ID NO:37) and PolyP is used.
  • SmPPK SEQ ID NO:37
  • the chemical driving force of the reaction with AcP as phosphate donor is significantly higher, so that the reaction equilibrium of RcIMK (SEQ ID NOT) is shifted more strongly to the product side.
  • EXAMPLE 4.4 Comparative synthesis of IPP under employment of an acetyl phosphate-based ATP-regeneration system or a polyphosphate-based ATP-regeneration system.
  • reaction mixtures were incubated at 30°C for 20 h, stopped by heating at 90 °C for 10 min and the resulting IPP quantified via HPLC, calibrated with an external standard.
  • the resulting yield was 80 % IPP with GkACK (SEQ ID NO:26) and 8 % IPP with SmPPK (SEQ ID NO:37).
  • the reaction was started by adding GsFPPS (SEQ ID NO:5) to a final concentration of 0,2 mg/ml.
  • the mixture was incubated at 30 °C for 4-6 h, stopped by adding one volume of abs. MeOH and the resulting FPP was quantified via HPLC, calibrated with an external standard.
  • EXAMPLE 6 General synthesis of sesquiterpenes starting from IOH under employment of a polyphosphate-based ATP-regeneration system
  • EXAMPLE 7 General synthesis of sesquiterpenes starting from POH under employment of a polyphosphate-based ATP-regeneration system
  • EXAMPLE 8 General synthesis of sesquiterpenes starting from IOH and POH under employment of a polyphosphate-based ATP-regeneration system
  • EXAMPLE 9 General synthesis of sesquiterpenes starting from IOH and acetyl phosphate under employment of a acetyl phosphate -based ATP-regeneration system
  • EXAMPLE 11 General synthesis of sesquiterpenes starting from IOH and POH under employment of a acetyl phosphate -based ATP-regeneration system

Abstract

The invention relates to process for isoprenoid production by an in-vitro synthetic platform involving at least the steps of phosphorylating a first hemiterpene alcohol with a first phosphate donor under catalysis of a first kinase thereby obtaining a first hemiterpene monophosphate and phosphorylating the latter with a second phosphate donor under catalysis of a second kinase thereby obtaining at least first hemiterpene diphosphate. The invention further relates to a composition comprising first hemiterpene diphosphate obtainable by the process according to the invention.

Description

In-vitro synthetic platform for the generation of isoprenoids
[0001] Priority is claimed of European patent application no. 21 152 479.8 and European patent application no. 21 152 478.0, both filed on January 20, 2021.
[0002] The invention relates to a process for isoprenoid production by an in-vitro synthetic platform involving at least the steps of phosphorylating a first hemiterpene alcohol with a first phosphate donor under catalysis of a first kinase thereby obtaining a first hemiterpene monophosphate and phosphorylating the latter with a second phosphate donor under catalysis of a second kinase thereby obtaining at least first hemiterpene diphosphate. The invention further relates to a composition comprising at least first hemiterpene diphosphate obtainable by the process according to the invention.
[0003] Due to their structural complexity and biological significance isoprenoids, including cannabinoids, terpenes and terpenoids, have gained great importance in a variety of industrially relevant applications for the production of pharmaceuticals, cosmetics, perfumes, flavors, animal feed supplements and nutraceuticals.
[0004] In particular, terpenes and terpenoids may be obtained from natural sources i.e. by extraction. However, a fundamental problem associated thereto regards the low availability, above all, due to the low amenability of such natural sources to large-scale production.
[0005] In-vivo approaches for obtaining terpenes and terpenoids have also been introduced. For example, Williams et. al (ACS Synth. Biol. 2019, 8, 2, 232-238) relates to an artificial alcohol-dependent hemiterpene biosynthetic pathway designed and coupled to several isoprenoid biosynthetic systems, affording lycopene and a prenylated tryptophan in robust yields. However, a core limitation related to such in-vivo approaches, concerns the cytotoxicity of the target product which lowers final yields and may render the whole process unpracticable on large scale.
[0006] For this reason in-vitro approaches are usually preferred. In this regard it is of note that despite the structural diversity of terpenoids, they are largely derived from two major diphosphorylated building blocks: isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Following their generation, such diphosphorylated building blocks may be subjected to sequential condensation under catalysis of specific prenyltransferases thereby resulting in geranyl pyrophosphate (GPP), famesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) which then may be converted to the more complex terpenoids under catalysis of appropriate terpene synthases. The resulting terpenoids may be then further functionalized either chemically or under enzymatic catalysis.
[0007] A number of approaches have been developed in this regard. For example, lacazio et. al. (ACS Omega 2019, 4, 4, 7838-7849) disclose a terpene mini -path (TMP) relying on a first phosphorylation by a promiscuous acid phosphatase (AP) coupled with an isopentenyl phosphate kinase (IPK). The in vitro and in vivo utility of said mini-path was demonstrated in the explorative synthesis of diketopiperazine tryprostatin B (TB). However, the used promiscuous acid phosphatase may lead to dephosphorylation side reaction of other phosphorylated species like ATP or to byproducts like IP and IPP making this kind of process economically unattractive.
[0008] WO 2011/009132 A2 relates to methods and compositions pertaining to the synthesis of isoprenoid diphosphates by using selected enzymes ( i.e. isopentyl monophosphate kinase (IMK)) and methods to identify amino acid substitutions within the IMKs which increase isoprenoid production rate. However, this method requires expensive starting materials such as isoprenoid diphosphate which direct impacts on the economic feasibility of the whole process.
[0009] WO 2019/023192 Al relates to herbicide-detoxifying enzymes, compositions containing one or more of the enzymes, and plant seeds treated with the enzymes.
[0010] WO 2019/232025 A2 relates to cells and cell-free systems for the conversion of isopentenols to isoprenoid precursors which are reacted with enzymes (choline kinase from S. cerevisiae) that phosphorylate short chain alcohol to produce the corresponding monophosphate, followed by phosphorylation of the latter to afford diphosphates upon reaction with a suitable enzyme (e.g. isopentenyl phosphate kinase (IPK)). The optional use of an isomerization enzyme such as an isopentenyl pyrophosphate isomerase (IDI) is also disclosed which is instrumental for the conversion of one or more synthetic precursors into the respective constitutional isomer.
[0011] WO 2020/150340 Al discloses methods for preparing isoprenoid subunits, as well as methods of employing these isoprenoid subunits for the synthesis of isoprenoids. A primary alcohol is reacted with an enzyme performing a first phosphorylation such as a phosphatase that exhibits bidirectional activity, a single enzyme or a system involving two independent kinases (i.e. a first and second kinase), in the presence of ATP to generate the corresponding diphosphates. From a suitability test of a set of diverse kinases for the first phosphorylation step, it is apparent that PhoN, a non-specific phosphatase from S. flexneri, shows a superior activity in the phosphorylation of DMAA/ISO. [0012] The processes of the prior art are not satisfactory in every respect and there is a demand for improved processes.
[0013] It is an object of the invention to provide improved processes for the enzymatic synthesis of terpenoids including but not limited to isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP). The processes should be efficient and provide the desired products in high yields with high specificity. The starting materials should be easily available at low costs making the overall process economically advantageous.
[0014] This object has been achieved by the subject-matter of the patent claims.
[0015] It has been surprisingly found that isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP) can be efficiently produced by enzymatic phosphorylation from the corresponding hemiterpene alcohols which phosphorylation can be greatly promoted by appropriate choice of the kinases involved in this step.
[0016] Further, it has been surprisingly found that employment of a phosphate regeneration system substantially enhances the yield of IPP and/or DMAPP thereby augmenting the overall efficiency of the process, also from an economic perspective.
[0017] A first aspect of the invention relates to a process for the preparation of a first hemiterpene diphosphate comprising the steps of:
(ai) phosphorylating a first hemiterpene alcohol with a first phosphate donor under catalysis of a first kinase thereby obtaining a first hemiterpene monophosphate and a first phosphate acceptor; and
(a2) phosphorylating the first hemiterpene monophosphate obtained in step (ai) with a second phosphate donor under catalysis of a second kinase thereby obtaining a first hemiterpene diphosphate and a second phosphate acceptor; wherein "terpene" encompasses terpenes as well as terpenoids according to IUPAC.
[0018] For the purpose of the specification, for the ease of definition and sake of conciseness, unless expressly stated otherwise, the term, prefix or suffix "terpene" is preferably not limited to "terpenes" according to IUPAC, but is to be interpreted in a broader meaning to additionally also encompass "terpenoids" according to IUPAC.
[0019] According to IUPAC, "terpenes" are hydrocarbons of biological origin having carbon skeletons formally derived from isoprene [CH2=C(CH3)CH=CH2], This class is subdivided into the C5 hemiterpenes, CIO monoterpenes, C15 sesquiterpenes, C20 diterpenes, C25 sesterterpenes, C30 triterpenes, C40 tetraterpenes (carotenoids) and C5n polyterpenes (see https://goldbook.iupac.org/terms/view/T06278). According to IUPAC, "terpenoids" are natural products and related compounds formally derived from isoprene units. They contain oxygen in various functional groups. This class is subdivided according to the number of carbon atoms in the same manner as are terpenes. The skeleton of terpenoids may differ from strict additivity of isoprene units by the loss or shift of a fragment, generally a methyl group (see https://goldbook.iupac.org/terms/view/T06279). Thus, according to IUPAC, "terpenes" are pure hydrocarbons, whereas "terpenoids" contain oxygen in various functional groups. Nonetheless, it is common practice to use the term "terpene" in the nomenclature of compounds containing oxygen in various functional groups, such as "terpene alcohol", which in accordance with IUPAC nomenclature, due to the presence of the alcohol as oxygen functional group, is not a terpene but a terpenoid according to IUPAC.
[0020] For the purpose of the specification, especially for the ease of definition and sake of conciseness, unless expressly stated otherwise, "terpene" refers to both, the pure hydrocarbons (terpenes according to IUPAC) and the derivatives contain oxygen in various functional groups (terpenoids according to IUPAC). When the term, prefix or suffix "terpene" is used, it preferably refers to a derivative containing oxygen in at least one of various functional groups.
[0021] The process according to the invention is drawn to the preparation of a first hemiterpene diphosphate. For the purpose of the specification, a "hemiterpene diphosphate" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC, more specifically as a hemiterpene or hemiterpenoid comprising up to five carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage. A hemiterpene diphosphate may for instance be obtained by reaction of pyrophosphoric acid (H4P2O7) with a hemiterpene alcohol, or analogously by reacting phosphoric acid (H3PO4) with a hemiterpene monophosphate.
[0022] Step (ai) of the process according to the invention involves phosphorylation of a hemiterpene alcohol. For the purpose of the specification, a "hemiterpene alcohol" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC, more specifically as a hemiterpene or hemiterpenoid comprising up to five carbons atoms as well as at least one alcoholic group within the hydrocarbon skeleton.
[0023] Step (a2) of the process according to the invention involves phosphorylation of a hemiterpene monophosphate. For the purpose of the specification, a "hemiterpene monophosphate" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC, more specifically as a hemiterpene or hemiterpenoid comprising up to five carbons atoms as well as one phosphate group. A hemiterpene monophosphate may for instance be obtained by reacting phosphoric acid (H3PO4) with a hemiterpene alcohol. [0024] For the purpose of the specification, "phosphorylation" refers to the attachment of a phosphate group to an organic molecule via a suitable mechanism for example by condensation, substitution or addition.
[0025] Steps (ai) and (a2) of the process according to the invention involve phosphorylation under catalysis of a kinase. For the purpose of the specification, a "kinase" is preferably to be interpreted as an enzyme that catalyzes the transfer of phosphate groups from a phosphate donor to specific substrates.
[0026] Steps (ai) and (a2) of the process according to the invention involve phosphorylation with a phosphate donor. For the purpose of the specification, a "phosphate donor" is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction.
[0027] Steps (ai) and (a2) of the process according to the invention involve phosphorylation with a phosphate donor obtaining a phosphate acceptor. For the purpose of the specification, a "phosphate acceptor" is preferably to be interpreted as a chemical species deriving from a dephosphorylation reaction of the corresponding phosphate donor, i.e. presenting at least one phosphate unit less than the corresponding phosphate donor. In that regard it is contemplated that the phosphate acceptor is preferably characterized by a molecular structure amenable to undergo addition of a phosphate unit thereby re- pristinating the corresponding phosphate donor.
[0028] In the meaning of this invention, the percent identity is calculated as: Sequence Identity [%] = number of Matches/ L x 100, wherein L is the number of aligned positions, i.e. identities and nonidentities (including gaps, if any). Identity is preferably calculated using BLASTP (see, for example, Alt- schul SF et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; or Altschul SF (2005) "Protein database searches using compositionally adjusted substitution matrices." FEBS J. 272:5101-5109), preferably with the following algorithm parameters: Matrix: BLOSUM62; Gap Costs: Existence: 11 Extension: 1, Expect threshold: 10 and Word size: 6. Results are filtered for sequences with more than 35 % query coverage. BlastP can be accessed online at the NCBI Homepage (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM= blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Other program settings can be adjusted as desired, for example using the following settings:
- Field "Enter Query Sequence": Query subrange: none
- Field "Choose Search Set": Database: non-redundant protein sequences (nr); optional parameters: none
- Field "Program Selection": Algorithm: blastp (protein-protein BLAST) - Algorithm parameters: Field "General parameters": Max target sequences: 20000; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0
- Algorithm parameters: Field "Scoring parameters": Matrix: BLOSUM62; Gap Costs: Existence: 11 Extension: 1; Compositional adjustments: Conditional compositional score matrix adjustment
- Algorithm parameters: Field "Filters and Masking": Filter: none; Mask: none
[0029] For the purpose of the specification, the wording "(deoxy)" shall preferably be understood as being entirely optional, i.e. covering both deoxy and non-deoxy variants of the same chemical substance.
[0030] For the purpose of the specification, "conversion" is preferably to be interpreted as the ratios between the molar amount of consumed reactant and the molar amount of reactant initially employed in the reaction.
[0031] For the purpose of the specification, the "first kinase" is preferably to be interpreted as an enzyme capable of catalyzing the monophosphorylation of an alcohol, said alcohol being preferably a hemiterpene alcohol, and even more preferably isoprenol and/or prenol.
[0032] Thus, in preferred embodiments, the first kinase is an enzyme capable of catalyzing the transfer of a phosphate group selected from the group consisting of:
- phosphotransferase with alcohol group as acceptor; preferably selected from the group consisting of ethanolamine kinase (EC 2.7.1.82), amide-alcohol kinase, ceramide kinase (EC 2.7.1.138), pantoate kinase (EC 2.7.1.169), undecaprenol kinase (EC 2.7.1.66), choline kinase (EC 2.7.1.32), glycerol kinase (EC 2.7.1.30), glycerone kinase (EC 2.7.1.29), mevalonate kinase (EC 2.7.1.30), and hydrox- yethylthiazole kinase (EC 2.7.1.50);
- phosphotransferases with a phosphate group as an acceptor (EC 2.7.4); preferably selected from the group consisting of polyphosphate kinase, phosphomevalonate kinase, adenylate kinase, nucleosidephosphate kinase, deoxycytidylate kinase, nucleoside-diphosphate kinase, phosphomethylpyrimidine kinase, guanylate kinase, dTMP kinase, nucleoside-triphosphate-adenylate kinase, (deoxy) adenylate kinase, T2-induced deoxynucleotide kinase, (deoxy) nucleoside-phosphate kinase, UMP/CMP kinase, thiamine-diphosphate kinase, thiamine-phosphate kinase, 3-phosphoglyceroyl- phosphate-polyphosphate phosphotransferase, famesyl-diphosphate kinase, 5 -methyldeoxy cytidine- 5'-phosphate kinase, dolichyl-diphosphate-polyphosphate phosphotransferase, inositol-hexakisphos- phate kinase, UMP kinase, ribose 1,5 -bisphosphate phosphokinase, diphosphoinositol-pentakisphos- phate kinase, (d)CMP kinase, isopentenyl phosphate kinase, [pyruvate, phosphate dikinase] -phosphate phosphotransferase, [pyruvate, water dikinase] -phosphate phosphotransferase, Kdo2-lipid A phosphotransferase, lipid A phosphoethanolamine transferase, [5-(aminomethyl)furan-3-yl]methyl phosphate kinase, famesyl phosphate kinase, yeast UMP kinase, polyphosphate- AMP phosphotransferase, and geranylgeranyl phosphate kinase;
- phosphotransferases with a carboxy group as acceptor (EC 2.7.2): acetate kinase (EC 2.7.2.1), acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10), glutamate 5-ki- nase (EC 2.7.2.11), glutamate 1-kinase (EC 2.7.2.13), branched-chain-fatty-acid kinase (EC 2.7.2.14), propionate kinase (EC 2.7.2.15), 2-phosphoglycerate kinase (EC 2.7.2. 16), [amino group carrier protein] -L-2 -aminoadipate 6-kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase( EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8).
[0033] In preferred embodiments, the first kinase is a hydroxyethylthiazole kinase from Escherichia coli (EC 2.7. 1.50).
[0034] Preferably, the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 1.
[0035] In a yet preferred embodiment, the first kinase is a choline kinase from Plasmodium falciparum (PfCK) (EC 2.7.1.32).
[0036] Preferably, the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:2. [0037] The first kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2. This means that the first kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the first kinase according to the invention may essentially consist of said amino acid sequence. When the first kinase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the first kinase is to be immobilized on a solid support, e.g. for purification purposes.
[0038] Preferably, step (ai) is performed in a liquid reaction medium and the first kinase is employed in a concentration of at least 0.0001 mg/ml.
[0039] Preferably, step (ai) is performed in a liquid reaction medium and the first kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, more preferably at most 10 mg/ml.
[0040] For the purpose of the specification, the first "hemiterpene alcohol" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to five carbons atoms as well as at least one alcoholic group within the hydrocarbon skeleton.
[0041] In this context, the first hemiterpene alcohol is preferably selected from the group consisting of isoprenol, 3-methyl-3-buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Methyl-2-propen-l-ol, 3 -methylene- 1 -pentanol, 2-hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-methyl-4-penten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-bu- ten-l-ol, 3-methyl-2-penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl-(2£)-penten-l-ol, (2E)-butene-2-methyl- 1,4-diol, and 3-phenyl-2-propen-l-ol.
[0042] Preferably, the first hemiterpene alcohol is isoprenol or prenol, and even more preferably isoprenol.
[0043] Preferably, the first hemiterpene alcohol is employed in a concentration of at least 0.1 mM; preferably of at least 0.5 mM, more preferably of at least 1.0 mM.
[0044] Preferably, step (ai) is performed in a liquid reaction medium and the first hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, more preferably at most 1000 mM. [0045] For the purpose of the specification, the "first phosphate donor" is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction.
[0046] In preferred embodiments, the first phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably the first phosphate donor is a nucleoside mono- or polyphosphate preferably selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glu- cose-6-phosphate, fructose- 1,6-bisphosphate, or 3 -phosphoglycerate.
[0047] Preferably, the first phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil and even more preferably of adenosine.
[0048] Preferably, the first phosphate donor is adenosine triphosphate (ATP).
[0049] In this context, step (ai) is preferably performed in a liquid reaction medium and the first phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, more preferably at least 0.01 mM.
[0050] Preferably, step (ai) is performed in a liquid reaction medium and the first phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, more preferably at most 10 mM.
[0051] In step (a2) of the process according to the invention, the first hemiterpene monophosphate obtained from step (ai) is reacted with a second phosphate donor under catalysis of a second kinase thereby obtaining a first hemiterpene diphosphate and a second phosphate acceptor.
[0052] For the purpose of the specification, the "second kinase" preferably refers to an enzyme capable of catalyzing the phosphorylation of a mono- or polyphosphorylated alcohol, preferably of a mono- or polyphosphorylated hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isopentenyl monophosphate and/or dimethylallyl monophosphate.
[0053] Thus, in preferred embodiments, the second kinase in an enzyme capable of catalyzing the transfer of a phosphate group, such as phosphotransferase with a phosphate group as acceptor. [0054] In yet preferred embodiments, the second kinase in an enzyme selected from the group consisting of polyphosphate kinase (EC 2.7.4. 1), phosphomevalonate kinase (EC 2.7.4.2), adenylate kinase (EC 2.7.4.3), nucleoside-phosphate kinase (EC 2.7.4.4), thiamine-diphosphate kinase (EC 2.7.4.15), thi- amone-phosphate kinase (EC 2.7.4.16), famesyl-diphosphate kinase (EC 2.7.4.18), isopentenyl phosphate kinase (EC 2.7.4.26), and famesyl phosphate kinase (EC 2.7.4.32), phosphotransferases with a carboxy group as acceptor (EC 2.7.2): acetate kinase (EC 2.7.2.1), acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10), glutamate 5-kinase (EC 2.7.2.11), glutamate 1-kinase (EC 2.7.2.13), branched-chain-fatty-acid kinase (EC 2.7.2.14), propionate kinase (EC 2.7.2.15), 2-phosphoglycerate kinase (EC 2.7.2. 16), [amino group carrier protein] -L-2 -aminoadipate 6- kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase (EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8), preferably a isopentenyl phosphate kinase (EC 2.7.4.26).
[0055] In this context, it is particularly preferred that said isopentenyl phosphate kinase is selected from an organism selected from the group consisting of Arabidopsis thaliana, Branchiostoma floridae, Meth- anolobus tindarius,Methanococcus vannielii, aus Roseiflexus castenholzii, Thermoplasma acidophilum, Methanothermobacter thermoautotropicus , Methanocaldococcus jannaschii, Haloferax volcanii, Sul- folobus solfataricus, Trichoplax adhaerens, or Methanosalsum zhilinae.
[0056] In preferred embodiments, the second kinase is a isopentenyl phosphate kinase from Roseiflexus castenholzii.
[0057] Preferably, the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:3.
[0058] In preferred embodiments, the second kinase is a isopentenyl phosphate kinase from Methanol- obus tindarius.
[0059] Preferably, the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:4.
[0060] In preferred embodiments, the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61 %, or at least 62%, or at least 63 %, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to the amino acid sequence of the first kinase.
[0061] The second kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of the first kinase, SEQ ID NO:3 or SEQ ID NO:4. This means that the second kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the second kinase according to the invention may essentially consist of said amino acid sequence. When the second kinase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-ter- minus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the second kinase is to be immobilized on a solid support, e.g. for purification purposes.
[0062] Preferably, step (ai) is performed in a liquid reaction medium and the second kinase is employed in a concentration of at least 0.0001 mg/ml.
[0063] Preferably, step (ai) is performed in a liquid reaction medium and the second kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, most preferably at most 10 mg/ml.
[0064] For the purpose of the specification, the "second phosphate donor" is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction. In this regard, the second phosphate donor preferably belongs to the same chemical category as the first phosphate donor, i.e. organic or inorganic monophosphate or polyphosphate, and is capable of donating a phosphate unit during the course of a reaction.
[0065] In this context, the second phosphate donor may not substantially differ from the first phosphate donor and preferably be a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3 -phosphoglycerate.
[0066] Preferably, the second phosphate donor is the same as the first phosphate donor.
[0067] In preferred embodiments, the second phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil and preferably of adenosine.
[0068] Preferably, this second phosphate donor is adenosine triphosphate.
[0069] In another embodiment, step (a2) is preferably performed in a liquid reaction medium and the second phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, most preferably at least 0.1 mM.
[0070] In other preferred embodiments, the second phosphate donor substantially differs from the first phosphate donor i.e. it has different molecular weights.
[0071] In this context, the second phosphate donor and the first phosphate donor might differ by at one methine group.
[0072] Preferably, the second phosphate donor and the first phosphate donor differ by at most one methine group.
[0073] In preferred embodiments, step (a2) is performed in a liquid reaction medium and the second phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, most preferably at most 10 mM.
[0074] In preferred embodiments the process according to the invention is characterized in that:
- the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least
92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 1; and/or
- the first hemiterpene alcohol is isoprenol; and/or
- the first and second phosphate donor is adenosine triphosphate; and/or
- the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:3.
[0075] Another aspect of the invention relates to a process for the preparation of a monoterpene diphosphate comprising the process for the preparation of a first hemiterpene diphosphate according to the invention as described above.
[0076] In this context, it is preferred that, besides steps (ai) and (a2) leading to the first hemiterpene diphosphate as described above, the process additionally comprises the steps of:
(as) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and
(b) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a monoterpene diphosphate synthase thereby obtaining the monoterpene diphosphate.
[0077] The above process is drawn to the preparation of a monoterpene diphosphate. For the purpose of the specification, a "monoterpene diphosphate" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to ten carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage. [0078] Step (b) of the process according to the invention involves linking under catalysis of a monoterpene diphosphate synthase. For the purpose of the specification, the "monoterpene diphosphate" synthase is preferably an enzyme capable of catalyzing the condensation of one unit of the first hemiterpene diphosphate obtained in step (a2) with one unit of the second hemiterpene diphosphate provided in step (as).
[0079] Preferably, the monoterpene diphosphate synthase is a geranyl diphosphate synthase (GPPS) (EC 2.5. 1.1) and/or the monoterpene diphosphate is a geranyl diphosphate (GPP).
[0080] It is contemplated that the second hemiterpene diphosphate is obtained in step (a2) and/or in step (ai). In that respect, the process may preferably entail performing step (as) as sub-step of steps (ai) and/or (a2) in an overall setting in which the second hemiterpene diphosphate is obtained concomitantly with the first hemiterpene monophosphate or diphosphate during the phosphorylation of the corresponding first hemiterpene alcohol or first hemiterpene monophosphate respectively, either in the same or in separate reaction environments.
[0081] In preferred embodiments the process according to the invention is characterized in that:
- the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
- the monoterpene diphosphate is geranyl pyrophosphate (GPP); and/or
- the monoterpene diphosphate synthase is a geranyl diphosphate synthase (GPPS) belonging to EC 2.5. 1.1.
[0082] Another aspect of the invention relates to a process for the preparation of a sesquiterpene diphosphate comprising
- the process for the preparation of a first hemiterpene diphosphate according to the invention as described above; and/or
- the process for the preparation of a monoterpene diphosphate according to the invention as described above.
[0083] In this context, it is preferred that, besides steps (ai) and (a2) leading to the first hemiterpene diphosphate as described above, the process additionally comprises the steps of:
(as) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and
(c) linking the first hemiterpene diphosphate obtained in step (a2) with the second hemiterpene diphosphate provided in step (as) under catalysis of a sesquiterpene diphosphate synthase thereby obtaining the sesquiterpene diphosphate. [0084] The above process is drawn to the preparation of a sesquiterpene diphosphate. For the purpose of the specification, a "sesquiterpene diphosphate" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to fifteen carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage.
[0085] Step (c) of the process according to the invention involves linking under catalysis of a sesquiterpene diphosphate synthase. For the purpose of the specification, the "sesquiterpene diphosphate synthase" is preferably an enzyme capable of catalyzing the sequential condensation of one or more units of the first hemiterpene diphosphate obtained in step (a2) with one unit of the second hemiterpene diphosphate provided in step (as).
[0086] It is contemplated that the second hemiterpene diphosphate may be obtained in step (as) and/or in step (ai). In that respect, the process may preferably entail performing step (as) as sub-step of steps (ai) and/or (as) in an overall setting in which the second hemiterpene diphosphate is obtained concomitantly with the first hemiterpene monophosphate or diphosphate during the phosphorylation of the corresponding first hemiterpene alcohol or first hemiterpene monophosphate respectively, either in the same or in separate reaction environments.
[0087] Preferably, the sesquiterpene diphosphate synthase is a famesyl diphosphate synthase (FPPS) (EC 2.5.1.10).
[0088] In preferred embodiments, the sesquiterpene diphosphate synthase is preferably an FPPS from an organismus selected from the group consisting of Geobacillus stearothermophilus, Bos Taurus, Gallus gallus, Pseudomonas aeruginosa, Rhizosolenia setigera, Ricinus communis, Lupinus albus, Homo sapiens, Artemisia spiciformi or Abies grandis.
[0089] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:5. [0090] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:6.
[0091] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:7.
[0092] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:8.
[0093] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:9.
[0094] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 10.
[0095] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 11.
[0096] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 12.
[0097] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 13.
[0098] Preferably, the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 14.
[0099] The sesquiterpene diphosphate synthase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. This means that the sesquiterpene diphosphate synthase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the sesquiterpene diphosphate synthase according to the invention may essentially consist of said amino acid sequence. When the sesquiterpene diphosphate synthase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the sesquiterpene diphosphate synthase is to be immobilized on a solid support, e.g. for purification purposes.
[0100] Preferably, the sesquiterpene diphosphate is famesyl diphosphate (FPP).
[0101] In preferred embodiments of the process according to the invention is characterized in that:
- the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
- the sesquiterpene diphosphate is famesyl pyrophosphate (FPP); and/or
- the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:5 or to SEQ ID NO:7 , or to SEQ ID NO:8.
[0102] Another aspect of the invention relates to a process for the preparation of a diterpene diphosphate comprising
- the process for the preparation of a first hemiterpene diphosphate according to the invention as described above; and/or
- the process for the preparation of a monoterpene diphosphate according to the invention as described above; and/or
- the process for the preparation of a sesquiterpene diphosphate according to the invention as described above.
[0103] In this context, it is preferred that, besides steps (ai) and (a2) leading to the first hemiterpene diphosphate, the process additionally comprises the steps of:
(as) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and
(d) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a diterpene diphosphate synthase thereby obtaining the diterpene diphosphate.
[0104] The above process is drawn to the preparation of a diterpene diphosphate. For the purpose of the specification, a "diterpene diphosphate" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to twenty carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage.
[0105] Step (d) of the process according to the invention involves linking under catalysis of a diterpene diphosphate synthase. For the purpose of the specification, the "sesquiterpene diphosphate synthase" is preferably to be interpreted as an enzyme capable of catalyzing the sequential condensation of one or more units of the first hemiterpene diphosphate obtained in step (a2) with one unit of the second hemiterpene diphosphate provided in step (as). [0106] It is contemplated that the second hemiterpene diphosphate may be obtained in step (a2) and/or in step (ai). In that respect, the process may preferably entail performing step (as) as sub-step of steps (ai) and/or (as) in an overall setting in which the second hemiterpene diphosphate is obtained concomitantly with the first hemiterpene monophosphate or diphosphate during the phosphorylation of the corresponding first hemiterpene alcohol or first hemiterpene monophosphate respectively, either in the same or in separate reaction environments.
[0107] Preferably, the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) (EC 2.5.1.29).
[0108] Preferably, the diterpene diphosphate is geranylgeranyl diphosphate (GGPP).
[0109] In preferred embodiments, the process of the invention is characterized in that:
- the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
- the diterpene diphosphate is geranylgeranyl pyrophosphate (GGPP); and/or
- the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) belonging to EC 2.5.1.29.
[0110] Another aspect of the invention relates to a process for the preparation of a tetraterpene comprising
- the process for the preparation of a first hemiterpene diphosphate according to the invention as described above; and/or
- the process for the preparation of a monoterpene diphosphate according to the invention as described above; and/or
- the process for the preparation of a sesquiterpene diphosphate according to the invention as described above; and/or
- the process for the preparation of a diterpene diphosphate according to the invention as described above.
[0111] In this context, it is preferred that, besides steps (as) and (d) leading to the diterpene diphosphate, the process additionally comprises the step of:
(e) linking at least two units of the diterpene diphosphate obtained in step (d) under catalysis of a tetraterpene synthase thereby obtaining the tetraterpene. [0112] The above process is drawn to the preparation of a tetraterpene. For the purpose of the specification, a "tetraterpene" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to forty carbons atoms as well as two phosphorus atoms connected by an oxygen atom to give an P-O-P linkage.
[0113] Step (e) of the process according to the invention involves linking under catalysis of a tetraterpene synthase. For the purpose of the specification, the "tetraterpene synthase" is preferably an enzyme capable of catalyzing the sequential condensation of two or more units of the diterpene diphosphate obtained in step (d).
[0114] Preferably, the tetraterpene synthase is a phytoene synthase (EC 2.5.1.32).
[0115] Preferably, the tetraterpene is phytoene.
[0116] In preferred embodiments, the process is characterized in that:
- the tetraterpene is phytoene; and/or
- the tetraterpene synthase is a phytoene synthase belonging to EC 2.5.1.32.
[0117] As described above, processes of the invention relate to the preparation of
(A) a first hemiterpene diphosphate (encompassing steps (ai) and (a2));
(B) a monoterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (b));
(C) a sesquiterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (c));
(D) a diterpene diphosphate (preferably encompassing steps (ai), (as), (as), and (d)); and
(E) a tetraterpene (preferably encompassing steps (ai), (as), (as), (d) and (e)).
[0118] In the following, various preferred embodiments of step (as) are described, that analogously apply to any of the above processes (B) through (E).
[0119] In preferred embodiments step (as) comprises the sub-steps of:
(as.i) phosphorylating a second hemiterpene alcohol differing from the first hemiterpene alcohol with a third phosphate donor under catalysis of a third kinase thereby obtaining a second hemiterpene monophosphate and a third phosphate acceptor; and
(as.2) phosphorylating the second hemiterpene monophosphate obtained in step (as.i) with a fourth phosphate donor under catalysis of a fourth kinase thereby obtaining the second hemiterpene diphosphate and a fourth phosphate acceptor. [0120] For the purpose of the specification, the "first phosphate acceptor", "second phosphate acceptor", "third phosphate acceptor" or "fourth phosphate acceptor" is preferably to be interpreted as a chemical species deriving from a dephosphorylation reaction of the corresponding phosphate donor, i.e. presenting at least one phosphate unit less than it.
[0121] In that regard it is contemplated that the first, second, third or fourth phosphate acceptor is preferably characterized by a molecular structure amenable to undergo addition of a phosphate unit thereby repristinating the corresponding phosphate donor.
[0122] For the purpose of the specification, the "second hemiterpene alcohol" is preferably to be interpreted as a terpene or a terpenoid according to IUPAC comprising up to five carbons atoms as well as at least one alcoholic group within the hydrocarbon skeleton.
[0123] Preferably, the second hemiterpene alcohol is a constitutional isomer of the first hemiterpene alcohol.
[0124] For the purpose of the specification, a "constitutional isomer" has the same meaning as it would be understood by a skilled artesian in the art i.e. a molecule presenting the same molecular formula of a parent molecule, however in a different special arrangement.
[0125] Preferably, the second hemiterpene alcohol is selected from the group consisting of isoprenol, 3-methyl-3-buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Me- thyl-2-propen-l-ol, 3 -methylene- 1 -pentanol, 2-hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-me- thyl-4-penten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-buten-l-ol, 3- methyl-2-penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl-(2£)-penten-l-ol, (2E)-butene-2 -methyl- 1,4-diol, 3- phenyl-2 -propen- 1 -ol.
[0126] Preferably, the second hemiterpene alcohol is isoprenol or prenol, and most preferably prenol.
[0127] Preferably, the second hemiterpene alcohol is employed in a concentration of at least 0. 1 mM; preferably of at least 0.5 mM, most preferably of at least 1.0 mM.
[0128] Preferably, the second hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, most preferably at most 1000 mM. [0129] For the purpose of the specification, the "third kinase" is preferably to be interpreted as an enzyme capable of catalyzing the phosphorylation of an alcohol, said alcohol being preferably a hemiterpene alcohol, and even more preferably isoprenol and/or prenol.
[0130] Preferably, the third kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to the amino acid sequence of the first kinase or second kinase; preferably to SEQ ID NO: 1.
[0131] The third kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of the first kinase, second kinase, or SEQ ID NO: 1. This means that the third kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the third kinase according to the invention may essentially consist of said amino acid sequence. When the third kinase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the third kinase is to be immobilized on a solid support, e.g. for purification purposes.
[0132] For the purpose of the specification, the "fourth kinase" preferably refers to an enzyme capable of catalyzing the phosphorylation of a mono- or polyphosphorylated alcohol, preferably of a mono- or polyphosphorylated hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isopentenyl monophosphate and/or dimethylallyl monophosphate.
[0133] Preferably, the fourth kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to the amino acid sequence of the first, second or third kinase; preferably to SEQ ID NO:3.
[0134] The fourth kinase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of the first kinase, second kinase, third kinase, or SEQ ID NO:3. This means that the fourth kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the fourth kinase according to the invention may essentially consist of said amino acid sequence. When the fourth kinase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the fourth kinase is to be immobilized on a solid support, e.g. for purification purposes.
[0135] For the purpose of the specification, the "third phosphate donor" is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction. In this regard, the third phosphate donor preferably belongs to the same chemical category as the first or second phosphate donor, i.e. organic or inorganic monophosphate or polyphosphate, and is capable of donating a phosphate unit during the course of a reaction.
[0136] Preferably, the third phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the third phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phos- phate, fructose- 1,6-bisphosphate, and 3 -phosphoglycerate.
[0137] Preferably, the third phosphate donor has the same molecular structure as the first and /or second phosphate donor.
[0138] For the purpose of the specification, the "fourth phosphate donor" is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit during the course of a reaction. In this regard, the fourth phosphate donor preferably belongs to the same chemical category as the first, second or third phosphate donor, i.e. organic or inorganic monophosphate or polyphosphate, and is capable of donating a phosphate unit during the course of a reaction. [0139] Preferably, the fourth phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phos- phate, fructose- 1,6-bisphosphate, 3 -phosphoglycerate.
[0140] Preferably, the fourth phosphate donor has the same molecular structure as the first and second and/or third phosphate donor.
[0141] Preferably, the molar ratio of the first hemiterpene alcohol relative to second hemiterpene alcohol is at least 1: 10, or at least 1:2, or preferably at least 1: 1, or most preferably at least 2: 1 or 3: 1.
[0142] Preferably, the molar ratio of the first hemiterpene alcohol relative second hemiterpene alcohol is at most 100: 1; preferably at most 10: 1 or at most 5: 1, or preferably at most 3: 1.
[0143] In preferred embodiments, the processes (B) through (E) according to the invention are characterized in that:
- at least one of steps (ai), (a2), (as.i), (a-, 2) is repeated at least once; and/or
- at least two of steps (ai), (a2), (as.i), (as.2) are performed in a single reactor; and/or
- at least steps (ai) and (a2) are performed simultaneously; and/or
- at least steps (as.i), (a-, 2) are performed simultaneously; and/or
- at least steps (ai) and (as.i), are performed simultaneously; and/or
- at least steps (ai) and (as.2), are performed simultaneously; and/or
- at least steps (a2) and (as.i), are performed simultaneously; and/or
- at least steps (a2) and (as.2), are performed simultaneously .
[0144] In preferred embodiments of the process according to the invention step (as) comprises the substep of
(as s) isomerizing the first hemiterpene diphosphate under catalysis of an isomerase thereby obtaining the second hemiterpene diphosphate.
[0145] For the purpose of the specification, an "isomerase" is preferably to be interpreted as an enzyme capable of altering the isomeric structure of a given initial isomeric substrate to afford a different isomeric substrate presenting the same molecular formula as compared with the initial substrate and different structural arrangement. [0146] Preferably, the isomerase is selected from the group consisting of steroid delta isomerase (EC 5.3.3.1), vinylacetyl-CoA delta isomerase (EC 5.3.3.3), muconolactone delta-isomerase (EC 5.3.3.4), 5- carboxymethyl-2-ydroxymuconate delta isomerase (EC 5.3.3.10), isopiperitenone delta-isomerase (EC 5.3.3.11), polyenoic fatty acid isomerase (EC 5.3.3.13), trans-2,3-dihydro-3-hydroxyanthranilate isomerase (EC 5.3.3.17), lutein isomerase (5.3.3.22) and isopentenyl diphosphate isomerase (EC 5.3.3.2) and even more preferably the isomerase is a isopentenyl diphosphate isomerases (EC 5.3.3.2).
[0147] In this context, it is preferred that the isomerase is a isopentenyl diphosphate isomerase from Schizosaccharomyces pombe, Claviceps purpurea, Phaffia rhodozyma, Homo sapiens, Sus scrofa, Gallus gallus, Solanum lycopersicum, Haematococcus lacustris, Gossypium barbadense, Escherichia coli, or Saccharomyces cerevisiae.
[0148] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 15.
[0149] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 16.
[0150] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 17.
[0151] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 18.
[0152] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 19.
[0153] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:20.
[0154] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least
87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least
98%, or at least 99%, or 100% to SEQ ID NO:21.
[0155] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:22.
[0156] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100 to SEQ ID NO:23.
[0157] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:24. [0158] Preferably, the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:25.
[0159] The Isomerase according to the invention comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24 SEQ ID NO:25. This means that the Isomerase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the Isomerase according to the invention may essentially consist of said amino acid sequence. When the Isomerase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the Isomerase is to be immobilized on a solid support, e.g. for purification purposes.
[0160] In preferred embodiments, the second hemiterpene diphosphate is selected from the group consisting of isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), 3-methyl-3-buten- 2-ol diphosphate, tiglic alcohol diphosphate, angelicic alcohol diphosphate, senecioic alcohol diphosphate and isovaleric alcohol diphosphate.
[0161] Preferably, the second hemiterpene diphosphate is a structural isomer; preferably a constitutional isomer of the first hemiterpene diphosphate.
[0162] Preferably, the second hemiterpene diphosphate is isopentenyl pyrophosphate (IPP) or dimethylallyl pyrophosphate (DMAPP).
[0163] Preferably, the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP).
[0164] In preferred embodiments, the processes (A) through (E) according to the invention are characterized in that: - at least steps (ai) and (a2) are performed simultaneously; and/or
- at least one of steps (ai), (a2), (as.i), (as.2), (as s) is repeated at least once; and/or
- at least two of steps (ai), (a2), (as.i), (as.2), (as s) are performed in a single reactor; and/or
- at least steps (as.i) and (a-, 2) are performed simultaneously; and/or
- at least steps (as.i) and (as s) are performed simultaneously; and/or
- at least steps (as.2) and (as s) are performed simultaneously; and/or
- at least steps (ai) and (as.i), are performed simultaneously; and/or
- at least steps (ai) and (a3.2), are performed simultaneously; and/or
- at least steps (ai) and (a ,). are performed simultaneously; and/or
- at least steps (a2) and (as.i), are performed simultaneously; and/or
- at least steps (a2) and (a3.2), are performed simultaneously; and/or
- at least steps (a2) and (a3.3), are performed simultaneously.
[0165] In preferred embodiments the process according to the invention is characterized in that:
- the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 1; and/or
- the first hemiterpene alcohol is isoprenol; and/or
- the first, second, third and fourth phosphate donor is adenosine triphosphate; and/or
- the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:3; and/or
- step (as) comprises the sub-steps of:
(as s) isomerizing the first hemiterpene diphosphate under catalysis of an isomerase thereby obtaining the second hemiterpene diphosphate; and/or
- the isomerase is a isopentenyl diphosphate isomerases an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 15, or to SEQ ID NO: 17; and/or
- the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP); and/or
- all steps (ai), (a2), (as.i), (as.z), (as s) are performed in a single reactor; and/or
- the third phosphate donor and fourth phosphate donor are adenosine triphosphate.
[0166] In a further preferred embodiment, any of the processes (A) to ( E) and all their preferred embodiments as described above, take place at a temperature of 2.0 °C to 99.5 °C, or 3.0°C to 95.0°C, or 4.0°C to 90.0°C, or 5.0°C to 85.0°C, or 15.0°C to 80.0°C, or 25.0°C to 75.0°C, or 28.0°C to 75.0°C, or 30.0°C to 60.0°C, or 33.0°C to 50.0°C, or 35.0°C to 45.0°C.
[0167] In a further preferred embodiment, any of the processes (A) to ( E) and all their preferred embodiments as described above, take place in a liquid reaction medium, wherein said liquid reaction medium is characterized by a pH in the range of 5.0 to 8.5, or 5.3 to 8.5, or 5.5 to 8.5, or 5.8 to 8.5, or 6.0 to 8.5, or 6.4 to 8.5, or 6.6 to 8.5, or 6.9 to 8.5, 7.5 to 8.5, or 8.0 to 8.5.
[0168] The processes (A) to (E) according to the invention as described above, and all their preferred embodiments as described above, provide target molecules that according to the invention may be advantageously used as starting materials or intermediates for the synthesis of further target molecules derived therefrom. [0169] Another aspect of the invention relates to a process for the preparation of a monoterpene comprising the process for the preparation of a monoterpene diphosphate according to the invention as described above.
[0170] In this context, it is preferred that, besides steps (as) and (b) leading to the monoterpene diphosphate according to the invention as described above, the process additionally comprises the step of:
(f) reacting a monoterpene diphosphate obtained in step (b) under catalysis of a monoterpene synthase thereby obtaining the monoterpene.
[0171] Forthe purpose of the specification, the "monoterpene synthase" is preferably to be interpreted as an enzyme capable of catalyzing the rearrangement and/or cyclization of GPP into a monoterpene i.e. an organic compound comprising up to 10 carbon atoms.
[0172] Preferably, the monoterpene synthase is a an selected from the group consisting of squalensyn- thase (EC 2.5.1.21), 1,8-cineol synthase, (EC 4.2.3.108), (-)-sabinene synthase (EC 4.2.3.109), (+)-sab- inene synthase (EC 4.2.3.110), 5-carene-synthase (EC 4.2.3.107) and (R)-limonene synthase (EC 4.2.3.20), preferably wherein the monoterpene synthase is (-)-sabinene synthase (EC 4.2.3.109), (+)- sabinene synthase (EC 4.2.3.110), 5-carene-synthase (EC 4.2.3.107).
[0173] Preferably, the monoterpene is selected from the group consisting of squalen 1,8-cineol, (-)- sabinene, (+)-sabinene, 5-carene and (R)-limonene, preferably wherein the monoterpene is squalen, (-)- sabinene, (+)-sabinene or 5-3-carene.
[0174] Another aspect of the invention relates to a process for the preparation of carvone comprising the process for the preparation of a monoterpene, particularly for the preparation of (R)-limonene, according to the invention as described above.
[0175] In this context, it is preferred that, besides step (f) leading to the monoterpene, particularly to (R)-limonene, according to the invention as described above, the process additionally comprises the step of:
(gi) reacting (R)-limonene obtained in step (f) under catalysis of a Limonene-6-hydroxylase (EC 1.14.13.48) thereby obtaining carvenol; and
(g2) reacting carvenol with a carvenol-dehydrogenase (EC 1.1.1.243) thereby obtaining carvone.
[0176] In preferred embodiments, the process according to the invention is characterized in that:
- at least one of steps (gi) or (g2) is repeated at least once; and/or at least one of steps (gi) or (g2) is performed in a single reactor; and/or steps (gi) or (g2) are performed simultaneously.
[0177] Another aspect of the invention relates to a process for the preparation of a cannanbinoide comprising the process for the preparation of a monoterpene diphosphate, particularly for the preparation of GPP according to the invention as described above.
[0178] In this context, it is preferred that, besides steps (as) and (b) leading to the monoterpene diphosphate, particularly to GPP according to the invention as described above, the process additionally comprises the step of:
(hi) reacting a monoterpene diphosphate, particularly GPP, obtained in step (b) with an olivetolic acid under catalysis of an aromatic prenyltransferase thereby obtaining cannabigerolic acid;
(h2) reacting cannabigerolic acid (CBG) with a THCA-synthase (EC 1.21.3.7) or CBDA-synthase (EC 1.21.3.8) thereby 9-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA); and
(h , ) subjecting THCA or CBDA to decarboxylation thereby obtaining Cannabidiol (CBD) or tetrahydrocannabinol (THC).
[0179] Preferably, the aromatic prenyltransferase is a an selected from the group consisting of aspul- vinone dimethylallyltransferase (EC 2.5.1.35), trihydroxypterocarpan dimethylallyltransferase (EC 2.5.1.36), 4-hydroxybenzoic acid oligoprenyltransferase (EC 2.5.1.39), naringenin 8-dimethylallyltrans- ferase (EC 2.5.1.70), 4-hydroxybenzoate geranyltransferase (EC 2.5.1.93), fumigaclavine A dimethylallyltransferase (EC 2.5.1.100), geranylpyrophosphate :olivetolate geranyltransferase (EC 2.5.1.102), 4- hydroxyphenylpyruvate: dimethylallyl transferase (EC 2.5.1.111), homogentisate geranylgeranyltransferase (EC 2.5.1.116), flaviolin linalyltransferase (EC 2.5.1.123), coumarin 8 -geranyltransferase (EC 2.5.1.138), umbelliferone 6-dimethylallyltransferase (EC 2.5.1.139).
[0180] In preferred embodiments, the process according to the invention is characterized in that:
- at least one of steps (hi) (h2) or (h ) is repeated at least once; and/or
- at least one of steps (hi) (h2) or (ha) is performed in a single reactor; and/or
- at least two of steps (hi) (h2) or (lv) are performed simultaneously.
[0181] Another aspect of the invention relates to a process for the preparation of a sesquiterpene comprising the process for the preparation of a sesquiterpene diphosphate according to the invention as described above. [0182] In this context, it is preferred that, besides steps (as) and (c) leading to the sesquiterpene diphosphate according to the invention as described above, the process additionally comprises the step of:
(i) reacting a sesquiterpene diphosphate obtained in step (c) under catalysis of a sesquiterpene synthase thereby obtaining the sesquiterpene.
[0183] Preferably, the sesquiterpene synthase is an enzyme capable of catalyzing the rearrangement and/or cyclization of FPP into a sesquiterpene i.e. an organic compound comprising up to 15 carbon atoms, preferably wherein the sesquiterpene synthase is a an enzyme selected from the group consisting of valencene synthase (EC 4.2.3.73), a-bisabolol-synthase (EC 4.2.3.138), (S)-beta-bisabolene synthase (EC 4.2.3.55), patchoulol synthase (EC 4.2.3.70), [3-elemene synthase (EC 4.2.3.x), 5-cadinene synthase (EC 4.2.3.13), (+)-endo-beta-bergamotene synthase (EC 4.2.3.53), (-)-endo-alpha-bergamo- tene synthase (EC 4.2.3.54), exo-alpha-bergamotene synthase (EC 4.2.3.81), zingiberene synthase (EC 4.2.3.65), longifolene synthase (4.2.3.58), a-santalene-synthase (EC 4.2.3.82), [3-santalene-synthase (EC 4.2.3.83).
[0184] Preferably, the sesquiterpene is selected from the group consisting of valencene, a-bisabolol, [3- bisabolene, patchoulol, [3-elemene, 5-cadinene, a-trans-bergamotene, [3-cis-bergamotene, [3-trans-berga- motene, zingiberene, longifolene, a-santalene, [3-santalene.
[0185] Another aspect of the invention relates to a process for the preparation of sesquiterpenoid comprising the process for the preparation of a sesquiterpene according to the invention as described above.
[0186] In this context, it is preferred that, besides step (i) leading to the sesquiterpene, according to the invention as described above, the process additionally comprises the step of:
(j) reacting the sesquiterpene obtained in step (i) under catalysis of a monooxygenase (EC 1. 14.x.x) thereby obtaining sesquiterpenoid.
[0187] Preferably, the sesquiterpenoid is selected from the group consisting of nootkatone, hemandul- cin, a-santalol, [3-santalol.
[0188] Another aspect of the invention relates to a process for the preparation of famesol comprising the process for the preparation of a sesquiterpene diphosphate, particularly for the preparation of FPP, according to the invention as described above.
[0189] In this context, it is preferred that, besides steps (as) and (c) leading to the sesquiterpene diphosphate, particularly FPP, according to the invention as described above, the process additionally comprises the step of: (k) reacting a sesquiterpene diphosphate, particularly FPP, obtained in step (c) under catalysis of a FPP-diphosphatase thereby obtaining farnesol.
[0190] Preferably, the FPP-diphosphatase is a phosphatase belonging to EC 3.6.1.x and/or EC 3.1.3.x.
[0191] Another aspect of the invention relates to a process for the preparation of a sclareol comprising the process for the preparation of a diterpene diphosphate, particularly for the preparation of GGPP, according to the invention as described above.
[0192] In this context, it is preferred that, besides steps (as) and (d) leading to the diterpene diphosphate, particularly to GGPP, according to the invention as described above, the process additionally comprises the step of:
(h) reacting a diterpene diphosphate, particularly GGPP obtained in step (d) under catalysis of a lab- denediol synthase (EC 4.2.1.133) thereby obtaining the labdenediol diphosphate (LDPP); and
(h) reacting labdenediol diphosphate obtained in step (h) under catalysis of a sclareol synthase (EC 4.2.3. 141) thereby obtaining the sclareol.
[0193] In preferred embodiments the process according to the invention is characterized in that:
- at least one of steps (h) or (I2) is repeated at least once; and/or
- at least one of steps (h) or (I2) is performed in a single reactor; and/or
- steps (h) or (I2) are performed simultaneously.
[0194] Another aspect of the invention relates to a process for the preparation of a tetraterpenoid, comprising the process for the preparation of a tetraterpene, particularly comprising the process for the preparation of phytoene according to the invention as described above.
[0195] In this context, it is preferred that, besides steps (e) leading to the tetraterpene, particularly to phytoene, according to the invention as described above, the process additionally comprises the step of:
(m) reacting phytoene obtained in step (e) under catalysis of one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ^-carotene desaturase (EC 1.3.99.26), lycopene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpenoid.
[0196] Preferably, the tetraterpenoid is selected from the group consisting of phytofluene, 8-carotene, lycopene, neurosporene and [3-carotene. [0197] Another aspect of the invention relates to a process for the preparation of ionone, comprising the process for the preparation of a tetraterpenoid, particularly comprising the process for the preparation of [3-carotene according to the invention as described above.
[0198] In this context, it is preferred that, besides steps (m) leading to the tetraterpenoid, particularly to -carotene, according to the invention as described above, the process additionally comprises the step of:
(n) reacting the tetraterpenoid, particularly p-carotene, obtained in step (m) under catalysis of a carotenoid dioxygenase (EC 1.13.11.71 thereby obtaining ionone).
[0199] In this context, although P-carotene constitutes a particularly preferred choice, other tetraterpenoids such as of phytoene, phytofluene, 8-carotene, lycopene, neurosporene represent suitable starting material from which ionone can be derived.
[0200] As described above, processes of the invention relate to the preparation of
(A) a first hemiterpene diphosphate (encompassing steps (ai) and (as));
(B) a monoterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (b));
(C) a sesquiterpene diphosphate (preferably encompassing steps (ai), (a2), (as), and (c));
(D) a diterpene diphosphate (preferably encompassing steps (ai), (as), (as), and (d));
(E) a tetraterpene (preferably encompassing steps (ai), (as), (as), (d) and (e));
(F) a monoterpene (preferably encompassing steps (ai), (as), (as), (b) and (f));
(G) carvone (preferably encompassing steps (ai), (as), (as), (b), (f), (gi) and (gs));
(H) a cannabinoide (preferably encompassing steps (ai), (as), (as), (b), (hi), (hs) and (hs));
(I) a sesquiterpene (preferably encompassing steps (ai), (as), (as), (c) and (i));
(J) a sequiterpenoid (preferably encompassing steps (ai), (as), (as), (c), (i) and (j));
(K) famesol (preferably encompassing steps (ai), (as), (as), (c) and (k));
(L) sclareol (preferably encompassing steps (ai), (as), (as), (d), (h) and (E);
(M) a tetraterpenoid (preferably encompassing steps (ai), (as), (as), (d), (e) and (m)); and
(N) ionone (preferably encompassing steps (ai), (as), (as), (d), (e), (m) and (n)).
[0201] In a further preferred embodiment, any of the processes (A) to ( N) and all their preferred embodiments as described above, take place at a temperature of 2.0 °C to 99.5 °C, or 3.0°C to 95.0°C, or 4.0°C to 90.0°C, or 5.0°C to 85.0°C, or 15.0°C to 80.0°C, or 25.0°C to 75.0°C, or 28.0°C to 75.0°C, or 30.0°C to 60.0°C, or 33.0°C to 50.0°C, or 35.0°C to 45.0°C.
[0202] In a further preferred embodiment, any of the processes (A) to ( N) and all their preferred embodiments as described above, preferably take place in a liquid reaction medium, wherein said liquid reaction medium is characterized by a pH in the range of 5.0 to 8.5, or 5.3 to 8.5, or 5.5 to 8.5, or 5.8 to 8.5, or 6.0 to 8.5, or 6.4 to 8.5, or 6.6 to 8.5, or 6.9 to 8.5, 7.5 to 8.5, or 8.0 to 8.5.
[0203] In particularly advantageous embodiments, the processes (A) through (N) according to the invention comprise a phosphate regeneration system enabling the phosphorylation of the first, second, third, and/or fourth phosphate acceptor thereby regenerating the corresponding phosphate acceptor.
[0204] In this context, any phosphate regeneration system known in the art can be employed. Examples of suitable phosphate regeneration systems may be polyphosphate kinase ATP regeneration systems, creatine kinase ATP regeneration systems, acetate kinase ATP regeneration systems, pyruvate kinase ATP regeneration systems, glucose-6-phosphate kinase ATP regeneration systems, fructose- 1,6- bisphosphate kinase ATP regeneration systems, 3 -phosphoglycerate kinase ATP regeneration systems.
[0205] Thus in preferred embodiments the process according to the invention may comprise the additional step of
(o) phosphorylating the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2), the third phosphate acceptor optionally obtained in step (as.i) and/or the fourth phosphate acceptor optionally obtained in step (a3.2) with a phosphorylating agent, optionally under catalysis of a fifth kinase, thereby obtaining first phosphate donor, second phosphate donor, third phosphate donor, and/or fourth phosphate donor, respectively.
[0206] For the purpose of the specification, a "phosphorylating agent" is preferably to be interpreted as a phosphoryl-containing compound, i.e. organic or inorganic monophosphate or polyphosphate, which is capable of donating a phosphate unit to the first, second, third or fourth phosphate acceptor during the course of a reaction.
[0207] Preferably, the phosphorylating agent is polyphosphate, phosphocreatine, acetyl phosphate, phosphopyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3 -phosphoglycerate; more preferably acetyl phosphate
[0208] For the purpose of the specification, the "fifth kinase" is preferably an enzyme capable of catalyzing the phosphorylation of the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2), the third phosphate acceptor optionally obtained in step (as.i), and/or fourth phosphate acceptor optionally obtained in step (a3.2).
[0209] Preferably, the fifth kinase is an acetate kinase belonging to EC 2.7.2.1 or EC 2.7.4.1.
[0210] Preferably, the fifth kinase is an acetate kinase from Geobacillus kaustophilus, Escherichia coli, Rhodococcus erythropolis, Acinetobacter baylyi, Butyrivibrio fibrisolvens, Geobacillus caldoxylosilyti- cus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus shackletonii or Sinorhizobium meliloti.
[0211] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:26.
[0212] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:27.
[0213] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least
87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least
98%, or at least 99%, or 100 SEQ ID NO:28.
[0214] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:29.
[0215] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:30.
[0216] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:31.
[0217] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least
87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least
98%, or at least 99%, or 100 % to SEQ ID NO:32.
[0218] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:33.
[0219] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:34.
[0220] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:35. [0221] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:36.
[0222] Preferably, the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:37
[0223] The fifth kinase according to the invention preferably comprises such an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, SEQ ID NO:35 SEQ ID NO:36 or SEQ ID NO:37. This means that the fifth kinase according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acid sequence, or that the fifth kinase according to the invention may essentially consist of said amino acid sequence. When the Fifth kinase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and/or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the fifth kinase is to be immobilized on a solid support, e.g. for purification purposes.
[0224] In preferred embodiments the process according to the invention is characterized in that:
- the phosphorylating agent is acetyl phosphate; and/or
- the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least
92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:26.
[0225] In a further preferred embodiment, any of the processes (A) to ( N) and all their preferred embodiments as described above, optionally including the phosphate regeneration system as described above, take place at a temperature of 2.0 °C to 99.5 °C, or 3.0°C to 95.0°C, or 4.0°C to 90.0°C, or 5.0°C to 85.0°C, or 15.0°Cto 80.0°C, or 25.0°Cto 75.0°C, or28.0°C to 75.0°C, or 30.0°C to 60.0°C, or 33.0°C to 50.0°C, or 35.0°C to 45.0°C.
[0226] In a further preferred embodiment, any of the processes (A) to ( N) and all their preferred embodiments as described above, optionally including the phosphate regeneration system as described above, take place in a liquid reaction medium, wherein said liquid reaction medium is characterized by a pH in the range of 5.0 to 8.5, or 5.3 to 8.5, or 5.5 to 8.5, or 5.8 to 8.5, or 6.0 to 8.5, or 6.4 to 8.5, or 6.6 to 8.5, or 6.9 to 8.5, 7.5 to 8.5, or 8.0 to 8.5.
[0227] Another aspect of the invention relates to a composition comprising (i) first hemiterpene diphosphate obtainable by the process according to the invention as described above in combination with (ii) a first kinase, (iii) a first phosphate donor, (iv) a second kinase, and (v) a second phosphate donor or any combination of the foregoing.
[0228] In preferred embodiments the composition further comprises (vi) a phosphorylating agent, and/or (vii) a fifth kinase, preferably wherein the phosphorylating agent is acetyl phosphate and/or wherein the fifth kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:26. [0229] In preferred embodiments the composition further comprises a (i) monoterpene diphosphate obtainable by the process of the invention as described above, in combination with a (ii) monoterpene diphosphate synthase.
[0230] Preferably, the composition according to the invention further comprises a (i) monoterpene obtainable by the process according to the invention as described above in combination with a (ii) monoterpene synthase.
[0231] In this context, the monoterpene is preferably selected from the group consisting of squalene 1,8-cineol, (-)-sabinene, (+)-sabinene, 5-carene and (R)-limonene, preferably wherein the monoterpene is squalene, (-)-sabinene, (+)-sabinene or 5-3-carene.
[0232] Preferably, the composition further comprises a (i) sesquiterpene diphosphate obtainable by the process according to the invention as described above in combination with a (ii) sesquiterpene diphosphate synthase.
[0233] Preferably, the composition further comprises a (i) sesquiterpene obtainable by the process according to the invention as described above in combination with a (ii) sesquiterpene synthase.
[0234] In this context, the sesquiterpene is preferably selected from the group consisting of valencene, a-bisabolol, [3-bisabolene, patchoulol, [3-elemene, 5-cadinene, a-trans-bergamotene, [3-cis-bergamotene, P-trans-bergamotene, zingiberene, longifolene, a-santalene, -santalene.
[0235] Preferably, the composition further comprises a (i) sesquiterpenoid obtainable by the process according to the invention as described above in combination with a (ii) monooxygenase (EC 1.14.x.x).
[0236] In this context, the sesquiterpenoid is preferably selected from the group consisting of noot- katone, hemandulcin, a-santalol, -santalol.
[0237] Preferably, the composition further comprises a (i) diterpene diphosphate obtainable by the process according to the invention as described above in combination with a (ii) diterpene diphosphate synthase.
[0238] Preferably, the composition further comprises (i) sclareol obtainable by the process according to the invention as described above in combination with a (ii) labdenediol diphosphate and a sclareol synthase (EC 4.2.3.141). [0239] Preferably, the composition further comprises a (i) tetraterpene obtainable by the process according to the invention as described above in combination with a (ii) tetraterpene synthase.
[0240] In this context, the tetraterpene is preferably phytoene.
[0241] Preferably, the composition further comprises a (i) tetraterpenoid obtainable by the process according to the invention as described above in combination with (ii) one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ^-carotene desaturase (EC 1.3.99.26), lyco- pene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpene
[0242] In this context, the tetraterpenoid is preferably selected from the group consisting of phyto- fluene, 8-carotene, lycopene, neurosporene and [3-carotene.
[0243] Preferably, the composition further comprises (i) ionone obtainable by the process according to the invention as described above in combination with a (ii) carotenoid dioxygenase (EC 1.13.11.71).
[0244] Preferred embodiments of the invention are summarized as clauses 1 to 181 hereinafter:
Clause 1 : A process for the preparation of a first hemiterpene diphosphate comprising the steps of: (ai) phosphorylating a first hemiterpene alcohol with a first phosphate donor under catalysis of a first kinase thereby obtaining a first hemiterpene monophosphate and a first phosphate acceptor; and (a2) phosphorylating the first hemiterpene monophosphate obtained in step (ai) with a second phosphate donor under catalysis of a second kinase thereby obtaining a first hemiterpene diphosphate and a second phosphate acceptor; wherein "terpene" encompasses terpenes as well as terpenoids according to IUPAC.
Clause 2: The process according to clause 1, wherein the first kinase is an enzyme capable of catalyzing the phosphorylation of an alcohol; preferably of a hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isoprenol and/or prenol.
Clause 3: The process of clauses 1 or 2, wherein the first kinase in an enzyme capable of catalyzing the transfer of a phosphate group; preferably wherein said first kinase is selected from the group consisting of:
- phosphotransferase with alcohol group as acceptor; preferably selected from the group consisting of ethanolamine kinase (EC 2.7.1.82), amide-alcohol kinase, ceramide kinase (EC 2.7.1.138), pantoate kinase (EC 2.7.1.169), undecaprenol kinase (EC 2.7.1.66), choline kinase (EC 2.7.1.32), glycerol kinase (EC 2.7.1.30), glycerone kinase (EC 2.7.1.29), mevalonate kinase (EC 2.7.1.30), and hydrox- yethylthiazole kinase (EC 2.7.1.50); and/or
- phosphotransferases with a phosphate group as an acceptor (EC 2.7.4); preferably selected from the group consisting of polyphosphate kinase, phosphomevalonate kinase, adenylate kinase, nucleoside- phosphate kinase, deoxycytidylate kinase, nucleoside-diphosphate kinase, phosphomethylpyrimidine kinase, guanylate kinase, dTMP kinase, nucleoside-triphosphate-adenylate kinase, (deoxy) adenylate kinase, T2-induced deoxynucleotide kinase, (deoxy)nucleoside-phosphate kinase, UMP/CMP kinase, thiamine-diphosphate kinase, thiamine-phosphate kinase, 3-phosphoglyceroyl- phosphate-polyphosphate phosphotransferase, famesyl-diphosphate kinase, 5 -methyldeoxy cytidine- 5'-phosphate kinase, dolichyl-diphosphate-polyphosphate phosphotransferase, inositol-hexakisphos- phate kinase, UMP kinase, ribose 1,5 -bisphosphate phosphokinase, diphosphoinositol-pentakisphos- phate kinase, (d)CMP kinase, isopentenyl phosphate kinase, [pyruvate, phosphate dikinase] -phosphate phosphotransferase, [pyruvate, water dikinase] -phosphate phosphotransferase, Kdo2-lipid A phosphotransferase, lipid A phosphoethanolamine transferase, [5-(aminomethyl)furan-3-yl]methyl phosphate kinase, famesyl phosphate kinase, yeast UMP kinase, polyphosphate- AMP phosphotransferase, and geranylgeranyl phosphate kinase; and/or
- Phosphotransferases with a carboxy group as acceptor (EC 2.7.2): acetate kinase (EC 2.7.2. 1), acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10), glutamate 5-ki- nase (EC 2.7.2.11), glutamate 1-kinase (EC 2.7.2.13), branched-chain-fatty-acid kinase (EC 2.7.2.14), propionate kinase (EC 2.7.2.15), 2-phosphoglycerate kinase (EC 2.7.2. 16), [amino group carrier protein] -L-2 -aminoadipate 6-kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase( EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8).
Clause 4: The process according to any of the preceding clauses, wherein the first kinase is a hydroxyethylthiazole kinase from Escherichia coli (EC 2.7.1.50).
Clause 5 : The process according to any of the preceding clauses, wherein the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 1. Clause 6: The process according to any of the preceding clauses, preferably according to any of clauses 1 to 3, wherein the first kinase is a choline kinase from Plasmodium falciparum (PfCK) (EC 2.7.1.32).
Clause 7: The process according to any of the preceding clauses, preferably according to any of clauses 1 to 3 or 6 wherein the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:2.
Clause 8: The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first kinase is employed in a concentration of at least 0.0001 mg/ml.
Clause 9: The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, more preferably at most 10 mg/ml.
Clause 10: The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is selected from the group consisting of isoprenol, 3-methyl-3-buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Methyl-2-propen-l-ol, 3 -methylene- 1 -pentanol, 2- hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-methyl-4-penten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-buten-l-ol, 3-methyl-2-penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl- (2E)-penten-l-ol, (2E)-butene-2 -methyl- 1,4-diol, and 3-phenyl-2-propen-l-ol.
Clause 11 : The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is isoprenol or prenol,
Clause 12: The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is isoprenol.
Clause 13: The process according to any of the preceding clauses, wherein the first hemiterpene alcohol is employed in a concentration of at least 0.1 mM; preferably of at least 0.5 mM, more preferably of at least 1.0 mM.
Clause 14: The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, more preferably at most 1000 mM.
Clause 15: The process according to any of the preceding clauses, wherein the first phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate preferably selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3-phospho- gly cerate.
Clause 16: The process according to any of the preceding clauses, wherein the first phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil. Clause 17: The process according to any of the preceding clauses, wherein the first phosphate donor is adenosine triphosphate.
Clause 18: The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, more preferably at least 0.01 mM.
Clause 19: The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the first phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, more preferably at most 10 mM.
Clause 20: The process according to any of the preceding clauses, wherein the second kinase is an enzyme capable of catalyzing the phosphorylation of a phosphorylated alcohol; preferably of a phosphorylated hemiterpene alcohol, and more preferably of catalyzing the phosphorylation of isopentenyl monophosphate and/or dimethylallyl monophosphate.
Clause 21 : The process according to any of the preceding clauses, wherein the second kinase in an enzyme capable of catalyzing the transfer of a phosphate group; preferably wherein said second kinase is a phosphotransferase with a phosphate group as acceptor; preferably selected from the group consisting of polyphosphate kinase (EC 2.7.4. 1), phosphomevalonate kinase (EC 2.7.4.2), adenylate kinase (EC 2.7.4.3), nucleoside-phosphate kinase (EC 2.7.4.4), thiamine-diphosphate kinase (EC 2.7.4.15), thi- amone-phosphate kinase (EC 2.7.4.16), famesyl-diphosphate kinase (EC 2.7.4.18), isopentenyl phosphate kinase (EC 2.7.4.26), and famesyl phosphate kinase (EC 2.7.4.32), phosphotransferases with a carboxy group as acceptor (EC 2.7.2): acetate kinase (EC 2.7.2.1), acetate kinase (diphosphate), (EC2.7.2.12), phosphoglycerate kinase (GTP) (EC2.7.2.10), glutamate 5-kinase (EC 2.7.2.11), glutamate 1-kinase (EC 2.7.2.13), branched-chain-fatty-acid kinase (EC 2.7.2.14), propionate kinase (EC 2.7.2.15), 2-phosphoglycerate kinase (EC 2.7.2. 16), [amino group carrier protein] -L-2 -aminoadipate 6- kinase (EC 2.7.2.17), carbamate kinase (EC 2.7.2.2), phosphoglycerate kinase (EC 2.7.2.3), aspartate kinase (EC 2.7.2.4), formate kinase( EC 2.7.2.6), (EC2.7.2.6), butyrate kinase (EC 2.7.2.7), acetylglutamate kinase (EC 2.7.2.8).
Clause 22: The process according to any of the preceding clauses, wherein the second kinase is a isopentenyl phosphate kinase (EC 2.7.4.26).
Clause 23: The process according to any of the preceding clauses, wherein the second kinase is a isopentenyl phosphate kinase from Arabidopsis thaliana, Branchiostoma floridae, Methanolobus tindarius, Methanococcus vannielii, aus Roseiflexus castenholzii, Thermoplasma acidophilum, Methanothermo- bacterthermoautotropicus, Methanocaldococcus jannaschii, Haloferax volcanii, Sulfolobus solfataricus, Trichoplax adhaerens, or Methanosalsum zhilinae.
Clause 24: The process according to any of the preceding clauses, wherein the second kinase is a isopentenyl phosphate kinase from Roseiflexus castenholzii.
Clause 25 : The process according to any of the preceding clauses, wherein the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:3.
Clause 26: The process according to any of the preceding clauses, preferably according to any of clauses 1 to 21 or 23, wherein the second kinase is a isopentenyl phosphate kinase from Methanolobus tindarius.
Clause 27: The process according to any of the preceding clauses, preferably according to any of clauses 1 to 21 or 23 or 26, wherein the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:4.
Clause 28: The process according to any of the preceding clauses, wherein the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to the amino acid sequence of the first kinase.
Clause 29: The process according to any of the preceding clauses, wherein step (a2) is performed in a liquid reaction medium and wherein the second kinase is employed in a concentration of at least 0.0001 mg/ml.
Clause 30: The process according to any of the preceding clauses, wherein step (a2) is performed in a liquid reaction medium and wherein the second kinase is employed in a concentration of at most 1000 mg/ml; preferably at most 100 mg/ml, most preferably at most 10 mg/ml. Clause 31 : The process according to any of the preceding clauses, wherein the second phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, or 3-phospho- glycerate.
Clause 32: The process according to any of the preceding clauses, wherein the second phosphate donor is a triphosphorylated conjugate of a ribose or a deoxyribose with a nucleobase selected from the group consisting of adenine, guanine, inosine, cytosine, thymine, and uracil.
Clause 33: The process according to any of the preceding clauses, wherein the second phosphate donor is adenosine triphosphate.
Clause 34: The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the second phosphate donor is employed in a concentration of at least 0.001 mM; preferably at least 0.005 mM, most preferably at least 0.1 mM.
Clause 35: The process according to any of the preceding clauses, wherein the second phosphate donor and the first phosphate donor have different molecular weights.
Clause 36: The process according to any of the preceding clauses, wherein the second phosphate donor and the first phosphate donor differ by at least one methine group.
Clause 37: The process according to any of the preceding clauses, wherein the second phosphate donor and the first phosphate donor differ by at most one methine group.
Clause 38: The process according to any of the preceding clauses, wherein step (ai) is performed in a liquid reaction medium and wherein the second phosphate donor is employed in a concentration of at most 1000 mM; preferably at most 100 mM, most preferably at most 10 mM.
Clause 39: The process according to any of the preceding clauses, wherein the second phosphate donor is the same as the first phosphate donor.
Clause 40: The process according to any of the preceding clauses, wherein
- the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 1; and/or
- the first hemiterpene alcohol is isoprenol; and/or
- the first and second phosphate donor is adenosine triphosphate; and/or - the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:3.
Clause 41: A process for the preparation of a monoterpene diphosphate comprising the process according to any of the preceding clauses.
Clause 42: The process according to any of the preceding clauses, preferably according to clause 41 additionally comprising the steps of:
(as) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and
(b) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a monoterpene diphosphate synthase thereby obtaining the monoterpene diphosphate.
Clause 43 : The process according to any of the preceding clauses, preferably according to clause 42, wherein the second hemiterpene diphosphate is obtained in step (as) and/or in step (ai).
Clause 44: The process according to any of the preceding clauses, preferably according to any of clauses 42 to 43, wherein the monoterpene diphosphate synthase is an enzyme capable of catalyzing the condensation of one unit of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
Clause 45: The process according to any of the preceding clauses, preferably according to any of clauses 42 to 44, wherein the monoterpene diphosphate synthase is a geranyl diphosphate synthase (GPPS) (EC 2.5.1.1).
Clause 46: The process according to any of the preceding clauses, preferably according to any of clauses 41 to 44, wherein the monoterpene diphosphate is a geranyl diphosphate (GPP).
Clause 47: The process according to any of the preceding clauses, preferably according to any of clauses 41 to 45 wherein
- the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
- the monoterpene diphosphate is geranyl pyrophosphate (GPP); and/or
- the monoterpene diphosphate synthase is a geranyl diphosphate synthase (GPPS) belonging to EC 2.5. 1.1. Clause 48: A process for the preparation of a sesquiterpene diphosphate comprising the process according to any of the preceding clauses, preferably according to any of clauses 1 to 40.
Clause 49: The process according to any of the preceding clauses, preferably according to clause 48 additionally comprising the steps of:
(as) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and
(c) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a sesquiterpene diphosphate synthase thereby obtaining the sesquiterpene diphosphate.
Clause 50: The process according to any of the preceding clauses, preferably according to clause 49, wherein the second hemiterpene diphosphate is obtained in step (as) and/or in step (ai).
Clause 51: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 50, wherein the sesquiterpene diphosphate synthase is an enzyme capable of catalyzing the sequential condensation of one or more units of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
Clause 52: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 51, wherein the sesquiterpene diphosphate synthase is a famesyl diphosphate synthase (FPPS) (EC 2.5.1.10).
Clause 53: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 52, wherein the sesquiterpene diphosphate synthase is an FPPS from Geobacillus stea- rothermophilus, Bos Taurus, Gallus gallus, Pseudomonas aeruginosa, Rhizosolenia setigera, Ricinus communis, Lupinus albus, Homo sapiens, Artemisia spiciformi or Abies grandis.
Clause 54: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:5.
Clause 55: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:6.
Clause 56: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:7.
Clause 57: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:8.
Clause 58: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:9.
Clause 59: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 10.
Clause 60: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 11.
Clause 61: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 12.
Clause 62: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 13.
Clause 63: The process according to any of the preceding clauses, preferably according to any of clauses 49 to 53, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 14.
Clause 64: The process according to any clauses 48 to 63, wherein the sesquiterpene diphosphate is famesyl diphosphate (FPP).
Clause 65: The process according to any of the preceding clauses, preferably according to any of clauses 48 to 64 wherein
- the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
- the sesquiterpene diphosphate is famesyl pyrophosphate (FPP); and/or
- the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:5 or to SEQ ID NO:7 , or to SEQ ID NO:8.
Clause 66: A process for the preparation of a diterpene diphosphate comprising the process according to any of the preceding clauses, preferably according to any of clauses 1 to 40.
Clause 67 : The process according to any of the preceding clauses, preferably according to clause 66 additionally comprising the steps of:
(as) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and (d) linking the first hemiterpene diphosphate obtained in step (a2) with the second hemiterpene diphosphate provided in step (as) under catalysis of a diterpene diphosphate synthase thereby obtaining the diterpene diphosphate.
Clause 68: The process according to any of the preceding clauses, preferably according to clause 67, wherein the second hemiterpene diphosphate is obtained in step (as) and/or in step (ai).
Clause 69: The process according to any of the preceding clauses, preferably according to any of clauses 67 to 68, wherein the diterpene diphosphate synthase is an enzyme capable of catalyzing the sequential condensation of one or more unit of the first hemiterpene diphosphate obtained in step (as) with one unit of the second hemiterpene diphosphate provided in step (as).
Clause 70: The process according to any of the preceding clauses, preferably according to any of clauses 67 to 69, wherein the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) (EC 2.5.1.29).
Clause 71: The process according to any of the preceding clauses, preferably according to any of clauses 66 to 70, wherein the diterpene diphosphate is geranylgeranyl diphosphate (GGPP).
Clause 72: The process according to any of the preceding clauses, preferably according to any of clauses 66 to 71, wherein
- the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or
- the diterpene diphosphate is geranylgeranyl pyrophosphate (GGPP); and/or
- the diterpene diphosphate synthase is a geranylgeranyl diphosphate synthase (GPPS) belonging to EC 2.5.1.29.
Clause 73: A process for the preparation of a tetraterpene comprising the process for the preparation of a diterpene diphosphate according to any of the preceding clauses, preferably according to any of clauses 66 to 72.
Clause 74: The process according to any of the preceding clauses, preferably according to clause 73 additionally comprising the step of:
(e) linking at least two units of the diterpene diphosphate obtained in step (d) under catalysis of a tetraterpene synthase thereby obtaining the tetraterpene.
Clause 75: The process according to any of the preceding clauses, preferably according to clause 74, wherein the tetraterpene synthase is an enzyme capable of catalyzing the sequential condensation of two or more units of the diterpene diphosphate obtained in step (d).
Clause 76: The process according to any of the preceding clauses, preferably according to any of clauses 74 to 75, wherein the tetraterpene synthase is a phytoene synthase (EC 2.5.1.32).
Clause 77: The process according to any of the preceding clauses, preferably according to any of clauses 73 to 76, wherein the tetraterpene is phytoene.
Clause 78: The process according to any of the preceding clauses, preferably according to any of clauses 73 to 77, wherein - the tetraterpene is phytoene; and/or
- the tetraterpene synthase is a phytoene synthase belonging to EC 2.5.1.32.
Clause 79: The process according to any of the preceding clauses, wherein step (as) comprises the substeps of
(as.i) phosphorylating a second hemiterpene alcohol differing from the first hemiterpene alcohol with a third phosphate donor under catalysis of a third kinase thereby obtaining a second hemiterpene monophosphate and a third phosphate acceptor;
(as.2) phosphorylating the second hemiterpene monophosphate obtained in step (as.i) with a fourth phosphate donor under catalysis of a fourth kinase thereby obtaining the second hemiterpene diphosphate and a fourth phosphate acceptor.
Clause 80: The process according to any of the preceding clauses, preferably according to clause 79, wherein the second hemiterpene alcohol is selected from the group consisting of isoprenol, 3-methyl-3- buten-2-ol, tiglic alcohol, angelicic alcohol, senecioic alcohol and isovaleric alcohol, 2-Methyl-2 -propen- l-ol, 3 -methylene- 1 -pentanol, 2-hexen-l-ol, 4-penten-l-ol, 3 -thiopheneethanol, 4-methyl-4-pen- ten-l-ol, 2-(Z)-hexen-l-ol, 2-butyn-l-ol, 3 -furanmethanol, 3-buten-l-ol, (2E)-buten-l-ol, 3 -methyl -2- penten-l-ol, 3-butyn-l-ol, 3,4-dimethyl-(2E)-penten-l-ol, (2E)-butene-2 -methyl- 1,4-diol, 3-phenyl-2- propen-l-ol.
Clause 81: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 80, wherein the second hemiterpene alcohol is preferably a constitutional isomer of the first hemiterpene alcohol.
Clause 82: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 81, wherein the second hemiterpene alcohol is isoprenol or prenol,
Clause 83: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 82, wherein the second hemiterpene alcohol is prenol.
Clause 84: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 83, wherein the second hemiterpene alcohol is employed in a concentration of at least 0.1 mM; preferably of at least 0.5 mM, most preferably of at least 1.0 mM.
Clause 85: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 84, wherein the second hemiterpene alcohol is employed in a concentration of at most 2000 mM; preferably at most 1500 mM, most preferably at most 1000 mM.
Clause 86: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 85, wherein the third kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to the amino acid sequence of the first kinase or second kinase; preferably to SEQ ID NO: 1.
Clause 87: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 86, wherein the fourth kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to the amino acid sequence of the first, second or third kinase; preferably to SEQ ID NO:3.
Clause 88: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 87, wherein the third phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the third phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6- phosphate, fructose- 1,6-bisphosphate, and 3 -phosphoglycerate.
Clause 89: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 88, wherein the third phosphate donor has the same molecular structure as the first and /or second phosphate donor.
Clause 90: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 89, wherein the fourth phosphate donor is selected from the group consisting of organic and/or inorganic mono- or polyphosphates; preferably wherein the first phosphate donor is a nucleoside mono- or polyphosphate, phosphoenolpyruvic acid, acetyl phosphate, phosphoenolpyruvate, glucose-6- phosphate, fructose- 1,6-bisphosphate, 3 -phosphoglycerate.
Clause 91: The process according to any of the preceding clauses, preferably according to any of clauses 78 to 90, wherein the fourth phosphate donor has the same molecular structure as the first and second and/or third phosphate donor.
Clause 92: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 91, wherein the molar ratio of the first hemiterpene alcohol relative to second hemiterpene alcohol is at least 1 : 10, or at least 1 : 2, or preferably at least 1 : 1 , or most preferably at least 2 : 1 or 3 : 1. Clause 93: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 92, wherein the molar ratio of the first hemiterpene alcohol relative second hemiterpene alcohol is at most 100: 1; preferably at most 10 : 1 or at most 5 : 1 , or preferably at most 3: 1.
Clause 94: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 93, wherein
- at least one of steps (ai), (a2), (as.i), (a-, 2) is repeated at least once; and/or
- at least two of steps (ai), (a2), (as.i), (a-, 2) are performed in a single reactor; and/or
- at least steps (ai) and (a2) are performed simultaneously; and/or
- at least steps (as.i), (a-, 2) are performed simultaneously; and/or
- at least steps (ai) and (as.i), are performed simultaneously; and/or
- at least steps (ai) and (as.2), are performed simultaneously; and/or
- at least steps (a2) and (as.i), are performed simultaneously; and/or
- at least steps (a2) and (as.2), are performed simultaneously.
Clause 95: The process according to any of the preceding clauses wherein step (as) comprises the substep of
(as s) isomerizing the first hemiterpene diphosphate under catalysis of an isomerase thereby obtaining the second hemiterpene diphosphate.
Clause 96: The process according to any of the preceding clauses, preferably according to clause 95, wherein the isomerase is selected from the group consisting of steroid delta isomerase (EC 5.3.3. 1), vinylacetyl-CoA delta isomerase (EC 5.3.3.3), muconolactone delta-isomerase (EC 5.3.3.4), 5-carbox- ymethyl-2-ydroxymuconate delta isomerase (EC 5.3.3.10), isopiperitenone delta-isomerase (EC 5.3.3.11), polyenoic fatty acid isomerase (EC 5.3.3.13), trans-2,3-dihydro-3-hydroxyanthranilate isomerase (EC 5.3.3.17), lutein isomerase (5.3.3.22) and isopentenyl diphosphate isomerase (EC 5.3.3.2). Clause 97: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 96, wherein the isomerase is a isopentenyl diphosphate isomerases (EC 5.3.3.2).
Clause 98: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 97, wherein the isomerase is a isopentenyl diphosphate isomerase from Schizosaccharo- myces pombe, Claviceps purpurea, Phaffia rhodozyma, Homo sapiens, Sus scrofa, Gallus gallus, Sola- num lycopersicum, Haematococcus lacustris, Gossypium barbadense, Escherichia coli, or Saccharomy- ces cerevisiae.
Clause 99: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least
87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least
98%, or at least 99%, or 100% to SEQ ID NO: 15.
Clause 100: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 16.
Clause 101: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 17.
Clause 102: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 18.
Clause 103: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least
71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least
87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least
98%, or at least 99%, or 100% to SEQ ID NO: 19.
Clause 104: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:20.
Clause 105: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:21.
Clause 106: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:22. Clause 107: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100 to SEQ ID NO:23.
Clause 108: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:24.
Clause 109: The process according to any of the preceding clauses, preferably according to any of clauses 95 to 98, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:25.
Clause 110: The process according to any of the preceding clauses, wherein the second hemiterpene diphosphate is selected from the group consisting of isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), 3-methyl-3-buten-2-ol diphosphate, tiglic alcohol diphosphate, angelicic alcohol diphosphate, senecioic alcohol diphosphate and isovaleric alcohol diphosphate.
Clause 111: The process according to any of the preceding clauses, wherein the second hemiterpene diphosphate is a structural isomer; preferably a constitutional isomer of the first hemiterpene diphosphate. Clause 112: The process according to any of the preceding clauses, wherein the second hemiterpene diphosphate is isopentenyl pyrophosphate (IPP) or dimethylallyl pyrophosphate (DMAPP).
Clause 113: The process according to any of the preceding clauses, wherein the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP).
Clause 114: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 113, wherein
- at least one of steps (ai), (a2), (as.i), (as.2), (as s) is repeated at least once; and/or
- at least two of steps (ai), (a2), (as.i), (as.2), (as s) are performed in a single reactor; and/or
- at least steps (ai) and (a2) are performed simultaneously; and/or
- at least steps (as.i) and (a-, 2) are performed simultaneously; and/or
- at least steps (as.i) and (as s) are performed simultaneously; and/or
- at least steps (a-, 2) and (as s) are performed simultaneously; and/or
- at least steps (ai) and (as.i), are performed simultaneously; and/or
- at least steps (ai) and (as.2), are performed simultaneously; and/or
- at least steps (ai) and (as s), are performed simultaneously; and/or
- at least steps (a2) and (as.i), are performed simultaneously; and/or
- at least steps (a2) and (as.2), are performed simultaneously; and/or
- at least steps (a2) and (as s), are performed simultaneously.
Clause 115: The process according to any of the preceding clauses, preferably according to any of clauses 79 to 114, wherein
- the first kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 1; and/or
- the first hemiterpene alcohol is isoprenol; and/or
- the first, second, third and fourth phosphate donor is adenosine triphosphate; and/or
- the second kinase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:3; and/or
- step (as) comprises the sub-steps of:
(as s) isomerizing the first hemiterpene diphosphate under catalysis of an isomerase thereby obtaining the second hemiterpene diphosphate; and/or
- the isomerase is a isopentenyl diphosphate isomerases an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO: 15, or to SEQ ID NO: 17; and/or
- the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP); and/or
- all steps (ai), (a2), (as.i), (as.z), (as s) are performed in a single reactor; and/or
- the third phosphate donor and fourth phosphate donor are adenosine triphosphate.
Clause 116: A process for the synthesis of a monoterpene comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a monoterpene diphosphate according to any clauses 41 to 47 or any of clauses 79 to 115.
Clause 117: The process according to any of the preceding clauses, preferably according to clause 116 additionally comprising the step of:
(f) reacting a monoterpene diphosphate obtained in step (b) under catalysis of a monoterpene synthase thereby obtaining the monoterpene.
Clause 118: The process according to any of the preceding clauses, preferably according to clause 117, wherein the monoterpene synthase is an enzyme capable of catalyzing the rearrangement and/or cyclization of GPP into a monoterpene i.e. an organic compound comprising up to 10 carbon atoms, preferably wherein the monoterpene synthase is a an selected from the group consisting of squalenesynthase (EC 2.5.1.21), 1,8-cineol synthase, (EC 4.2.3.108), (-)-sabinene synthase (EC 4.2.3.109), (+)-sabinene synthase (EC 4.2.3.110), 5-carene-synthase (EC 4.2.3.107) and (R)-limonene synthase (EC 4.2.3.20), preferably wherein the monoterpene synthase is (-)-sabinene synthase (EC 4.2.3. 109), (+)-sabinene synthase (EC 4.2.3.110), 5-carene-synthase (EC 4.2.3.107). Clause 119: The process according to any of the preceding clauses, preferably according to any of clauses 116 to 118, wherein the monoterpene is selected from the group consisting of squalene 1,8- cineol, (-)-sabinene, (+)-sabinene, 5-carene and (R)-limonene, preferably wherein the monoterpene is squalene, (-)-sabinene, (+)-sabinene or 5-3-carene.
Clause 120: A process for the synthesis of a carvone comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a monoterpene according to any clauses 116 to 119.
Clause 121: The process according to any of the preceding clauses, preferably according to clause 119 additionally comprising the steps of:
(gi) reacting (R)-limonene obtained in step (f) under catalysis of a Limonene-6-hydroxylase (EC 1.14.13.48) thereby obtaining carvenol;
(g2) reacting carvenol with a carvenol-dehydrogenase (EC 1.1.1.243) thereby obtaining carvone.
Clause 122: The process according to any of the preceding clauses, preferably according to clause 121, wherein
- at least one of steps (gi) or (g2) is repeated at least once; and/or
- at least one of steps (gi) or (g2) is performed in a single reactor; and/or
- steps (gi) or (g2) are performed simultaneously.
Clause 123: A process for the synthesis of a cannanbinoide comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a monoterpene diphosphate according to any clauses 41 to 47 or any of clauses 78 to 114.
Clause 124: The process according to any of the preceding clauses, preferably according to clause 123 additionally comprising the steps of:
(hi) reacting a monoterpene, particularly GPP diphosphate obtained in step (b) with an olivetolic acid under catalysis of an aromatic prenyltransferase thereby obtaining cannabigerolic acid;
(h2) reacting cannabigerolic acid (CBG) with a THCA-synthase (EC 1.21.3.7) or CBDA-synthase (EC 1.21.3.8) thereby 9-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA); and
(h , ) subjecting THCA or CBDA to decarboxylation thereby obtaining Cannabidiol (CBD) or tetrahydrocannabinol (THC).
Clause 125: The process according to any of the preceding clauses, preferably according to clause 124 wherein the aromatic prenyltransferase is a an selected from the group consisting of aspulvinone dimethylallyltransferase (EC 2.5.1.35), trihydroxypterocarpan dimethylallyltransferase (EC 2.5.1.36), 4- hydroxybenzoic acid oligoprenyltransferase (EC 2.5.1.39), naringenin 8 -dimethylallyltransferase (EC 2.5.1.70), 4-hydroxybenzoate geranyltransferase (EC 2.5.1.93), fumigaclavine A dimethylallyltransferase (EC 2.5.1.100), geranylpyrophosphate: olivetolate geranyltransferase (EC 2.5.1.102), 4-hydroxy- phenylpyruvate: dimethylallyl transferase (EC 2.5.1.111), homogentisate geranylgeranyltransferase (EC 2.5.1.116), flaviolin linalyltransferase (EC 2.5.1.123), coumarin 8 -geranyltransferase (EC 2.5.1.138), umbelliferone 6-dimethylallyltransferase (EC 2.5.1.139).
Clause 126: The process according to any of the preceding clauses, preferably according to clause 124, wherein
- at least one of steps (hi) (I12) or (h ) is repeated at least once; and/or
- at least one of steps (hi) (I12) or (I13) is performed in a single reactor; and/or
- at least two of steps (hi) (I12) or (lv) are performed simultaneously.
Clause 127: A process for the synthesis of a sesquiterpene comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a sesquiterpene diphosphate according to any clauses 48 to 65 or any of clauses 79 to 115.
Clause 128: The process according to any of the preceding clauses, preferably according to clause 127 additionally comprising the step of:
(i) reacting a sesquiterpene diphosphate obtained in step (c) under catalysis of a sesquiterpene synthase thereby obtaining the sesquiterpene.
Clause 129: The process according to any of the preceding clauses, preferably according to clause 127 and/or 128, wherein the sesquiterpene synthase is an enzyme capable of catalyzing the rearrangement and/or cyclization of FPP into a sesquiterpene i.e. an organic compound comprising up to 15 carbon atoms, preferably wherein the sesquiterpene synthase is a an enzyme selected from the group consisting of valencene synthase (EC 4.2.3.73), a-bisabolol-synthase (EC 4.2.3.138), (S)-beta-bisabolene synthase (EC 4.2.3.55), patchoulol synthase (EC 4.2.3.70), p-elemene synthase (EC 4.2.3.x), 5-cadinene synthase (EC 4.2.3.13), (+)-endo-beta-bergamotene synthase (EC 4.2.3.53), (-)-endo-alpha-bergamo- tene synthase (EC 4.2.3.54), exo-alpha-bergamotene synthase (EC 4.2.3.81), zingiberene synthase (EC 4.2.3.65), longifolene synthase (4.2.3.58), a-santalene-synthase (EC 4.2.3.82), P-santalene-synthase (EC 4.2.3.83).
Clause 130: The process according to any of the preceding clauses, preferably according to any of clauses 127 to 129, wherein the sesquiterpene is selected from the group consisting of valencene, a- bisabolol, P-bisabolene, patchoulol, P-elemene, 5-cadinene, a-trans-bergamotene, P-cis-bergamotene, P- trans-bergamotene, zingiberene, longifolene, a-santalene, P-santalene.
Clause 131: A process for the synthesis of a sesquiterpenoid comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a sesquiterpene according to any clauses 127 to 130.
Clause 132: The process according to any of the preceding clauses, preferably according to clause 131 additionally comprising the steps of:
(j) reacting the sesquiterpene obtained in step (i) under catalysis of a monooxygenase (EC 1. 14.x.x) thereby obtaining sesquiterpenoid. Clause 133: The process according to any of the preceding clauses, preferably according to any of clauses 131 to 132, wherein the sesquiterpenoid is selected from the group consisting of nootkatone, hemandulcin, a-santalol, P-santalol.
Clause 134: A process for the synthesis of a famesol comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a sesquiterpene diphosphate according to any clauses 48 to 65 or any of clauses 79 to 115.
Clause 135: The process according to any of the preceding clauses, preferably according to clause 134 additionally comprising the steps of:
(k) reacting a sesquiterpene diphosphate obtained in step (c) under catalysis of a FPP-diphosphatase thereby obtaining fame sol.
Clause 136: The process according to any of the preceding clauses, preferably according to clause 135 wherein the FPP-diphosphatase is a phosphatase belonging to EC 3.6.1.x and/or EC 3.1.3.x.
Clause 137: A process for the synthesis of a sclareol comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a diterpene diphosphate according to any clauses 66 to 72 or any of clauses 79 to 115.
Clause 138: The process according to any of the preceding clauses, preferably according to clause 137 additionally comprising the step of:
(h) reacting a diterpene diphosphate, particularly GGPP, obtained in step (d) under catalysis of a labdenediol synthase (EC 4.2.1.133) thereby obtaining the labdenediol diphosphate (LDPP); and
(h) reacting labdenediol diphosphate obtained in step (h) under catalysis of a sclareol synthase (EC 4.2.3. 141) thereby obtaining the sclareol.
Clause 139: The process according to any of the preceding clauses, preferably according to any of clauses 137 to 138, wherein
- at least one of steps (h) or (I2) is repeated at least once; and/or
- at least one of steps (h) or (I2) is performed in a single reactor; and/or
- steps (h) or (I2) are performed simultaneously.
Clause 140: A process for the synthesis of a tetraterpenoid comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a tetraterpene according to any clauses 73 to 78 or any of clauses 79 to 115.
Clause 141: The process according to any of the preceding clauses, preferably according to clause 140 additionally comprising the step of:
(m) reacting phytoene obtained in step (e) under catalysis of one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ^-carotene desaturase (EC 1.3.99.26), lycopene-a-cyclase (EC 5.5.1.18), lycopene P-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpene. Clause 142: The process according to any of the preceding clauses, preferably according to clause 141, wherein the tetraterpenoid is selected from the group consisting of phytofluene, 8-carotene, lycopene, neurosporene and P-carotene.
Clause 143: A process for the synthesis of ionone comprising the process according to any of the preceding clauses, preferably comprising the process for the synthesis of a tetraterpenoid according to any clauses 140 to 142.
Clause 144: The process according to any of the preceding clauses, preferably according to clause 143 wherein the process comprises the additional step of:
(n) reacting the tetraterpenoid obtained in step (m) under catalysis of a carotenoid dioxygenase (EC 1.13.11.71 thereby obtaining ionone) .
Clause 145: The process according to any of the preceding clauses, preferably according to clause 144, wherein the tetraterpenoid is selected from the group consisting of phytoene, phytofluene, 8-carotene, lycopene, neurosporene and P-carotene.
Clause 146: The process according to any of the preceding clauses which comprises the additional step of
(o) phosphorylating the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2), the third phosphate acceptor optionally obtained in step (as.i) and/or the fourth phosphate acceptor optionally obtained in step (a3.2) with a phosphorylating agent, optionally under catalysis of a fifth kinase, thereby obtaining first phosphate donor, second phosphate donor, third phosphate donor, and/or fourth phosphate donor, respectively.
Clause 147: The process according to any of the preceding clauses, preferably according to clause 146, wherein the phosphorylating agent is acetyl phosphate, polyphosphate, phosphocreatine, acetyl phosphate, phosphopyruvate, glucose-6-phosphate, fructose- 1,6-bisphosphate, 3 -phosphoglycerate, preferably wherein the phosphorylating agent is acetyl phosphate
Clause 148: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 147, wherein the fifth kinase is an enzyme capable of catalyzing the phosphorylation of the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2) and/or the third phosphate acceptor optionally obtained in step (as.i).
Clause 149: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 148, wherein the fifth kinase is an acetate kinase belonging to EC 2.7.2. 1 or EC 2.7.4.1.
Clause 150: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 149, wherein the fifth kinase is an acetate kinase from Geobacillus kaustophilus, Escherichia coli, Rhodococcus erythropolis, Acinetobacter baylyi, Butyrivibrio fibrisolvens, Geobacillus cald- oxylosilyticus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus pu- milus, Bacillus shackletonii or Sinorhizobium meliloti. Clause 151: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:26.
Clause 152: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:27.
Clause 153: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100 SEQ ID NO:28.
Clause 154: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least
97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:29.
Clause 155: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:30.
Clause 156: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:31.
Clause 157: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100 % to SEQ ID NO:32.
Clause 158: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least
81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least
86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least
97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:33.
Clause 159: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% SEQ ID NO:34.
Clause 160: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:35.
Clause 161: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:36. Clause 162: The process according to any of the preceding clauses, preferably according to any of clauses 146 to 150, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:37.
Clause 163: The process according to any of the preceding clauses, preferably according to any of clauses to clauses 146 to 162, wherein
- the phosphorylating agent is acetyl phosphate; and/or
- the fifth kinase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:26.
Clause 164: The process according to any of the preceding clauses, wherein the process take place at a temperature of 2.0 °C to 99.5 °C, or 3.0°C to 95.0°C, or 4.0°C to 90.0°C, or 5.0°C to 85.0°C, or 15.0°C to 80.0°C, or 25.0°Cto 75.0°C, or 28.0°Cto 75.0°C, or 30.0°C to 60.0°C, or 33.0°C to 50.0°C, or 35.0°C to 45.0°C.
Clause 165: The process according to any of the preceding clauses, wherein the process take place in a liquid reaction medium, wherein said liquid reaction medium is characterized by a pH in the range of 5.0 to 8.5, or 5.3 to 8.5, or 5.5 to 8.5, or 5.8 to 8.5, or 6.0 to 8.5, or 6.4 to 8.5, or 6.6 to 8.5, or 6.9 to 8.5, 7.5 to 8.5, or 8.0 to 8.5.
Clause 166: A composition comprising a (i) first hemiterpene diphosphate obtainable by the process according to any of the proceeding clauses in combination with (ii) a first kinase, (iii) a first phosphate donor, (iv) a second kinase, and (v) a second phosphate donor or any combination of the foregoing.
Clause 167: The composition according to clause 166 further comprising a (i) monoterpene diphosphate obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 41 to 47 or any of clauses 79 to 115 in combination with a (ii) monoterpene diphosphate synthase. Clause 168: The composition according to any of clauses 166 or 167 further comprising a (i) monoterpene obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 116 to 119 or any of clauses 79 to 115 in combination with a (ii) monoterpene synthase.
Clause 169: The process according to clause 167, wherein the monoterpene is selected from the group consisting of squalene 1,8-cineol, (-)-sabinene, (+)-sabinene, 5-carene and (R)-limonene, preferably wherein the monoterpene is squalene, (-)-sabinene, (+)-sabinene or 5-3-carene.
Clause 170: The composition according to any of clauses 166 to 169 further comprising a (i) sesquiterpene diphosphate obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 48 to 65 or any of clauses 79 to 115 in combination with a (ii) sesquiterpene diphosphate synthase.
Clause 171: The composition according to clause 170 further comprising a (i) sesquiterpene obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 127 to 130 or any of clauses 79 to 115 in combination with a (ii) sesquiterpene synthase.
Clause 172: The composition according to clause 171, wherein the sesquiterpene is selected from the group consisting of valencene, a-bisabolol, P-bisabolene, patchoulol, P-elemene, 5-cadinene, a-trans- bergamotene, P-cis-bergamotene, P-trans-bergamotene, zingiberene, longifolene, a-santalene, P-san- talene.
Clause 173: The composition according to any of clauses 166 to 171 further comprising a (i) sesquit- erpenoid obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 131 to 133 or any of clauses 79 to 115 in combination with a (ii) monooxygenase (EC 1.14.x.x).
Clause 174: The composition according to clause 173, wherein the sesquiterpenoid is selected from the group consisting of nootkatone, hemandulcin, a-santalol, P-santalol.
Clause 175: The composition according to any of clauses 166 to 174 further comprising a (i) diterpene diphosphate obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 66 to 72 or any of clauses 79 to 115 in combination with a (ii) diterpene diphosphate synthase.
Clause 176: The composition according to clause 175 further comprising a (i) sclareol obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 137 to 139 in combination with a (ii) labdenediol diphosphate and a sclareol synthase (EC 4.2.3.141).
Clause 177: The composition according to any of clauses 166 to 176 further comprising a (i) tetraterpene obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 73 to 78 or any of clauses 79 to 115 in combination with a (ii) tetraterpene synthase.
Clause 178: The composition according to any clause 177, wherein the tetraterpene is phytoene.
Clause 179: The composition according to any of clauses 166 to 178 further comprising a (i) tetraterpenoid obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 140 to 142 or any of clauses 79 to 115 in combination with (ii) one or more enzyme selected from the group consisting of phytoene desaturase (EC 1.3.99.31), ^-carotene desaturase (EC 1.3.99.26), lycopene-a-cyclase (EC 5.5.1.18), lycopene [3-cyclase (EC 5.5.1.19) thereby obtaining the tetraterpene Clause 180: The composition according to clause 179, wherein the tetraterpenoid is selected from the group consisting of phytofluene, 8-carotene, lycopene, neurosporene and [3-carotene.
Clause 181: The composition according to any of clauses 166 to 180 further comprising a (i) ionone obtainable by the process according to any of the preceding clauses, preferably according to any of clauses 143 to 145 or any of clauses 79 to 115 in combination with a (ii) carotenoid dioxygenase (EC 1.13.11.71).
[0245] The following examples further illustrate the invention but are not to be construed as limiting its scope:
EXAMPLE 1: Preparation of enzymes and activity measurements
EXAMPLE 1.1: Production of Enzymes
[0246] Expression of Hydroxyethylthiazole kinase from Escherichia coli (EcHTK, SEQ ID NO: 1, wild-type gene, UniProtKB/Swiss-Prot: WP 001195564.1); Isopentenyl phosphate kinase from Ro- seiflexus castenholzii (RcIMK, SEQ ID NO:3, codon-optimized gene, UniProtKB/Swiss-Prot: WP_012122512.1); prenyltransferase from Geobacillus stearothermophilus (GsFPPS, SEQ ID NO:5, codon-optimized gene, UniProtKB/Swiss-Prot: WP_033016440.1); Isopentenyl-diphosphate isomerase from Schizosaccharomyces pombe (SpIDI, SEQ ID NO: 15, UniProtKB/Swiss-Prot: NP_595164.2); acetyl phosphate kinase from Geobacillus kaustophilus (GkACK, SEQ ID NO:26, codon-optimized gene, UniProtKB/Swiss-Prot: WP_011232259.1, SEQ ID NO:26), SmPPK (SEQ ID NO:37, wild-tape gene, UniProtKB/Swiss-Prot: WP_010968631.1); Glycerol kinase from Escherichia coli (EcGK, SEQ ID NO:38, wild-type gene, UniProtKB/Swiss-Prot: EGI08362.1); Dihydroxyacetone kinase from Escherichia coli (EcDHK, SEQ ID NO:39, wild-tape gene, UniProtKB/Swiss-Prot: EGI 10746.1); Mevalonate kinase from Saccharomyces cerevisiae (ScMVK, SEQ ID NO:40, UniProtKB/Swiss-Prot: AHY76662.1); Isopentenyl phosphate kinase from Thermoplasma acidophilum (TalMK, SEQ ID NO:41, UniProtKB/Swiss-Prot: WP_010900530.1); Thiamine pyrophosphokinase from Saccharomyces cerevisiae (ScTPK, SEQ ID NO:42, UniProtKB/Swiss-Prot: NP 014786.1); Thiamine pyrophosphokinase from Bacillus subtilis (BsTPK, SEQ ID NO:43, UniProtKB/Swiss-Prot: WP_003245470.1); Thiamine pyrophosphokinase from Arabidopsis thaliana (AtTPK, SEQ ID NO:44, UniProtKB/Swiss-Prot: NP_850424.1); Choline kinase from Saccharomyces cerevisiae (ScCK, SEQ ID NO:45, UniProtKB/Swiss-Prot: NP_013234.1); Choline kinase from Streptococcus pneumoniae (SpCK, SEQ ID NO:46, UniProtKB/Swiss-Prot: WP_000411210.1); Choline/ethanolamine kinase from Trypanosoma bruceii (TbCEK, SEQ ID NO:47, UniProtKB/Swiss-Prot: XP_844788.1); Choline kinase from Plasmodium falciparum (PfCK, SEQ ID NO:2, UniProtKB/Swiss-Prot: XP_001348193.1).
[0247] Cloning: The genes were cloned into the expression vector pLElA18 (derivative of pRSF-lb, Novagen) containing an N-terminal Hise-tag for purification of the enzymes. The resulting plasmids were each used for transformation of E. coli BL21(DE3) cells.
[0248] Expression: All enzymes were expressed by inoculating ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 pg/ml) with a fresh overnight culture. Cultures were grown at 37 °C. Expression of the gene was induced at logarithmic phase by IPTG (0.1 mM) and carried out overnight at 30 °C. [0249] Preparation and purification of Hise-tagged enzymes: Cells were harvested by centrifugation and suspended in a buffer containing 100 mM Tris-HCl-buffer pH 7,5, 500 mM NaCl, 10 mM Imidazole, 10% (v/v) glycerol, 2 mM MgC’T. 0.5 mg/ml lysozyme and 20 U/ml NuCLEANase (c-LEcta GmbH). Cell lysis was achieved by sonication. Cell free extract containing soluble enzyme was separated from the debris by centrifugation. The target enzymes were affinity purified from supernatant using Ni Sepharose™ 6 Fast Flow resin (GE Healthcare) and a gravity flow column according to manufacturer's manual. The eluted solution was rebuffered into 100 mM Tris-HCl-buffer pH 7,5, 150 mM NaCl, 10% (v/v) glycerol using PD-10 desalting columns according to the manufacturer (GE Healthcare). Aliquots of the purified enzyme solution were shock frozen in liquid nitrogen and stored at -20 °C.
[0250] Activity measurements: For the determination of the standard activity of EcHTK (SEQ ID NO: 1), the synthesis of IP (isoprenol) from IOH (isoprenol) and ATP was assayed. An EcHTK (SEQ ID NO: 1) unit (U) corresponds to the synthesis of 1 pmol IP per minute from 100 mM IOH and 1,25 mM ATP in 200 mM Tris-HCL buffer pH 8.5, 50 mM MgCl2, 150 mM PolyP, 0. 1 mg/ml SmPPK (SEQ ID NO:37) at 30 °C. Reaction progress was determined discontinuously by stopping reaction after a given reaction time through heating at 90 °C for 10 min and quantifying the amount of synthesized IP via HPLC calibrated with an external standard.
[0251] For the determination of the standard activity of RcIMK (SEQ ID NO:3), the synthesis of IPP from IP and ATP was assayed. An RcIMK (SEQ ID NO :3) unit (U) corresponds to the synthesis of 1 pmol IPP per minute from 50 mM IP and 5 mM ATP in 200 mM Tris-HCL buffer pH 8.5, 20 mM MgC’T. 100 mM AcP, 5 U/ml GkACK (SEQ ID NO:26) at 30 °C. Reaction progress was determined discontinuously by stopping reaction after a given reaction time through heating at 90 °C for 10 min and quantifying the amount of synthesized IP via HPLC calibrated with an external standard.
[0252] For the determination of the standard activity of GkACK (SEQ ID NO:26), the synthesis of ATP from 1 mM ADP, with 10 mM MgC’T and 10 mM acetyl phosphate in 50 mM Tris-Cl pH7.5 was determined. The reaction mixture was incubated for 10 min at 30 °C, stopped by heating at 95°C for 10 min. The resulting ATP was quantified with Hexokinase/Glucose-6-phosphate dehydrogenase. An GkACK (SEQ ID NO:26) unit (U) corresponds to the synthesis of 1 pmol ATP per minute.
[0253] For the determination of the standard activity of GsFPPS (SEQ ID NO:5), the synthesis of FPP from IPP and DMAPP was assayed. A GsFPPS unit (U) corresponds to the synthesis of 1 pmol FPP per minute from 1 mM IPP and 1 mM DMAPP in 50 mM Tris-HCL buffer pH 7.5, 0.40 mM MgC’T at 30 °C. Reaction progress was determined discontinuously by stopping reaction after a given reaction time by adding one volume of abs. MeOH and quantifying the amount of synthesized IP via HPLC calibrated with an external standard. EXAMPLE 2: Screening of different kinase for the synthesis of the IP and DMAP
[0254] The screening of different kinases for activity with IOH or POH was done with 50 mM substrate in 100 mM Tris-HCl pH 7.5, with 12.5 mM MgC’E. 5 mM ATP and 0.1 mg/ml kinase. IP or DMAP forming was determined by stopping reaction after incubation overnight at 37 °C by adding one volume of abs. MeOH and quantifying the amount of synthesized IP via HPLC, calibrated with an external standard.
[0255] The following table shows the outcome of the screening experiments in terms of the integrated area of the HPLC signal.
[0256] From this initial screening two enzymes showed particularly high conversion of IOH and POH as substrates, namely EcHTK (SEQ ID NO: 1) and PfCK (SEQ ID NO:2).
EXAMPLE 3: Synthesis of IPP and DMAPP under employment of a polyphosphate-based ATP- regeneration system.
EXAMPLE 3.1: Synthesis of IPP
[0257] Synthesis of IPP from IOH was done in 200 mM Tris-HCl, pH 8.5 with 100 mM IOH, 5 mM ATP, 75 mM MgCl2, 1 mg/ml EcHTK (SEQ ID NO: 1) 0.1 mg/ml RcIMK (SEQ ID NO :3), 0.1 mg/ml SmPPK (SEQ ID NO: 37) and 300 mM polyphosphate. The reaction mixture was incubated at 30°C for 20 h, stopped by heating at 90 °C for 10 min and the resulting IPP quantified via HPLC, calibrated with an external standard. The resulting yield was 50 % IP and 10 % IPP. The process can be performed either as one-pot synthesis as described here, or in separate reaction steps, producing IP and in a second step IPP.
EXAMPLE 3.2: Synthesis of DMAPP - under employment of an Isomerase [0258] For isomerization of isopentenyl diphosphate (IPP) from example 3.1 to dimethylallyl diphosphate DMAPP a reaction containing 50 mM IPP, 5 mM MgCT and 0.2 mg/ml SpIDI (SEQ ID NO: 15) in 50 mM Tris-HCl, pH 7.5 was used. The reaction was incubated at 30 °C for 5 h to give Dimethylallyl pyrophosphate DMAPP. The isomerization from IPP to DMAPP can either be performed in a separate step, as described here. Or it can be coupled to the reaction from IOH to IPP described in example 3.1.
EXAMPLE 3.3: Synthesis of DMAPP - by phosphorylation of POH
[0259] Synthesis of DMAPP from POH was done in 200 mM Tris-HCl, pH 8.5 with 100 mM POH, 5 mM ATP, 75 mM MgC12, 2 mg/ml EcHTK (SEQ ID NOT) 0.1 mg/ml RcIMK (SEQ ID NO :3), 0.1 mg/ml SmPPK (SEQ ID NO:37) and 300 mM polyphosphate. The reaction mixture was incubated at 30°C for 20 h, stopped by heating at 90 °C for 10 min and the resulting DMAPP quantified via HPLC, calibrated with an external standard. The resulting yield was 23 % DMAP and 12 % mM DMAPP. The process can be performed either as one-pot synthesis as described here, or in separate reaction steps, producing first DMAP and in a second step DMAPP.
EXAMPLE 4: Synthesis of IPP and DMAPP - under employment of a acetyl phosphate -based ATP-regeneration system
EXAMPLE 4.1: Synthesis of IPP
[0260] Synthesis of IPP from IOH was done in 200 mM Tris-HCl, pH 8.5 with 100 mM IOH, 5 mM ATP, 60 mM MgC12, 1 mg/ml EcHTK, 0.1 mg/ml RcIMK (SEQ ID NOT), 2.35 U/ml GkACK (SEQ ID NO:26) and 300 mM acetyl phosphate. The reaction mixture was incubated at 30°C for 20 h, stopped by heating at 90 °C for 10 min and the resulting IPP quantified via HPLC, calibrated with an external standard. The resulting yield was 7 % IP and 93 % IPP. The process can be performed either as one-pot synthesis as described here, or in separate reaction steps, producing first the IP and in a second step IPP.
[0261] By using a acetyl phosphate-based ATP-regeneration system with GkACK (SEQ ID NO:26) in combination with AcP, the IPP yield can be significantly increased as compared to the case in which a polyphosphate-based ATP-regeneration system with SmPPK (SEQ ID NO:37) and PolyP is used. One reason for this is that the SmPPK (SEQ ID NO:37) overphosphorylates IPP and in this way a large part of the product is lost. On the other hand, the chemical driving force of the reaction with AcP as phosphate donor is significantly higher, so that the reaction equilibrium of RcIMK (SEQ ID NOT) is shifted more strongly to the product side. EXAMPLE 4.2: Synthesis of DMAPP - under employment of an Isomerase
[0262] For isomerization of IPP from example 4.1 to DMAPP a reaction containing 50 mM IPP, 5 mM MgC12 and 0.2 mg/ml SpIDI (SEQ ID NO: 15) in 50 mM Tris-HCl, pH 7.5 was used. The reaction was incubated at 30 °C for 5 h to give DMAPP. The isomerization from IPP to DMAPP can either be performed in a separate step, as described here. Or it can be coupled to the reaction from IOH to IPP described in example 4.1.
[0263] As experimentally observed for the synthesis of its synthetic precursor IPP (see example 3.1 and 4.1), it can be reasonably expected that by using a acetyl phosphate-based ATP -regeneration system with GkACK (SEQ ID NO:26) in combination with AcP, the DMAPP yield can be significantly increased as compared to the case in which a polyphosphate-based ATP -regeneration system with SmPPK (SEQ ID NO: 37) and PolyP is used instead.
EXAMPLE 4.3: Synthesis of DMAPP - by phosphorylation of POH
[0264] Synthesis of DMAPP from POH was done in 200 mM Tris-HCl, pH 8.5 with 100 mM POH, 5 mM ATP, 60 mM MgC12, 2 mg/ml EcHTK (SEQ ID NOT) , 0.1 mg/ml RcIMK (SEQ ID NOT), 2.35 U/ml GkACK (SEQ ID NO:26) and 300 mM acetyl phosphate. The reaction mixture was incubated at 30°C for 20 h, stopped by heating at 90 °C for 10 min and the resulting DMAPP quantified via HPLC, calibrated with an external standard. The resulting yield was 4 % DMAP and 46 % DMAPP. The process can be performed either as one-pot synthesis as described here, or in separate reaction steps, producing first DMAP and in a second step DMAPP.
[0265] By using a acetyl phosphate-based ATP -regeneration system with GkACK (SEQ ID NO:26) in combination with AcP, the DMAPP yield can be significantly increased as compared to the case in which a polyphosphate-based ATP-regeneration system with SmPPK (SEQ ID NO:37) and PolyP is used. One reason for this is that the SmPPK (SEQ ID NO:37) overphosphorylates IPP and in this way a large part of the product is lost. On the other hand, the chemical driving force of the reaction with AcP as phosphate donor is significantly higher, so that the reaction equilibrium of RcIMK (SEQ ID NOT) is shifted more strongly to the product side.
EXAMPLE 4.4: Comparative synthesis of IPP under employment of an acetyl phosphate-based ATP-regeneration system or a polyphosphate-based ATP-regeneration system.
[0266] In order to demonstrate the superiority of the acetyl phosphate-based ATP-regeneration system over the polyphosphate-based ATP-regeneration system, SmPPK (SEQ ID NO:37) and GkACK (SEQ ID NO:26) were subjected to a comparative test for the synthesis of IPP from IOH under identical conditions.
[0267] To that end, two independent experiments were carried out in a 200 mM Tris-HCl, pH 8.5 buffer containing 100 mM IOH, 5 mM ATP, 60 mM MgCl2, 1.0 mg/ml EcHTK (SEQ ID NO: 1) and 0.1 mg/ml RcIMK (SEQ ID NO:3).
[0268] In one experiment, 0.002 mg/mL (corresponding to 2.35 U/ml) GkACK (SEQ ID NO:26) was used in combination with 300 mM acetyl phosphate. In a second experiment, 0.002 mg/mL SmPPK (SEQ ID NO:37) was used in combination with 300 mM polyphosphate.
[0269] The reaction mixtures were incubated at 30°C for 20 h, stopped by heating at 90 °C for 10 min and the resulting IPP quantified via HPLC, calibrated with an external standard. The resulting yield was 80 % IPP with GkACK (SEQ ID NO:26) and 8 % IPP with SmPPK (SEQ ID NO:37).
EXAMPLE 5: General enzymatic synthesis of FPP starting from IPP and DMAPP from Example 2 or 3
[0270] A reaction solution containing 15 mM DMAPP and 35 mM IPP with 5 mM MgC12 in 50 mM Tris-HCl buffer, pH 7.5 was preheated to 30 °C. The reaction was started by adding GsFPPS (SEQ ID NO:5) to a final concentration of 0,2 mg/ml. The mixture was incubated at 30 °C for 4-6 h, stopped by adding one volume of abs. MeOH and the resulting FPP was quantified via HPLC, calibrated with an external standard.
EXAMPLE 6: General synthesis of sesquiterpenes starting from IOH under employment of a polyphosphate-based ATP-regeneration system
[0271] A reaction solution containing 100 mM IOH in 200 mM Tris-HCl buffer pH 8.5, with 75 mM MgC12, 5 mM ATP, 300 mM PolyP, 0.1 mg/ml SmPPK (SEQ ID NO:37) ,1 mg/ml EcHTK (SEQ ID NO: 1), 0.1 mg/ml RcIMK (SEQ ID NO:3), 0.2 mg/ml SpIDI (SEQ ID NO: 15), 0.2 mg/ml GsFPPS (SEQ ID NO:5) and 0.1-10 mg/ml of a sesquiterpene synthase is overlaid with cyclohexane and incubated for 20 h at 30°C. Samples are taken over time and the amount of the desired sesquiterpene is quantified by GC-FID, using an external standard.
EXAMPLE 7: General synthesis of sesquiterpenes starting from POH under employment of a polyphosphate-based ATP-regeneration system [0272] A reaction solution containing 100 mM POH in 200 mM Tris-HCl buffer pH 8.5, with 75 mM MgC12, 5 mM ATP, 300 mM PolyP, 0.1 mg/ml SmPPK (SEQ ID NO:37) 2 mg/ml EcHTK (SEQ ID NOT), 0.1 mg/ml RcIMK (SEQ ID NO:3), 0.2 mg/ml SpIDI (SEQ ID NO: 15), 0.2 mg/ml GsFPPS (SEQ ID NO:5) and 0.1-10 mg/ml of a sesquiterpene synthase is overlaid with cyclohexane and incubated for 20 h at 30°C. Samples are taken over time and the amount of the desired sesquiterpene is quantified by GC-FID, using an external standard.
EXAMPLE 8: General synthesis of sesquiterpenes starting from IOH and POH under employment of a polyphosphate-based ATP-regeneration system
[0273] A reaction solution containing 30 mM POH and 60 mM IOH in 200 mM Tris-HCl buffer pH 8.5, with 75 mM MgC12, 5 mM ATP, 300 mM PolyP, 0.1 mg/ml SmPPK (SEQ ID NO:37), 2 mg/ml EcHTK (SEQ ID NOT), 0.1 mg/ml RcIMK (SEQ ID NOT), 0.2 mg/ml GsFPPS (SEQ ID NO:5) and 0.1-10 mg/ml of a sesquiterpene synthase is overlaid with cyclohexane and incubated for 20 h at 30°C. Samples are taken over time and the amount of the desired sesquiterpene is quantified by GC-FID, using an external standard.
EXAMPLE 9: General synthesis of sesquiterpenes starting from IOH and acetyl phosphate under employment of a acetyl phosphate -based ATP-regeneration system
[0274] A reaction solution containing 100 mM IOH in 200 mM Tris-HCl buffer pH 8.5, with 60 mM MgC12, 5 mM ATP, 300 mM AcP, 2,35 U/ml GkACK (SEQ ID NO:26), 1 mg/ml EcHTK (SEQ ID NOT), 0.1 mg/ml RcIMK (SEQ ID NOT), 0.2 mg/ml SpIDI (SEQ ID NO: 15), 0.2 mg/ml GsFPPS (SEQ ID NO:5) and 0.1-10 mg/ml of a sesquiterpene synthase is overlaid with cyclohexane and incubated for 20 h at 30°C. Samples are taken over time and the amount of the desired sesquiterpene is quantified by GC-FID, using an external standard.
[0275] It is expected that by using a acetyl phosphate-based ATP-regeneration system with GkACK (SEQ ID NO:26) in combination with AcP, the final sesquiterpenes yield can be significantly increased as compared to the case in which a polyphosphate-based ATP-regeneration system with SmPPK (SEQ ID NO:37) and PolyP is used. One reason for this is that the SmPPK (SEQ ID NO:37) overphosphorylates IPP and in this way a large part of the product is lost. On the other hand, the chemical driving force of the reaction with AcP as phosphate donor is significantly higher, so that the reaction equilibrium of RcIMK (SEQ ID NOT) is shifted more strongly to the product side. EXAMPLE 10: General synthesis of sesquiterpenes starting from POH under employment of a acetyl phosphate -based ATP-regeneration system
[0276] A reaction solution containing 100 mM POH in 200 mM Tris-HCl buffer pH 8.5, with 60 mM MgC12, 5 mM ATP, 300 mM AcP, 2.35 U/ml GkACK (SEQ ID NO:26), 2 mg/ml EcHTK (SEQ ID NO: 1), 0.1 mg/ml RcIMK (SEQ ID NO:3), 0.2 mg/ml SpIDI (SEQ ID NO: 15), 0.2 mg/ml GsFPPS (SEQ ID NO:5) and 0.1-10 mg/ml of a sesquiterpene synthase is overlaid with cyclohexane and incubated for 20 h at 30°C. Samples are taken over time and the amount of the desired sesquiterpene is quantified by GC-FID, using an external standard.
[0277] It is expected that by using a acetyl phosphate-based ATP-regeneration system with GkACK (SEQ ID NO:26) in combination with AcP, the final sesquiterpenes yield can be significantly increased as compared to the case in which a polyphosphate-based ATP-regeneration system with SmPPK (SEQ ID NO:37) and PolyP is used. One reason for this is that the SmPPK (SEQ ID NO:37) overphosphorylates IPP and in this way a large part of the product is lost. On the other hand, the chemical driving force of the reaction with AcP as phosphate donor is significantly higher, so that the reaction equilibrium of RcIMK (SEQ ID NO:3) is shifted more strongly to the product side.
EXAMPLE 11: General synthesis of sesquiterpenes starting from IOH and POH under employment of a acetyl phosphate -based ATP-regeneration system
[0278] A reaction solution containing 30 mM POH and 60 mM IOH in 200 mM Tris-HCl buffer pH 8.5, with 60 mM MgC12, 5 mM ATP, 300 mM AcP, 2.35 U/ml (SEQ ID NO:26), 2 mg/ml EcHTK (SEQ ID NO: 1), 0.1 mg/ml RcIMK (SEQ ID NO:3), 0.2 mg/ml GsFPPS (SEQ ID NO:5) and 0.1-10 mg/ml of a sesquiterpene synthase is overlaid with cyclohexane and incubated for 20 h at 30°C. Samples are taken over time and the amount of the desired sesquiterpene is quantified by GC-FID, using an external standard.
[0279] It is expected that by using a acetyl phosphate-based ATP-regeneration system with GkACK (SEQ ID NO:26) in combination with AcP, the final sesquiterpenes yield can be significantly increased as compared to the case in which a polyphosphate-based ATP-regeneration system with SmPPK (SEQ ID NO:37) and PolyP is used. One reason for this is that the SmPPK (SEQ ID NO:37) overphosphorylates IPP and in this way a large part of the product is lost. On the other hand, the chemical driving force of the reaction with AcP as phosphate donor is significantly higher, so that the reaction equilibrium of RcIMK (SEQ ID NO:3) is shifted more strongly to the product side.

Claims

82 Patent claims:
1. A process for the preparation of a first hemiterpene diphosphate comprising the steps of:
(ai) phosphorylating a first hemiterpene alcohol with a first phosphate donor under catalysis of a first kinase thereby obtaining a first hemiterpene monophosphate and a first phosphate acceptor; and
(a2) phosphorylating the first hemiterpene monophosphate obtained in step (ai) with a second phosphate donor under catalysis of a second kinase thereby obtaining a first hemiterpene diphosphate and a second phosphate acceptor.
2. The process according to claim 1, wherein the first kinase is an enzyme capable of catalyzing the phosphorylation of an alcohol; preferably of a hemiterpene alcohol; more preferably of isoprenol and/or prenol.
3. The process according to claim 1 or 2, wherein the first kinase is a hydroxyethylthiazole kinase from Escherichia coli (EC 2.7.1.50).
4. The process according to any of the preceding claims, wherein the first kinase is an enzyme comprising a primary sequence having an identity of at least 75% to SEQ ID NO: 1.
5. The process according to any of the preceding claims, wherein the first hemiterpene alcohol is isoprenol.
6. The process according to any of the preceding claims, wherein the second kinase is a isopentenyl phosphate kinase (EC 2.7.4.26).
7. The process according to any of the preceding claims, wherein the second kinase is an enzyme comprising a primary sequence having an identity of at least 90% to SEQ ID NO:3.
8. The process according to any of the preceding claims, wherein the second kinase is a isopentenyl phosphate kinase from Methanolobus tindarius.
9. The process according to any of the preceding claims, wherein the first kinase is an enzyme comprising a primary sequence having an identity of at least 75% to SEQ ID NO: 1; and/or the first hemiterpene alcohol is isoprenol; and/or 83 the first and second phosphate donor is adenosine triphosphate; and/or the second kinase is an enzyme comprising a primary sequence having an identity of at least 75% to SEQ ID N0:3. A process for the preparation of a sesquiterpene diphosphate comprising the process according to any of the preceding claims. The process according to claim 10, which additionally comprises the steps of:
(as) providing a second hemiterpene diphosphate differing from the first hemiterpene diphosphate; and
(c) linking the first hemiterpene diphosphate obtained in step (as) with the second hemiterpene diphosphate provided in step (as) under catalysis of a sesquiterpene diphosphate synthase thereby obtaining the sesquiterpene diphosphate. The process according to claim 11, wherein the sesquiterpene diphosphate synthase is a famesyl diphosphate synthase (FPPS) (EC 2.5.1.10). The process according to claim 11 or 12, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:5. The process according to claim 11 or 12, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or 84 at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:7. The process according to claim 11 or 12, wherein the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity at least 60%, or at least 61%, or at least 62%, or at least 63%, or at least 64%; preferably at least 65%, or at least 66%, or at least 67%, or at least 68%, or at least 69%; more preferably at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%; still more preferably at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%; yet more preferably at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%; even more preferably at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%; most preferably at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%; and in particular at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% to SEQ ID NO:8. The process according to any claims 10 to 15, wherein the sesquiterpene diphosphate is famesyl diphosphate (FPP). The process according to any of claims 10 to 16, wherein the first hemiterpene diphosphate is isopentenyl pyrophosphate (IPP); and/or the sesquiterpene diphosphate is famesyl pyrophosphate (FPP); and/or the sesquiterpene diphosphate synthase is an enzyme comprising a primary sequence having an identity of at least 80% to SEQ ID NO:5 or to SEQ ID NO:7, or to SEQ ID NO:8. The process according to any of claims 11 to 17, wherein step (as) comprises the sub-steps of (as.i) phosphorylating a second hemiterpene alcohol differing from the first hemiterpene alcohol with a third phosphate donor under catalysis of a third kinase thereby obtaining a second hemiterpene monophosphate and a third phosphate acceptor; and
(as.2) phosphorylating the second hemiterpene monophosphate obtained in step (as.i) with a fourth phosphate donor under catalysis of a fourth kinase thereby obtaining the second hemiterpene diphosphate and a fourth phosphate acceptor. The process according to any of claims 18, wherein the second hemiterpene alcohol is prenol. The process according to any of claims 18 to 19, wherein the third kinase is an enzyme comprising a primary sequence having an identity of at least 90% to SEQ ID NO: 1. 85 The process according to any of claims 18 to 20, wherein the fourth kinase is an enzyme comprising a primary sequence having an identity of at least 90% to SEQ ID NO:3. The process according to any of claims 11 to 21, wherein step (as) comprises the sub-step of
(as s) isomerizing the first hemiterpene diphosphate under catalysis of an isomerase thereby obtaining the second hemiterpene diphosphate. The process according to claim 22, wherein the isomerase is a isopentenyl diphosphate isomerases (EC 5.3.3.2). The process according to any of claims 22 to 23, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 90% to SEQ ID NO: 15. The process according to any of claims 22 to 23, wherein the isomerase is an enzyme comprising a primary sequence having an identity of at least 90% to SEQ ID NO: 17. The process according to any of claims 22 to 25, wherein the first kinase is an enzyme comprising a primary sequence having an identity of at least 80% to SEQ ID NO: 1; and/or the first hemiterpene alcohol is isoprenol; and/or the first, second, third and fourth phosphate donor is adenosine triphosphate; and/or the second kinase is an enzyme comprising a primary sequence having an identity of at least 80% to SEQ ID NO:3; and/or step (as) comprises the sub-steps of:
(as s) isomerizing the first hemiterpene diphosphate under catalysis of an isomerase thereby obtaining the second hemiterpene diphosphate; and/or the isomerase is a isopentenyl diphosphate isomerases an enzyme comprising a primary sequence having an identity of at least 90% to SEQ ID NO: 15, or to SEQ ID NO: 17; and/or the second hemiterpene diphosphate is dimethylallyl pyrophosphate (DMAPP); and/or all steps (ai), (a2), (as.i), (as.2), (as s) are performed in a single reactor; and/or the third phosphate donor and fourth phosphate donor are adenosine triphosphate. A process for the synthesis of a sesquiterpene comprising the process for the synthesis of a sesquiterpene diphosphate according to any claims 10 to 26. 86 The process according to claim 27, which additionally comprises the step of:
(i) reacting a sesquiterpene diphosphate obtained in step (c) under catalysis of a sesquiterpene synthase thereby obtaining the sesquiterpene. A process for the synthesis of a sesquiterpenoid comprising the process for the synthesis of a sesquiterpene according to any claims 27 to 28. The process according to claim 29, which additionally comprises the step of:
(j) reacting the sesquiterpene obtained in step (i) under catalysis of a monooxygenase (EC 1.14.x.x) thereby obtaining sesquiterpenoid. The process according to any of the preceding claims, which additionally comprises the step of
(o) phosphorylating the first phosphate acceptor obtained in step (ai), the second phosphate acceptor obtained in step (a2), the third phosphate acceptor optionally obtained in step (as.i) and/or the fourth phosphate acceptor optionally obtained in step (a3.2) with a phosphorylating agent, optionally under catalysis of a fifth kinase, thereby obtaining first phosphate donor, second phosphate donor, third phosphate donor, and/or fourth phosphate donor, respectively. The process according to claim 31, wherein the fifth kinase is an enzyme comprising a primary sequence having an identity of at least 80 % to SEQ ID NO:26. The process according to claim 31 or 32, wherein the phosphorylating agent is acetyl phosphate; and/or the fifth kinase is an enzyme comprising a primary sequence having an identity at least 80 % to SEQ ID NO:26. A composition comprising a
(i) first hemiterpene diphosphate obtainable by the process according to any of the proceeding claims in combination with
(ii) a first kinase,
(iii) a first phosphate donor,
(iv) a second kinase, and 87
(v) a second phosphate donor, or any combination of the foregoing. The composition of claim 34, which further comprises
(vi) a phosphorylating agent being acetyl phosphate, and/or
(vii) a fifth kinase being an enzyme comprising a primary sequence having an identity of at least 80 % to SEQ ID NO:26. The composition according to any of claims 34 to 35, which further comprises a (i) sesquiterpene obtainable by the process according to claim 27 or 28 in combination with a (ii) sesquiterpene synthase. The composition according to any of claims 34 to 36, which further comprises a (i) sesquiterpe- noid obtainable by the process according to claim 29 or 30 in combination with a (ii) monooxygenase (EC 1.14.x.x).
EP22700507.1A 2021-01-20 2022-01-19 In-vitro synthetic platform for the generation of isoprenoids Pending EP4281570A1 (en)

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