ACID ESTER

The invention provides an improved method of isolating the 3-(E)-isomer of an unsaturated carboxylic acid from a mixture of corresponding (E/Z)isomers. More partic- ularly, the present invention relates to an improved method for the biocatalytic preparation of (3E,7E)-homofarnesylic acid; as well as a novel biocatalytic method for the improved preparation of homofarnesol, in particular of (3E,7E)-homofarnesol and homofarnesol preparations having an increased content of (3E,7E)-homofarnesol (also designated as "all-E-homofarnesol"). The present invention also relates to methods of preparing (-)-ambrox by applying (3E,7E)-homofarnesylic acid or (3E,7E)-homofarnesol as obtained according to the invention as starting material.

Background of the Invention:

Ambrox® is the tradename of the enantiomerically pure compound (-)-Ambrox (3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphtho [2,1 -b] furan) which is used as valuable fragrance. Naturally occurring (-)-Ambrox is a constituent of ambra, a digestive product of pot whales.

(-)-Ambrox may be synthesized by applying chemical and/or of enzymatic reaction steps (see Scheme 1 )

cherrical reduction

according to known rrethods

3E;7E-horrofamesylic acid

3E;7E-horrofamesol enzyrre-catalysed enzyrre-catalysed

enant selective BP16156 10 enanfoselective WCX201(yi39719 cyclisation of polyenes cyclisation of polyenes

(-)-Arrbrox

Sclaredide

Scheme 1 (3E,7E)-homofarnesylic acid or (3E,7E)-homofarnesol stereochemical^ pure form are the most preferred starting materials form enzyme- bases synthetic routes, as they allow the biosynthesis of (-)-ambrox with the correct stereochemistry.

Mixtures of the isomers (3E,7E)- and (3Z,7E)-homofarnesylic acid are available. However, it is very difficult to separate such mixture of isomers in view of the high similarity between said two isomers. Separation by means of classical methods like distillation and chromatography is, therefore, quite laborious.

It is, therefore an object of the present invention to provide methods which allow a simpler access to stereoisomerically pure (3E,7E)-homofarnesylic acid and/or (3E,7E)-homofarnesol, or at least to preparations with an increased content of (3E,7E)- homofarnesylic acid or (3E,7E)-homofarnesol.

Summary of the Invention:

The above-mentioned problems could, surprisingly, be solved by providing en- zymatically catalyzed methods of selectively preparing 3E-iomers of unsaturated car- boxylic acids, as exemplified by the 3E,7Eisomer of homofarnesylic acid, by applying certain lipase (E.C. 3.1 .1 .3) enzymes.

Surprisingly, it has been found in a first particular aspect of the invention that by applying the enzymatic activity of a lipase, as for example a lipase from Candida ant- arctica, the (3E,7E)-isomer of the free homofarnesylic acid is much more rapidly esteri- fied in the presence of an alcohol than the corresponding (3Z,7E)-isomer. According to this first aspect of the invention, mixtures of (3E,7E)-homofarnesylic acid ester and free 3Z,7E-homofarnesylic acid are obtained. In view of the significant chemical differences between acid and ester, a very efficient and simple separation via extraction or distilla- tion is now possible. Chemical saponification of the thus isolated (3E,7E)- homofarnesylic acid ester yields the desired free 3E,7E)-homofarnesylic acid

Surprisingly, it has also been found in a second particular aspect of the invention that by applying the enzymatic activity of a lipase, as for example a lipase from Candida antarctica, the (3E,7E)-isomer of the homofarnesylic acid ester is much more rap- idly saponified in the presence of water than the corresponding (3Z,7E)-isomer. According to this second aspect of the invention, mixtures of (3E,7E)-homofarnesylic acid and 3Z,7E-homofarnesylic acid ester are obtained. In view of the significant chemical differences between acid and ester, a very efficient and simple separation via extraction or distillation is now possible.

The following Scheme 2 illustrates said two aspects of the present invention: 3Z,7E- acid

enzyme-catalysed esterification

3E.7E- and 3Z,7E-homofarnesylic acid 3E,7E-homofarnesylic acid ester esterification saponification

enzyme-catalysed saponification

3Z,7E-homofarnesylic acid ester

Scheme 2 Detailed Description of the Invention:

1. General Definitions:

In the absence of information to the contrary the following general definitions shall apply:

„Homofarnesylic acid" or „homofarnesoic acid" are synonyms for "(3£,7E)-

4,8,12-trimethyltrideca-3,7,1 1 -trienic acid" or "(3Z,7E)-4,8,12-trimethyltrideca-3,7,1 1 - trienic acid" or mixtures of said E/Z isomers.

"Sclareolid" is used as synonym for "(3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyl- 1 ,4,5,5a,7,8,9,9b-octahydrobenzo[e]benzofuran-2-one".

Left-handed sclareolide shows the following structural formula:

„Ambrox", „Ambroxan" and „Ambroxid" are used as synonyms. They include all stereoisomeric forms, such as, in particular, (+) Ambrox, 3a-epi-(-)Ambrox, 9b-epi-(-) Ambrox and in particular (-) Ambrox.

According to the invention, the term "lipase" means enzymes of class E.C. 3.1 .1 .3 according to the IUBMB enzyme nomenclature (http://www.iubmb.unibe.ch; http://www.chem.amul.ac.uk/iubmb/enzvme/).

According to a special embodiment of the method according to the invention, the lipase is lipase B, the gene product of CALB from Candida antarctica. The CALB gene was described previously (Uppenberg J., Hansen, M.T., Patkar, S., Jones, A., Structure 2: 293-308 (1994)) and its nucleotide or protein sequence was deposited under the access numbers Z30645 and CAA83122.1 at GenBank. Unless designated more precisely, here CALB means a nucleotide sequence with this access number. Another example of a triacylglycerol lipase is lipase B from Pseudozyma tsukubaensis (Suen, W.C., Zhang, N., Xiao, L, Madison, V., Zaks, A. Protein Eng. Des. Sel. 17(2): 133-40 (2004)).

For the purposes of the present invention, "cyclases" are generally enzymes or enzyme mutants, which in particular exhibit the activity of a homofarnesylic acid cyclase and/or of a homofarnesol cyclase. As enzymes with the activity of a homofarnesylic acid cyclase or homofarnesol cyclase are intramolecular transferases from the subclass of the isomerases; i.e. proteins with the EC number EC 5.4 are suitable. (Enzyme code according to Eur. J. Biochem. 1999, 264, 610-650). In particular, these are members of the class EC 5.4.99.17. Suitable enzymes having the activity of a homofarnesylic acid cyclase or homofarnesol cyclase are, in particular, those cyclases which also effect the cyclization of homofarnesylic acid to sclareolide and / or of squalene to hopene (hence also sometimes referred to as "SHC" or squalene- hopene cyclase) and which are described extensively in the International Patent Application WO2010139719, which is incorporated herein by reference. Mutants thereof are, for example, described in WO 2012/066059, which is expressly incorporated herein by reference.

The term "cyclase activity" describes an enzyme activity determined with a "reference substrate under standard conditions", and which describes the formation of a cyclic product from a non-cyclic substrate. Standard conditions are e.g. substrate concentrations of 10 mM to 0.2 M, especially 1 5 to 100 mM, e.g. about 20 to 25 mM; at pH 4 to 8, and at temperatures of e.g. 15 to 30 or 20 to 25 °C. The determination can be carried out with recombinant cyclase-expressing cells, digested cyclase-expressing cells, fractions thereof or enriched or purified cyclase enzyme. In particular, a reference substrate is a (3E,7E)-homofarnesylic acid.

"Yield" and / or the "conversion rate" of a reaction according to the invention is determined over a defined period of, for example, 4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours, in which the reaction takes place. In particular, the reaction is carried out under precisely defined conditions, for example at 25, 30, 40, 50 or 60 ° C.

An "enzymatically catalyzed" or "biocatalytic" method means that said method is performed under the catalytic action of an enzyme, including enzyme mutants, as herein defined. Thus the method can either be performed in the presence of said enzyme in isolated (purified, enriched) or crude form or in the presence of a cellular system, in particular, natural or recombinant microbial cells containing said enzyme in active form, and having the ability to catalyze the conversion reaction as disclosed herein.

The terms "selectively converting" or "increasing the selectivity" in general means that a particular stereoisomeric form, i.p. the 3E-form, of an unsaturated carboxylic acid as herein defined, is converted in a higher proportion or amount (compared on a molar basis) than the corresponding 3Z-form, either during the entire course of said reaction (i.e. between initiation and termination of the reaction), at a certain point of time of said reaction, or during an "interval" of said reaction. In particular, said selectivity may be observed during an "interval" corresponding 1 to 99 %, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15 to 75%, 20 to 70%, 25 to 65%, 30 to 60, or 40 to 50% conversion of the initial amount of the substrate. Said higher proportion or amount may, for example, be expressed in terms of:

a higher maximum yield of the 3E-isomer observed during the entire course of the reaction or said interval thereof;

- a higher relative amount of the 3E-isomer at a defined % degree of conversion value of the substrate; and/or

an identical relative amount of the 3E-isomer at a higher % degree of conversion value;

each of which preferably being observed relative to a reference method, said reference method being performed under otherwise identical condition with chemical means, as for example chemical esterification or chemical ester cleavage.

The term "about" indicates a potential variation of ± 25% of the stated value, in particular ± 15%, ±10 %, preferably ± 5%, ± 2% or ± 1 %. The term "substantially" describes a range of values of from about 80 to 100%, such as, for example, 85-99.9%, in particular 90 to 99.9%, preferably 95 to 99.9%, or 98 to 99.9% Especially 99 to 99.9%.

"Predominantly" refers to a proportion in the range of above 50%, as for example in the range of 51 to 100%, preferably in the range of 75 to 99,9%; more particularly 85 to 98,5%, like 95 to 99%.

Because of the reversibility of enzymatic reactions, the present invention relates to the enzymatic reactions described herein in both directions of reaction.

"Functional mutants" of herein described enzymes include the "functional equiva- lents" of such enzymes as defined below.

The term "stereoisomers" includes in particular conformational isomers.

Included in general are, according to the invention, all stereoisomeric forms of the compounds described herein, such as constitutional isomers and, in particular, stereoisomers and mixtures thereof, e.g. optical isomers, or geometric isomers such as E and Z isomers, and combinations thereof. If several asymmetric centers are present in one molecule, the invention encompasses all combinations of different conformations of these asymmetry centers, e.g. enantiomeric pairs

..Stereoselectivity" describes the ability to produce a particular stereoisomer of a compound in a stereoisomerically pure form or to specifically convert a particular stere- oisomer in an enzymatically catalyzed method as described herein out of a plurality of stereoisomers. More specifically, this means that a product of the invention is enriched with respect to a specific stereoisomer. This may be quantified via the purity %ee- parameter calculated according to the formula: %ee = [XA-XB]/[ XA+XB] ^* 100, wherein X _A and X _B represent the molar ratio (Molenbruch) of the stereoisomers A and B.

Έ-stereoselectivity" or Έ-selectivity" describes the ability to produce an E-lsomer of an at least one-fold unsaturated at a particular C=C-double bond in an E-isomerically pure or essentially pure or enriched form or to specifically or essentially specifically convert an E-isomer in an enzymatically catalyzed method as described herein out of a plurality of other isomers or a mixture of E-and Z-isomers at said particular position of the double-bond within s The above definition for stereoselectivity and its calculation applies in analogy also to the term "enantioselecitivity"

Stereoisomeric or enantiomeric purities of at least 90 %ee, like at least 95 %ee, or at lest 98 %ee, or at east 99 %ee or more may be obtained according to the inven- tion.

Unless otherwise stated, the following general chemical definitions apply:

The term "carboxylic acid" encompasses both the free acid and the salt form thereof, e.g. their alkali metal or alkaline earth metal salts. This applies accordingly to all carboxylic acids mentioned herein, in particular homofarnesylic acid.

A linear or branched, saturated or non-saturated, "hydrocarbyl" residue according to the present invention particularly refers to linear or branched, alkyl or alkenyl residues.

An "alkyl" residue comprises CrC ₂ o-alkyl radicals which are linear or branched radicals having from 1 to 20 carbon atoms or CrC ₄ -alkyl radicals or C ₄ -C ₂ o-alkyl radicals. Examples thereof are:

Ci-C ₄ -alkyl radicals selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, 2- butyl, isobutyl or ie f-butyl,

CrC ₆ -alkyl radicals selected from the Ci-C ₄ -alkyl radicals as defined above, pen- tyl, 1 -methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1 -ethylpropyl, hex- yl, 1 ,1 -dimethylpropyl, 1 ,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3- methylpentyl, 4-methylpentyl, 1 ,1 -dimethylbutyl, 1 ,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1 -ethylbutyl, 2-ethylbutyl, 1 ,1 ,2- trimethylpropyl, 1 ,2,2-trimethylpropyl, 1 -ethyl-1 -methylpropyl, 1 -ethyl-2-methylpropyl; - C ₇ -C ₂ o-alkyl radicals which are linear or branched radicals having from 7 to 20 carbon atoms; examples thereof being selected from heptyl, octyl, nonyl, decyl, un- decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, non- adecyl, eicosyl, and their constitutional isomers, as for example 4,8-dimethynonyl.

An "alkenyl" residue comprises C ₂ -C ₂ o-alkenyl radicals which are mono- or poly- unsaturated, in particular 1 -, 2-, 3- or 4-fold, preferably 1 - 2- or 3-fold unsaturated linear or branched hydrocarbon radicals having from 2 to 20 carbon atoms.

Examples of mono-nsatureated C ₂ -C ₂₀ -alkenyl residues, with a position of the double bond in any position whithin the hydrocarbon chain are vinyl, 2-propene-1 -yl, 1 - methylprop-2-ene-1 -yl, 2-butene-1 -yl, 3-butene-1 -yl, n-pentenyl, n-hexenyl, n-heptenyl, n-octenyl, n-nonenyl, n-decenyl, n-undecenyl, n-dodecenyl, n-tridecenyl, n- tetradecenyl, n-pentadecenyl, n-hexadecenyl, n-heptadecenyl, n-octadecenyl, oleyl, n- nonadecenyl, n-eicosenyl;

Examples of di- or tri-unsatureated C4-C2o-alkenyl residues with two or three double, preferably non-cumulated and preferably non. conjugated bounds ina any position of the hydrocarbon chain are sind n-butadienyl, n-pentadienyl, n-hexadienyl, n- heptadienyl, n-octadienyl, n-octatrienyl, n-nonadienyl, n-nonatrienyl, n-decadienyl, n- decatrienyl, n-undecadienyl, n-undecatrienyl, n-dodecadienyl, n-dodecatrienyl, n-tridecadienyl, n-tridecatrienyl, n-tetradecadienyl n-tetradecatrienyl, n-pentadecadienyl, n-pentadecatrienyl, n-hexadecadienyl, n-hexadecatrienyl, n-heptadecadienyl, n-heptadecatrienyl, n-octadecadienyl, n-octadecatrienyl, n- nonadecadienyl, n-nonadecatrienyl, n-eicosadienyl, n-eicosatrienyl, and the constitutional isomers thereof, as for example 4,8-dimethynona-3,7-dienyl.

Each double bond within the above C ₂ -C ₂ o-alkenyl residue may, unless otherwise stated, take the E- or the Z-configuration, and independetly of the other double bond in the case of polyunsaturation.

Non-limiting examples of "optionally substituted" residues as defined herein comprise 1 , 2, 3, 4, 5 or 6, preferably 1 or 2 identical or different substituents like, HO, SH, NH ₂ , N0 ₂ , halogen, like F, CI, Br, J; lower alkyl, lower alkoxy, lower alkylthio, lower alkyl, lower alkenyl, lower alkynyl or hydroxyl-lower alkyl, as defined above.

"Lower alkyl" refers to CrC ₄ -alkyl radicals as defined above.

"Lower alkoxy" preferably refers to the CrC ₄ -alkoxy analogues of the above- mentioned lower alkyl radicals.

"Lower alkylthio" preferably refers to the CrC ₄ -alkthio analogues of the above- mentioned lower alkyl radicals. Examples are methylthio, ethylthio, propylthio, iso- propylthio, butylthio, sec-butylthio, isobutylthio and tert-butylthio.

"Lower alkenyl" comprises C ₂ -C ₄ -alkenyl radicals as defined above.

"Lower alkynyl" comprises the alkynyl homologues of the above "lower alkeny" radicals.

The term "hydroxy lower-alkyl" refers to CrC ₄ -hydroxyalkyl which is a linear or branched alkyl radical having from 1 to 4 carbon atoms, in which at least one hydrogen atom, for example 1 or 2 of the hydrogen atoms, is/are replaced by a hydroxyl group. Examples thereof are hydroxymethyl, 2-hydroxy-1 -ethyl, 2- and 3-hydroxy-1 -propyl, 2-, 3- and 4-hydroxy-1 -butyl, and their constitutional isomers.

Herein disclosed are parameter ranges of different degree of preference for a particular parameter. Within the scope of the present disclosure is also any combina- tion of parameter ranges of different degree of preference for any combination of two or more parameter referred to herein.

Particular Embodiments of the Invention:

The present invention provides the following particular embodiments

1 . A method for isolating the 3-(E)-isomer of an unsaturated carboxylic acid compound of the general formula (I)

(I)

wherein

R ¹ is H or a straight chain or branched, saturated or unsaturated, optionally substituted Ci-C ₂ o hydrocarbyl residue, preferably a saturated, non- substituted straight chain Ci-C ₂ o hydrocarbyl residue;

R ³ is H or a CrC ₄ -hydrocarbyl residue, preferably H or methyl; R ² is a straight chain or branched, saturated or unsaturated, optionally substituted, CrC ₂ o-hydrocarbyl residue, preferably a non-saturated, branched chain C ₂ -Ci ₆ -hydrocarbyl residue;

with the proviso that, if R ³ is a CrC ₄ -hydrocarbyl residue, R ² represents an hydrocarbyl residue containing at least one additional carbon atom; from a mixture of isomers comprising the 3-(E)- and 3-(Z)-isomer of said carboxylic acid compound, whereby said mixture of isomers is subjected to a lipase (E.C. 3.1 .1.3) catalyzed enzymatic conversion reaction, which lipase preferentially, in particular stereoselectively, converts said 3-(E)-isomer, and the conversion product of said 3-(E)-isomer is isolated from said reaction mixture.

2. The method of embodiment 1 , wherein said lipase catalyzes a 3-(E)- stereoselective conversion reaction. The method of one of the preceding embodiments, wherein the lipase is applied in free or immobilized form.

The method of one of the preceding embodiments, wherein said lipase is a natural or recombinantly produced, optionally genetically modified (mutated) enzyme. The method of one of the preceding embodiments, wherein said lipase originates from Candida sp., in particular Candida antarctica.

The method of embodiment 5, wherein said lipase is Candida antarctica. lipase B (CALB) comprising an amino acid sequence of SEQ ID NO:330 or a mutant thereof having a sequence identity of at least 60 % to SEQ ID NO:330 and retaining said 3-(E)-selectivity.

The method of one of the preceding embodiments, wherein said conversion reaction comprises an enzymatic esterification reaction of an acid of the formula (la);

(la)

wherein

R ² and R ³ are as defined above;

and wherein the 3-(E)-ester is predominantly formed; and wherein an aliphatic alkanol R ¹ OH, wherein R ¹ is as defined above, preferably a straight chain or branched, saturated CrC ₂ o alkyl preferably Ci-C ₄ alkyl residue, optionally in the presence of an organic solvent which does not disturb or even inhibit lipase activity.

The method of one of the embodiments 1 to 6, wherein said conversion reaction comprises an enzymatic ester cleavage reaction of an ester of the formula (lb);

(lb)

wherein

R ¹ is H or a straight chain or branched, saturated or unsaturated C1-C20-, in particular C4-C20-, hydrocarbyl residue;

and R ² and R ³ are as defined above;

and wherein the 3-(E)-acid is predominantly formed; and wherein the reaction is performed in the presence of water, and optionally in the presence of an organic solvent, which does not disturb or even inhibit lipase activity.

The method of one of the preceding embodiments, wherein said conversion reaction is performed in an organic solvent or aqueous-organic solvent.

The method of embodiment 9, wherein said organic solvent is selected from aliphatic or cycloaliphatic hydrocarbons, like hydrocarbons having at least 5 carbon atoms, in particular hexane, cyclohexane, heptane, octane; aromatic hydrocarbons, in particular mononuclear aromatic compounds, like benzene, xylene and toluene; and aliphatic ethers, like MTBE, di-iso-propyl ether. Preferably, suitable organic solvents should be able to form a 2-phase system with water.

The method of one of the preceding embodiments, wherein said carboxylic acid compound is a 3-(E)/7-(E)- homofarnesoic acid compound of formula (II)

(II) wherein R ¹ is as defined above. A method of preparing an unsaturated 3-(E)-carboxylic acid of the general formula (la):

(la)

wherein

R ² and R ³ are as defined above;

wherein

a) an isomer mixture, comprising the 3-(E)- and the 3-(Z)- isomer of said car- boxylic acid of formula (la) is subjected to an enzymatic esterification reaction in the presence of an alkanol of the formula R ¹ OH, wherein R ¹ is a straight chain or branched, saturated or unsaturated CrC ₂ o-hydrocarbyl residue, preferably a straight chain or branched, saturated Ci-C ₂ o alkyl, preferably Ci-C ₄ alkyl residue; and in the presence of a lipase enzyme as defined in one of the embodiments 1 to 6;

b) said 3-(E)-carboxylic ester as formed in step a) is isolated, for example by transferring the non-reacted acid isomer in an aqueous phase (as acid salt) and recovering the ester by means of an organic solvent which was either present during the conversion or which was added afterwards; and

c) said isolated ester of step b) is saponified to the corresponding 3-(E)- car- boxylic acid of formula (la).

A method of preparing an unsaturated 3-(E)-carboxylic acid of the general formula (la):

(la)

wherein

R ² and R ³ are as defined above;

wherein

a) an isomer mixture, comprising the 3-(E)- and the 3-(Z)- isomer of a carbox- ylic acid ester of formula (lb)

wherein

R ¹ is a straight chain or branched, saturated or unsaturated C1-C2 ₀ - hydrocarbyl residue, preferably a straight chain or branched, saturated C C ₂ o alkyl, preferably Ci-C ₄ alkyl, residue; and

R ² and R ³ are as defined above; is subjected to an enzymatic ester cleavage reaction in the presence of a lipase enzyme as defined in one of the embodiments 1 to 6, and preferably in the presence of water and optionally an organic solvent;

b) said 3-(E)-carboxylic acid as formed in step a) is isolated, preferably as free acid from the aqueous phase of the reaction mixture..

The method of embodiment 12 or 13, wherein an organic solvent as defined one of the embodiments 9 and 10 is applied. 15. The method of one of the preceding embodiments, wherein said 3-(E)-isomer of an unsaturated carboxylic acid is 3-(E)/7-(E)-homofarnesoic acid.

16. The method of one of the preceding embodiments wherein said isomer mixture comprises a mixture of 3-(E)/7-(E)-homofarnesoic acid and 3-(Z)/7-(E)- homofarnesoic acid; or a mixture of 3-(E)/7-(E)-homofarnesoic acid ester and 3- (Z)/7-(E)-homofarnesoic acid ester of an alkanol of the formula R ¹ OH, wherein R ¹ is a straight chain or branched, saturated or unsaturated CrC ₂ o-hydrocarbyl residue.

17. The method of embodiment 16 for preparing 3-(E)/7-(E)-homofarnesoic acid wherein

a) an isomer mixture, comprising 3-(E)/7-(E)-homofarnesoic acid and 3-(Z)/7- (E)-homofarnesoic acid is subjected to an enzymatic esterification reaction in the presence of an alkanol of the formula R ¹ OH, wherein R ¹ is a straight chain or branched, saturated or unsaturated CrC ₂ o-hydrocarbyl residue, preferably a straight chain or branched, saturated Ci-C ₂ o alkyl, preferably Ci-C ₄ alkyl residue; and in the presence of a lipase enzyme as defined in one of the embodiments 1 to 6 in a solvent as defined in one of the embodiments 9 and 10;

b) said 3-(E)/7-(E)-homofarnesoic acid ester as formed in step a) is separated from unreacted acid (i.p. 3-(Z)/7-(E)-homofarnesoic acid), in particular by distillation or, preferably, extraction; and

c) said isolated 3-(E)/7-(E)-homofarnesoic acid ester is saponified to 3-(E)/7- (E)-homofarnesoic acid.

18. The method of embodiment 16 for preparing 3-(E)/7-(E)-homofarnesoic acid wherein a) an isomer mixture, comprising 3-(E)/7-(E)-homofarnesoic acid ester and 3- (Z)/7-(E)-homofarnesoic acid ester is subjected to an enzymatic ester cleavage reaction in the presence of a lipase enzyme as defined in one of the embodiments 1 to 6 in a solvent as defined in one of the embodiments 9 and 10, and preferably in the presence of water; and b) said 3-(E)/7-(E)-homofarnesoic acid as formed in step a) is separated from unreacted ester (i.p. 3-(Z)/7-(E)-homofarnesoic acid ester), in particular by distillation or, preferably, extraction.

A method of preparing (-)-ambrox of the formula (III)

(III)

comprising the steps of

a) obtaining said 3-(E)/7-(E)-homofarnesoic acid by applying a method as de- fined in anyone of the embodiments 1 to 18;

b) chemically reducing 3-(E)/7-(E)-homofarnesoic acid to 3-(E)/7-(E)- homofarnesol and

c) enzymatically cycling of 3-(E)/7-(E)-homofarnesol to (-)-ambrox.

A method of preparing (-)-ambrox of the formula (III)

(III)

comprising the steps of

a) obtaining said 3-(E)/7-(E)-homofarnesoic acid by applying a method as defined in anyone of the embodiments 1 to 18,

b) enzymatically cycling of 3-(E)/7-(E)-homofarnesoic acid to sclareolide of the formula (IV)

(IV)

and

c) chemically converting said sclareolide to (-)-ambrox.

The method of embodiment 19 or 20, wherein the enzymatic cyclization is performed in the presence of an intramolecular transferase (E.C. 5.4), in particular a homofarnesoic acid cyclase (E.C. 5.4.99.17), in particular a squalene-hopene cyclase (E.C. 5.4.99.17), showing homofarnesoic acid cyclase activity.

The method of embodiment 21 , wherein said cyclase is a natural or recombinant- ly produced, optionally genetically modified (mutated) squalene-hopene cyclase (SHC).

The method of embodiment 22, wherein said cyclase is a squalene hopene cyclase originating from Methylococcus capsalatus, Rhodopseudomonas palus- tris, Bradyrhizobium japonicum, Frankia spec, Streptomyces coelicolor or, preferably, Zymomonas mobilis.

The method of embodiment 23, wherein said cyclase is from Zymomonas mobilis, comprising an amino acid sequence according to SEQ ID NO:2 or an amino acid sequence having at least 60 % sequence identity to SEQ ID NO:2.

3-(E)/7-(E)-homofarnesoic acid alkyl ester, wherein said alkyl group is selected from C2-C10, in particular C ₃ -C ₈ -alkyl, as for example, ethyl, n-propyl, isopropyl, n- butyl, 2-butyl, isobutyl or ie f-buty; as well as pentyl, hexyl, heptyl, octyl, and their constitutional isomers; preferably in the form of the substantially stereoisomerical- ly pure 3-(E)/7-(E)-form, and in particular as prepared according to a method as defined herein above. In general cyclases applicable in said aspect of the invention are SHCs which are listed below by making reference to their wild type sequence (SEQ ID NO's and Genbank- numbers are stated) and their microbial source.

SJD DB SEQ ID NO Organism Gl-Nr. der Re- ferenz Se- quenzen

s1 seq_ ID 2 Zymomonas mobilis AAV90172.1

s20 seq_ JD 3 Streptomyces coelicolor CAB39697.1

s91 1 seq_ ID 4 Acetobacter pasteurianus BAH99456.1

s2 seq_ JD 5 Bradyrhizobium sp. ABQ33590.1

s940 seq_ JD 6 Zymomonas mobilis EER62728.1

s949 seq_ JD 7 Acidithiobacillus caldus EET25937.1

s167 seq_ JD 8 A cidithioba cillus ferrooxidans ACH84004.1

s41 seq_ JD 9 Acidobacterium capsulatum AC034244.1

s36 seq_ JD 10 Acidothermus cellulolyticus ABK53469.1

s83 seq_ JD 1 1 Adiantum capillus-veneris BAF93209.1

s143 seq_ JD 12 Ajellomyces capsulatus EDN09769.1

s995 seq_ JD 13 Ajellomyces capsulatus EER40510.1

s163 seq_ JD 14 Ajellomyces capsulatus EEH02950.1

s13 seq_ JD 15 Alicyclobacillus acidocaldarius EED08231.1

s14 seq_ JD 16 Alicyclobacillus acidocaldarius P33247.4

S1 193 seq_ JD 17 Alicyclobacillus acidocaldarius AAT70690.1

s21 seq_ JD 18 Alicyclobacillus acidoterrestris CAA61950.1

S1 189 seq_ JD 19 Alicyclobacillus acidoterrestris AAT70691.1

s51 seq_ JD 20 Anabaena variabilis ABA24268.1

s76 seq_ JD 21 Anaeromyxobacter sp. ABS28257.1

s159 seq_ JD 22 Aspergillus clavatus EAW07713.1

s131 seq_ JD 23 Aspergillus flavus EED48353.1

s176 seq_ JD 24 Aspergillus fumigatus EDP50814.1

s126 seq_ JD 25 Aspergillus fumigatus EAL84865.1

s178 seq_ JD 26 Aspergillus fumigatus EAL86291.2

s121 seq_ JD 27 Aspergillus niger CAK43501.1

s1 15 seq_ JD 28 Aspergillus niger CAK45506.1

s124 seq_ JD 29 Aspergillus oryzae BAE63941.1

s1 19 seq_ JD 30 Azotobacter vinelandii EAM0761 1.1

s223 seq_ JD 31 Bacillus amyloliquefaciens ABS74269.1

s221 seq_ JD 32 Bacillus anthracis AAP27368.1

s976 seq_ JD 33 Bacillus cereus EEK66523.1 SJD DB SEQ ID NO Organism Gl-Nr. der Re- ferenz Se- quenzen s225 seq_ D 34 Bacillus cereus EAL12758.1 s972 seq_ D 35 Bacillus cereus EEL44583.1 s977 seq_ D 36 Bacillus cereus EEK43841.1 s985 seq_ D 37 Bacillus cereus EEK82938.1 s988 seq_ D 38 Bacillus cereus EEK99528.1 s981 seq_ D 39 Bacillus cereus EEK77935.1 s987 seq_ D 40 Bacillus cereus EEL81079.1 s960 seq_ D 41 Bacillus cereus EEK88307.1 s979 seq_ D 42 Bacillus cereus EEL63943.1 s974 seq_ D 43 Bacillus cereus EEL59884.1 s956 seq_ D 44 Bacillus cereus EEL69857.1 s951 seq_ D 45 Bacillus cereus EEL92663.1 s986 seq_ D 46 Bacillus cereus EEL49968.1 s227 seq_ D 47 Bacillus cereus AAU16998.1 s224 seq_ D 48 Bacillus cereus AAS42477.1 s212 seq_ D 49 Bacillus cereus ACK95843.1 s289 seq_ D 50 Bacillus coahuilensis 205373680 s219 seq_ D 51 Bacillus cytotoxicus ABS22481.1 s230 seq_ D 52 Bacillus licheniformis AAU23777.1 s955 seq_ D 53 Bacillus my co ides EEL98438.1 s990 seq_ D 54 Bacillus my co ides EEM04821 .1 s989 seq_ D 55 Bacillus pseudomycoides EEM16144.1 s247 seq_ D 56 Bacillus pumilus ABV62529.1 s250 seq_ D 57 Bacillus pumilus EDW21 137.1 s249 seq_ D 58 Bacillus sp. EAR64404.1 s218 seq_ D 59 Bacillus sp. EDL66148.1 s241 seq_ D 60 Bacillus subtil is Q796C3.1 s284 seq_ D 61 Bacillus subtil is AAB84441.1 s215 seq_ D 62 Bacillus thuringiensis ABK86448.1 s984 seq_ D 63 Bacillus thuringiensis EEM21409.1 s957 seq_ ID 64 Bacillus thuringiensis EEM82653.1 s980 seq_ ID 65 Bacillus thuringiensis EEM52372.1 s961 seq_ ID 66 Bacillus thuringiensis EEM27851.1 s969 seq_ ID 67 Bacillus thuringiensis EEM40716.1 s959 seq_ ID 68 Bacillus thuringiensis EEM46814.1 s965 seq_ ID 69 Bacillus thuringiensis EEM94969.1 s202 seq_ ID 70 Bacillus weihenstephanensis ABY44436.1 S_ID DB SEQ ID NO Organism Gl-Nr. der Re- ferenz Se- quenzen s63 seq_ D 71 Bacterium Ellin514 P P CC7 9C 1 s72 seq_ D 72 Bacterium Ellin514 EEF59508.1 s87 seq_ D 73 Beijerinckia indica ACB96717.1 s69 seq_ D 74 Blastopirellula marina EAQ81955.1 s543 seq_ D 75 Blastopirellula marina EAQ78122.1 s156 seq_ D 76 Bradyrhizobium japonicum CAA60250.1 s938 seq_ D 77 Acetobacter pasteurianus BAH98349.1 s3 seq_ D 78 Bradyrhizobium sp. CAL79893.1 s201 seq_ D 79 Brevibacillus brevis BAH44778.1 s148 seq_ D 80 Burkholderia ambifaria EDT05097.1 s158 seq_ D 81 Burkholderia ambifaria EDT37649.1 s149 seq_ D 82 Burkholderia ambifaria ACB68303.1 s100 seq_ D 83 Burkholderia ambifaria EDT42454.1 s146 seq_ D 84 Burkholderia cenocepacia EAY66961.1 s139 seq_ D 85 Burkholderia cenocepacia ACA95661.1 s147 seq_ D 86 Burkholderia cenocepacia CAR57099.1 s95 seq_ D 87 Burkholderia cenocepacia CAR56694.1 s102 seq_ D 88 Burkholderia dolosa EAY7131 1 .1 s941 seq_ D 89 Burkholderia glumae ACR32572.1 s945 seq_ D 90 Burkholderia glumae ACR30752.1 s132 seq_ D 91 Burkholderia graminis EDT12320.1 s104 seq_ D 92 Burkholderia mallei ABM48844.1 s140 seq_ ID 93 Burkholderia multivorans ABX19650.1 s1 16 seq_ ID 94 Burkholderia multivorans ABX16859.1 s91 seq_ ID 95 Burkholderia oklahomensis 167567074 s1 1 1 seq_ ID 96 Burkholderia phymatum ACC73258.1 s127 seq_ ID 97 Burkholderia phytofirmans ACD21317.1 s120 seq_ ID 98 Burkholderia pseudomallei EEC32728.1 s137 seq_ ID 99 Burkholderia sp. EEA03553.1 s144 seq_ D 100 Burkholderia sp. ABB06563.1 s98 seq_ D 101 Burkholderia sp. ABB10136.1 s944 seq_ ID 102 Burkholderia sp. CCGE1002 EFA54357.1 s89 seq_ ID 103 Burkholderia thailandensis 167840988 s1 13 seq_ ID 104 Burkholderia thailandensis 167617352 s154 seq_ ID 105 Burkholderia ubonensis 167589807 s93 seq_ ID 106 Burkholderia ubonensis 167584986 s96 seq_ ID 107 Burkholderia vietnamiensis AB056791.1 SJD DB SEQ ID NO Organism Gl-Nr. der e- Ferenz Se- quenzen s150 seq_ JD 108 Burkholderia xenovorans ABE35912.1 s54 seq_ JD 109 Candidatus Koribacter ABF40741.1 s171 seq_ JD 1 10 Candidatus Kuenenia CAJ71215.1 s79 seq_ JD 1 1 1 Candidatus Solibacter ABJ82180.1 s99 seq_ JD 1 12 Candidatus Solibacter ABJ82254.1 s917 seq_ JD 1 13 Catenulispora acidiphila ACU75510.1 s65 seq_ JD 1 14 Chthoniobacter flavus EDY15838.1 s637 seq_ JD 1 15 Chthoniobacter flavus E DY22035. i s38 seq_ JD 1 16 Crocosphaera watsonii EAM53094.1 s186 seq_ JD 1 17 Cupriavidus taiwanensis CAQ72562.1 s32 seq_ JD 1 18 Cyanothece sp. ACB53858.1 s40 seq_ JD 1 19 Cyanothece sp. ACK71719.1 s30 seq_ JD 120 Cyanothece sp. EDY02410.1 s29 seq_ JD 121 Cyanothece sp. ACK66841.1 s47 seq_ JD 122 Cyanothece sp. EDX97382.1 s35 seq_ JD 123 Cyanothece sp. EAZ91809.1 s39 seq_ JD 124 Cyanothece sp. ACL45896.1 s925 seq_ JD 125 Cyanothece sp. PCC 8802 ACV02092.1 s64 seq_ JD 126 Desulfovibrio salexigens EEC62384.1 s74 seq_ JD 127 Dryopteris crassirhizoma BAG68223.1 s59 seq_ JD 128 Frankia alni CAJ61 140.1 s48 seq_ JD 129 Frankia alni CAJ60090.1 s56 seq_ JD 130 Frankia sp. ABD10207.1 s60 seq_ JD 131 Frankia sp. ABW 15063.1 s31 seq_ JD 132 Frankia sp. ABW14125.1 s948 seq_ JD 133 Frankia sp. Eul1c EFA59873.1 s919 seq_ JD 134 Frankia sp. Eul1c EFA59089.1 s628 seq_ JD 135 Gemmata obscuriglobus 168700710 s209 seq_ JD 136 Geobacillus sp. EED61885.1 s206 seq_ JD 137 Geobacillus sp. EDY05760.1 s964 seq_ JD 138 Geobacillus sp. Y412MC52 EEN95021.1 s993 seq_ JD 139 Geobacillus sp. Y412MC61 ACX79399.1 s205 seq_ JD 140 Geobacillus thermodenitrificans AB067242.1 s15 seq_ JD 141 Geobacter bemidjiensis ACH40355.1 s8 seq_ JD 142 Geobacter lovleyi ACD95949.1 s62 seq_ JD 143 Geobacter metallireducens ABB30662.1 s12 seq_ JD 144 Geobacter metallireducens ABB33038.1 SJD DB SEQ ID NO Organism Gl-Nr. der e-

Ferenz Se- quenzen s73 seq_ JD 145 Geobacter sp. ACM21577.1 s10 seq_ JD 146 Geobacter sp. EDV72707.1 s1 1 seq_ JD 147 Geobacter sp. ACM22003.1 s913 seq_ JD 148 Geobacter sp. M18 EET34621.1 s914 seq_ JD 149 Geobacter sp. M21 ACT16952,1 s58 seq_ JD 150 Geobacter sulfurreducens AAR36453.1 s7 seq_ JD 151 Geobacter sulfurreducens AAR34018.1 s9 seq_ JD 152 Geobacter uraniireducens ABQ25226.1 s46 seq_ JD 153 Gloeobacter violaceus BAC91998.1 s67 seq_ JD 154 Gluconacetobacter diazotrophicus ACI51585.1 s165 seq_ JD 155 Gluconacetobacter diazotrophicus CAP55563.1 s68 seq_ JD 156 Gluconobacter oxydans AAW61994.1 s80 seq_ JD 157 Granulibacter bethesdensis ABI63005.1 s937 seq_ JD 158 Hyphomicrobium denitrificans EET65847.1 s932 seq_ JD 159 Leptospirillum ferrodiazotrophum EES53667.1 s24 seq_ JD 160 Leptospirillum rubarum EAY57382.1 s25 seq_ JD 161 Leptospirillum sp. EDZ38599.1 s174 seq_ JD 162 Magnaporthe grisea EDK02551.1 s153 seq_ JD 163 Magnetospirillum magnetotacti- 46203107

cum

s49 seq_ JD 164 Methylacidiphilum infernorum ACD82457.1 s169 seq_ JD 165 Methylobacterium chloromethani- ACK83067.1

cum

s75 seq_ JD 166 Methylobacterium chloromethani- ACK86232.1

cum

s946 seq_ JD 167 Methylobacterium extorquens CAX24364.1 s141 seq_ JD 168 Methylobacterium nodulans ACL61886.1 s152 seq_ JD 169 Methylobacterium populi ACB79998.1 s162 seq_ JD 170 Methylobacterium radiotolerans ACB27373.1 s180 seq_ JD 171 Methylobacterium sp. ACA2061 1 .1 s175 seq_ JD 172 Methylocella silvestris ACK52150.1 s181 seq_ JD 173 Methylococcus capsulatus CAA71098.1 s55 seq_ JD 174 Microcystis aeruginosa CA086472.1 s101 seq_ JD 175 Neosartorya fischeri EAW20752.1 s129 seq_ JD 176 Nitrobacter hamburgensis ABE63461.1 s161 seq_ JD 177 Nitrobacter sp. EAQ34404.1 s160 seq_ JD 178 Nitrobacter winogradskyi ABA05523.1 SJD DB SEQ ID NO Organism Gl-Nr. der e- Ferenz Se- quenzen s157 seq_ JD 179 Nitrococcus mobilis EAR22397.1 s164 seq_ JD 180 Nitrosococcus oceani ABA57818.1 s170 seq_ JD 181 Nitrosomonas europaea CAD85079.1 s173 seq_ JD 182 Nitrosomonas eutropha ABI59752.1 s943 seq_ JD 183 Nitrosomonas sp. AL212 EET32702.1 s142 seq_ JD 184 Nitrosospira multiformis ABB75845.1 s52 seq_ JD 185 Nostoc punctiforme ACC84529.1 s45 seq_ JD 186 Nostoc sp. BAB72732.1 s122 seq_ JD 187 Oligotropha carboxidovorans ACI93782.1 s233 seq_ JD 188 Paenibacillus sp. EDS49994.1 s991 seq_ JD 189 Paenibacillus sp. JDR-2 ACS99948.1 s950 seq_ JD 190 Paenibacillus sp. oral taxon 786 EES74793.1

S1280 seq_ JD 191 Paramecium tetraurelia 145542269 s71 seq_ JD 192 Pelobacter carbinolicus ABA87701.1 s5 seq_ JD 193 Pelobacter carbinolicus ABA87615.1 s66 seq_ JD 194 Pelobacter propionicus ABK98395.1 s16 seq_ JD 195 Pelobacter propionicus ABK9881 1.1 s136 seq_ JD 196 Penicillium chrysogenum CAP99707.1 s936 seq_ JD 197 Planctomyces limnophilus EE067214.1

S1 158 seq_ JD 198 Planctomyces limnophilus EE068341.1 s526 seq_ JD 199 Planctomyces maris E D ^" 1 s992 seq_ JD 200 Polypodiodes niponica BAI48071.1 s942 seq_ JD 201 Polypodiodes niponica BAI48070.1

S1202 seq_ JD 202 Populus trichocarpa EEF12098.1 s168 seq_ JD 203 Ralstonia eutropha AAZ64302.1 s190 seq_ JD 204 Ralstonia eutropha CAJ96989.1 s81 seq_ JD 205 Ralstonia metallidurans ABF1 1015.1 s1 10 seq_ JD 206 Ralstonia metallidurans ABF1 1268.1 s123 seq_ JD 207 Rhizobium sp. P55348.1 s657 seq_ JD 208 Rhodopirellula baltica CAD74517.1 s4 seq_ JD 209 Rhodopseudomonas palustris ABJ08391.1 s130 seq_ JD 210 Rhodopseudomonas palustris CAA71 101.1 s155 seq_ JD 21 1 Rhodopseudomonas palustris ABD06434.1 s97 seq_ JD 212 Rhodopseudomonas palustris ABD87279.1 s135 seq_ JD 213 Rhodopseudomonas palustris ACF02757.1 s84 seq_ JD 214 Rhodospirillum rubrum ABC20867.1

S1279 seq_ JD 215 Rubrobacter xylanophilus ABG05671.1 SJD DB SEQ ID NO Organism Gl-Nr. der e-

Ferenz Se- quenzen s915 seq_ D 216 Saccharomonospora viridis ACU97316.1 s42 seq_ D 217 Saccharopolyspora erythraea CAM03596.1 s82 seq_ D 218 Schizosaccharomyces japonicus EEB08219.1 s923 seq_ D 219 Sphaerobacter thermophilus ACZ39437.1 s924 seq_ D 220 Streptomyces albus 239983547 s23 seq_ D 221 Streptomyces avermitilis BAC69361.1 s44 seq_ D 222 Acaryochloris marina ABW29816.1 s921 seq_ D 223 Streptomyces filamentosus 239945642 s934 seq_ D 224 Streptomyces flavogriseus EEW7081 1.1 s920 seq_ D 225 Streptomyces ghanaensis 239927462 s922 seq_ D 226 Streptomyces griseoflavus 256812310 s28 seq_ D 227 Streptomyces griseus BAG17791.1 s926 seq_ D 228 Streptomyces hygroscopicus 256775136 s916 seq_ D 229 Streptomyces lividans 256783789 s33 seq_ D 230 Streptomyces peucetius ACA52082.1 s27 seq_ D 231 Streptomyces pristinaespiralis EDY61772.1 s933 seq_ D 232 Streptomyces scabiei CBG68454.1 s37 seq_ D 233 Streptomyces sp. EDX25760.1 s34 seq_ D 234 Streptomyces sp. EDY46371.1 s931 seq_ D 235 Streptomyces sp. AA4 256668250 s918 seq_ D 236 Streptomyces sp. C 256770952 s929 seq_ D 237 Streptomyces sp. Mg1 254385931 s928 seq_ D 238 Streptomyces sp. SPB74 254379682 s930 seq_ D 239 Streptomyces sp. SPB78 256680470 s26 seq_ D 240 Streptomyces sviceus EDY55942.1 s927 seq_ D 241 Streptomyces viridochromogenes 256805984 s61 seq_ D 242 Synechococcus sp. EDX84551.1 s935 seq_ D 243 Synechococcus sp. PCC 7335 254422098 s53 seq_ D 244 Synechocystis sp. BAA17978.1 s22 seq_ D 245 Syntrophobacter fumaroxidans ABK18414.1 s6 seq_ D 246 Syntrophobacter fumaroxidans ABK17672.1 s912 seq_ D 247 Teredinibacter turnerae ACR13362.1 s57 seq_ D 248 Thermosynechococcus elongatus BAC09861.1 s43 seq_ ID 249 Trichodesmium erythraeum ABG50159.1

S1 178 seq_ ID 250 Uncultured organism ACA58560.1

S1 176 seq_ ID 251 Uncultured organism ABL07557.1

S1 165 seq_ ID 252 Uncultured organism ACA58559.1 S_ID DB SEQ ID NO Organism Gl-Nr. der Re- ferenz Se- quenzen s1166 seq_ D253 Uncultured organism ACA58558.1

S1168 seq_ D254 Uncultured organism ABL07560.1

S1169 seq_ D255 Uncultured organism ABL07565.1

S1170 seq_ D256 Uncultured organism ABL07566.1

S1 67 seq_ D257 Uncultured organism ACA58545.1 s1171 seq_ D258 Uncultured organism ACA58535.1

S1180 seq_ D259 Uncultured organism ACA58549.1

S1179 seq_ D260 Uncultured organism ACA58554.1 s1181 seq_ D261 Uncultured organism ACA58555.1

S1182 seq_ D262 Uncultured organism ACA58556.1

S1235 seq_ D263 Uncultured organism ACA58530.1

S1188 seq_ D264 Uncultured organism ACA58534.1

S1237 seq_ D265 Uncultured organism ACA58552.1

S1223 seq_ D266 Uncultured organism ABL07558.1

S1200 seq_ D267 Uncultured organism ABL07542.1

S1236 seq_ D268 Uncultured organism ACA58539.1

S1238 seq_ D269 Uncultured organism ACA58537.1

S1233 seq_ D270 Uncultured organism ACA58543.1

S1173 seq_ D271 Uncultured organism ABL07553.1

S1241 seq_ D272 Uncultured organism ABL07540.1

S1242 seq_ D273 Uncultured organism ABL07544.1

S1225 seq_ D274 Uncultured organism ACA58557.1

S1183 seq_ D275 Uncultured organism ACA58520.1

S1197 seq_ D276 Uncultured organism ACA58524.1

S1185 seq_ D277 Uncultured organism ACA58522.1

S1190 seq_ D278 Uncultured organism ACA58525.1

S1187 seq_ D279 Uncultured organism ACA58523.1

S1184 seq_ D280 Uncultured organism ACA58521.1

S1204 seq_ D281 Uncultured organism ACA58547.1

S1221 seq_ D282 Uncultured organism ACA58544.1 s1198 seq_ D283 Uncultured organism ACA58546.1

S1226 seq_ D284 Uncultured organism ACA58527.1

S1227 seq_ ID 285 Uncultured organism ABL07537.1

S1232 seq_ ID 286 Uncultured organism ACA58510.1

S1230 seq_ ID 287 Uncultured organism ACA58538.1

S1229 seq_ ID 288 Uncultured organism ACA58542.1

S1231 seq_ ID 289 Uncultured organism ACA58540.1 SJD DB SEQ ID NO Organism Gl-Nr. der Re- ferenz Se- quenzen

S1207 seq_ D290 Uncultured organism ABL07564.1

S1212 seq_ D291 Uncultured organism ABL07563.1

S1208 seq_ D292 Uncultured organism ABL07562.1

S1209 seq_ D293 Uncultured organism ABL07559.1

S1214 seq_ D294 Uncultured organism ABL07556.1

S1216 seq_ D295 Uncultured organism ACA58528.1

S1219 seq_ D296 Uncultured organism ACA58536.1

S1192 seq_ D297 Uncultured organism ABL07533.1

S1195 seq_ D298 Uncultured organism ABL07536.1

S1174 seq_ D299 Uncultured organism ABL07545.1 s1186 seq_ D300 Uncultured organism ABL07548.1

S1196 seq_ D301 Uncultured organism ACA58561.1

S1172 seq_ D302 Uncultured organism ABL07555.1

S1194 seq_ D303 Uncultured organism ABL07541.1

S1211 seq_ D304 Uncultured organism ABL07554.1

S1220 seq_ D 305 Uncultured organism ABL07547.1

S1203 seq_ D306 Uncultured organism ABL07550.1

S1199 seq_ D307 Uncultured organism ABL07551.1

S1228 seq_ D308 Uncultured organism ACA58509.1

S1201 seq_ D309 Uncultured organism ACA58514.1

S1205 seq_ D310 Uncultured organism ABL07543.1

S1206 seq_ D311 Uncultured organism ABL07534.1

S1177 seq_ D312 Uncultured organism ABL07546.1

S1210 seq_ D313 Uncultured organism ABL07535.1

S1175 seq_ D314 Uncultured organism ABL07552.1

S1191 seq_ D 315 Uncultured organism ABL07549.1

S1222 seq_ D316 Uncultured organism ACA58553.1

S1244 seq_ D317 Uncultured organism ABL07539.1

S1213 seq_ D318 Uncultured organism ACA58532.1

S1239 seq_ D319 Uncultured organism ACA58548.1

S1215 seq_ D320 Uncultured organism ABL07561.1

S1240 seq_ D321 Uncultured organism ACA58533.1

S1234 seq_ D 322 Uncultured organism ABL07538.1

S1224 seq_ ID 323 Uncultured organism ACA58541.1

S1217 seq_ ID 324 Uncultured organism ACA58529.1 s596 seq_ ID 325 Verrucomicrobium spinosum 171910093 s70 seq_ ID 326 Acidiphilium cryptum ABQ30890.1 SEQ ID NO:2 is the amino acid sequence of a cyclase which is also known as Zm- SHC-1 . 3. Enzymes and enzyme mutants according to the invention

The present invention is not limited to the use of the specifically disclosed lipases and cyclases, but also extends to functional equivalents thereof.

"Functional equivalents" or analogs of the concretely disclosed enzymes are, within the scope of the present invention, various polypeptides thereof, which moreover possess the desired biological function or activity, e.g. enzyme activity.

For example, "functional equivalents" means enzymes, which, in a test used for enzymatic activity, display at least a 1 to 10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower activity of an enzyme, as defined herein.

"Functional equivalents", according to the invention, also means in particular mu- tants, which, in at least one sequence position of the amino acid sequences stated above, have an amino acid that is different from that concretely stated, but nevertheless possess one of the aforementioned biological activities. "Functional equivalents" thus comprise the mutants obtainable by one or more, as for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15, amino acid additions, substitutions, insertions, deletions and/or inversions, where the stated changes can occur in any sequence position, provided they lead to a mutant with the profile of properties according to the invention. Functional equivalence is in particular also provided if the reactivity patterns coincide qualitatively between the mutant and the unchanged polypeptide, i.e. if for example the same substrates are converted at a different rate. Examples of suitable amino acid substitutions are shown in the following table:

Original residue Examples of substitution

Ala Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Asn ; Gin lie Leu; Val

Leu lie; Val

Lys Arg ; Gin ; Glu

Met Leu ; lie

Phe Met ; Leu ; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp ; Phe

Val lie; Leu

"Functional equivalents" in the above sense are also "precursors" of the polypeptides described, as well as "functional derivatives" and "salts" of the polypeptides.

"Precursors" are in that case natural or synthetic precursors of the polypeptides with or without the desired biological activity.

The expression "salts" means salts of carboxyl groups as well as salts of acid addition of amino groups of the protein molecules according to the invention. Salts of carboxyl groups can be produced in a known way and comprise inorganic salts, for example sodium, calcium, ammonium, iron and zinc salts, and salts with organic ba- ses, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like. Salts of acid addition, for example salts with inorganic acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and oxalic acid, are also covered by the invention.

"Functional derivatives" of polypeptides according to the invention can also be produced on functional amino acid side groups or at their N-terminal or C-terminal end using known techniques. Such derivatives comprise for example aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, produced by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups, produced by reaction with acyl groups. "Functional equivalents" naturally also comprise polypeptides that can be obtained from other organisms, as well as naturally occurring variants. For example, areas of homologous sequence regions can be established by sequence comparison, and equivalent enzymes can be determined on the basis of the concrete parameters of the invention.

"Functional equivalents" also comprise fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention, which for example display the desired biological function.

"Functional equivalents" are, moreover, fusion proteins, which have one of the polypeptide sequences stated above or functional equivalents derived there from and at least one further, functionally different, heterologous sequence in functional N- terminal or C-terminal association (i.e. without substantial mutual functional impairment of the fusion protein parts). Non-limiting examples of these heterologous sequences are e.g. signal peptides, histidine anchors or enzymes.

"Functional equivalents" that are also included according to the invention are homologues of the concretely disclosed proteins. These possess percent identity values as stated above. Said values refer to the identity with the concretely disclosed amino acid sequences, and may be calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448.

The % identity values may also be calculated from BLAST alignments, algorithm blastp (protein-protein BLAST) or by applying the Clustal setting as given below.

A percentage identity of a homologous polypeptide according to the invention means in particular the percentage identity of the amino acid residues relative to the total length of one of the amino acid sequences concretely described herein.

In the case of a possible protein glycosylation, "functional equivalents" according to the invention comprise proteins of the type designated above in deglycosylated or glycosylated form as well as modified forms that can be obtained by altering the glycosylation pattern.

Such functional equivalents or homologues of the proteins or polypeptides ac- cording to the invention can be produced by mutagenesis, e.g. by point mutation, lengthening or shortening of the protein.

Such functional equivalents or homologues of the proteins according to the invention can be identified by screening combinatorial databases of mutants, for example shortening mutants. For example, a variegated database of protein variants can be produced by combinatorial mutagenesis at the nucleic acid level, e.g. by enzymatic ligation of a mixture of synthetic oligonucleotides. There are a great many methods that can be used for the production of databases of potential homologues from a degenerated oligonucleotide sequence. Chemical synthesis of a degenerated gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene can then be ligated in a suitable expression vector. The use of a degenerated genome makes it possible to supply all sequences in a mixture, which code for the desired set of potential protein sequences. Methods of synthesis of degenerated oligonucleotides are known to a person skilled in the art (e.g. Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 1 1 :477).

In the prior art, several techniques are known for the screening of gene products of combinatorial databases, which were produced by point mutations or shortening, and for the screening of cDNA libraries for gene products with a selected property. These techniques can be adapted for the rapid screening of the gene banks that were produced by combinatorial mutagenesis of homologues according to the invention. The techniques most frequently used for the screening of large gene banks, which are based on a high-throughput analysis, comprise cloning of the gene bank in expression vectors that can be replicated, transformation of the suitable cells with the resultant vector database and expression of the combinatorial genes in conditions in which de- tection of the desired activity facilitates isolation of the vector that codes for the gene whose product was detected. Recursive Ensemble Mutagenesis (REM), a technique that increases the frequency of functional mutants in the databases, can be used in combination with the screening tests, in order to identify homologues (Arkin and Your- van (1992) PNAS 89:781 1 -7815; Delgrave et al. (1993) Protein Engineering 6(3):327- 331 ).

4. Coding nucleic acid sequences

The invention also relates to nucleic acid sequences that code for enzymes and mutants as defined herein.

The present invention also relates to nucleic acids with a certain degree of "identity" to the sequences specifically disclosed herein. "Identity" between two nucleic acids means identity of the nucleotides, in each case over the entire length of the nucleic acid.

For example the identity may be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 Apr; 5(2): 151 -1 ) with the following settings:

Multiple alignment parameter:

Gap opening penalty 10

Gap extension penalty 10

Gap separation penalty range 8

Gap separation penalty off

% identity for alignment delay 40

Residue specific gaps off

Hydrophilic residue gap off

Transition weighing 0

Pairwise alignment parameter:

FAST algorithm on

K-tuple size 1

Gap penalty 3

Window size 5

Number of best diagonals 5

Alternatively the identity may be determined according to Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13):3497-500, the web page: http://www.ebi.ac.Uk/Tools/clustalw/index.html# and the following settings

DNA Gap Open Penalty 15.0

DNA Gap Extension Penalty 6.66

DNA Matrix Identity

Protein Gap Open Penalty 10.0

Protein Gap Extension Penalty 0.2

Protein matrix Gonnet

Protein/DNA ENDGAP -1

Protein/DNA GAPDIST 4

All the nucleic acid sequences mentioned herein (single-stranded and double- stranded DNA and RNA sequences, for example cDNA and mRNA) can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. Chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.

The invention also relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, e.g. cDNA and mRNA), coding for one of the above polypeptides and their functional equivalents, which can be obtained for example using artificial nucleotide analogs.

The invention relates both to isolated nucleic acid molecules, which code for polypeptides or proteins according to the invention or biologically active segments thereof, and to nucleic acid fragments, which can be used for example as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention.

The nucleic acid molecules according to the invention can in addition con- tain non-translated sequences from the 3' and/or 5' end of the coding genetic region.

The invention further relates to the nucleic acid molecules that are complementary to the concretely described nucleotide sequences or a segment thereof.

The nucleotide sequences according to the invention make possible the production of probes and primers that can be used for the identification and/or cloning of ho- mologous sequences in other cellular types and organisms. Such probes or primers generally comprise a nucleotide sequence region which hybridizes under "stringent" conditions (see below) on at least about 12, preferably at least about 25, for example about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or of a corresponding antisense strand.

An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and can moreover be substantially free from other cellular material or culture medium, if it is being produced by recombinant techniques, or can be free from chemical precursors or other chemicals, if it is being synthesized chemically.

A nucleic acid molecule according to the invention can be isolated by means of standard techniques of molecular biology and the sequence information supplied according to the invention. For example, cDNA can be isolated from a suitable cDNA library, using one of the concretely disclosed complete sequences or a segment thereof as hybridization probe and standard hybridization techniques (as described for exam- pie in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, a nucleic acid molecule comprising one of the disclosed sequences or a segment thereof, can be isolated by the polymerase chain reaction, using the oligonucleotide primers that were constructed on the ba- sis of this sequence. The nucleic acid amplified in this way can be cloned in a suitable vector and can be characterized by DNA sequencing. The oligonucleotides according to the invention can also be produced by standard methods of synthesis, e.g. using an automatic DNA synthesizer.

Nucleic acid sequences according to the invention or derivatives thereof, homo- logues or parts of these sequences, can for example be isolated by usual hybridization techniques or the PCR technique from other bacteria, e.g. via genomic or cDNA libraries. These DNA sequences hybridize in standard conditions with the sequences according to the invention.

"Hybridize" means the ability of a polynucleotide or oligonucleotide to bind to an almost complementary sequence in standard conditions, whereas nonspecific binding does not occur between non-complementary partners in these conditions. For this, the sequences can be 90-100% complementary. The property of complementary sequences of being able to bind specifically to one another is utilized for example in Northern Blotting or Southern Blotting or in primer binding in PCR or RT-PCR.

Short oligonucleotides of the conserved regions are used advantageously for hybridization. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization. These standard conditions vary depending on the nucleic acid used (oligonucleotide, longer fragment or complete sequence) or depending on which type of nucleic acid - DNA or RNA - is used for hybridization. For example, the melting temperatures for DNA:DNA hybrids are approx. 10°C lower than those of DNA:RNA hybrids of the same length.

For example, depending on the particular nucleic acid, standard conditions mean temperatures between 42 and 58°C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC = 0.15 M NaCI, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide, for example 42°C in 5 x SSC, 50% formamide. Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1 x SSC and temperatures between about 20°C to 45°C, preferably between about 30°C to 45°C. For DNA:RNA hybrids the hybridization conditions are advantageously 0.1 x SSC and temperatures between about 30°C to 55°C, preferably between about 45°C to 55°C. These stated temperatures for hybridization are examples of calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant genetics textbooks, for example Sambrook et al., 1989, and can be calculated using formulae that are known by a person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrids or the G + C content. A person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, I RL Press at Oxford University Press, Oxford; Brown (ed), 1991 , Essential Molecular Biology: A Practical Approach, I RL Press at Oxford University Press, Oxford.

"Hybridization" can in particular be carried out under stringent conditions. Such hybridization conditions are for example described in Sambrook, J., Fritsch, E.F., Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31 -9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6.

"Stringent" hybridization conditions mean in particular: Incubation at 42°C overnight in a solution consisting of 50% formamide, 5 x SSC (750 mM NaCI, 75 mM tri- sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt Solution, 10% dextran sulfate and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing of the filters with O. l x SSC at 65°C.

The invention also relates to derivatives of the concretely disclosed or derivable nucleic acid sequences.

Thus, further nucleic acid sequences according to the invention can be derived from the sequences specifically disclosed herein and can differ from it by addition, substitution, insertion or deletion of individual or several nucleotides, and furthermore code for polypeptides with the desired profile of properties.

The invention also encompasses nucleic acid sequences that comprise so-called silent mutations or have been altered, in comparison with a concretely stated se- quence, according to the codon usage of a special original or host organism, as well as naturally occurring variants, e.g. splicing variants or allelic variants, thereof.

It also relates to sequences that can be obtained by conservative nucleotide substitutions (i.e. the amino acid in question is replaced by an amino acid of the same charge, size, polarity and/or solubility).

The invention also relates to the molecules derived from the concretely disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms can exist between individuals within a population owing to natural variation. These natural variations usually produce a variance of 1 to 5% in the nucleotide sequence of a gene.

Derivatives of nucleic acid sequences according to the invention mean for example allelic variants, having at least 60% homology at the level of the derived amino acid, preferably at least 80% homology, quite especially preferably at least 90% homology over the entire sequence range (regarding homology at the amino acid level, reference should be made to the details given above for the polypeptides). Advanta- geously, the homologies can be higher over partial regions of the sequences.

Furthermore, derivatives are also to be understood to be homologues of the nucleic acid sequences according to the invention, for example animal, plant, fungal or bacterial homologues, shortened sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence. For example, homologues have, at the DNA level, a homology of at least 40%, preferably of at least 60%, especially preferably of at least 70%, quite especially preferably of at least 80% over the entire DNA region given in a sequence specifically disclosed herein.

Moreover, derivatives are to be understood to be, for example, fusions with promoters. The promoters that are added to the stated nucleotide sequences can be modi- fied by at least one nucleotide exchange, at least one insertion, inversion and/or deletion, though without impairing the functionality or efficacy of the promoters. Moreover, the efficacy of the promoters can be increased by altering their sequence or can be exchanged completely with more effective promoters even of organisms of a different genus.

5. Constructs according to the invention

The invention also relates to expression constructs, containing, under the genetic control of regulatory nucleotide sequences, a nucleotide sequence coding for a polypeptide or fusion protein according to the invention; as well as vectors comprising at least one of these expression constructs. "Expression unit" means, according to the invention, a nucleic acid with expression activity, which comprises a promoter as defined herein and, after functional association with a nucleic acid that is to be expressed or a gene, regulates the expression, i.e. the transcription and the translation of this nucleic acid or of this gene. In this con- text, therefore, it is also called a "regulatory nucleic acid sequence". In addition to the promoter, other regulatory elements may be present, e.g. enhancers.

"Expression cassette" or "expression construct" means, according to the invention, an expression unit, which is functionally associated with the nucleic acid that is to be expressed or the gene that is to be expressed. In contrast to an expression unit, an expression cassette thus comprises not only nucleic acid sequences, which regulate transcription and translation, but also the nucleic acid sequences, which should be expressed as protein as a result of the transcription and translation.

The terms "expression" or "overexpression" describe, in the context of the invention, the production or increase of intracellular activity of one or more enzymes in a microorganism, which are encoded by the corresponding DNA. For this, it is possible for example to insert a gene in an organism, replace an existing gene by another gene, increase the number of copies of the gene or genes, use a strong promoter or use a gene that codes for a corresponding enzyme with a high activity, and optionally these measures can be combined.

Preferably such constructs according to the invention comprise a promoter 5'- upstream from the respective coding sequence, and a terminator sequence 3'- downstream, and optionally further usual regulatory elements, in each case functionally associated with the coding sequence.

A "promoter", a "nucleic acid with promoter activity" or a "promoter sequence" mean, according to the invention, a nucleic acid that, functionally associated with a nucleic acid that is to be transcribed, regulates the transcription of this nucleic acid.

"Functional" or "operative" association means, in this context, for example the sequential arrangement of one of the nucleic acids with promoter activity and of a nucleic acid sequence that is to be transcribed and optionally further regulatory elements, for example nucleic acid sequences that enable the transcription of nucleic acids, and for example a terminator, in such a way that each of the regulatory elements can fulfill its function in the transcription of the nucleic acid sequence. This does not necessarily require a direct association in the chemical sense. Genetic control sequences, such as enhancer sequences, can also exert their function on the target sequence from more remote positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence that is to be transcribed is positioned behind (i.e. at the 3' end) the promoter sequence, so that the two sequences are bound covalently to one another. The distance between the promoter sequence and the nucleic acid sequence that is to be expressed transgenically can be less than 200 bp (base pairs), or less than 100 bp or less than 50 bp.

Apart from promoters and terminators, examples of other regulatory elements that may be mentioned are targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, replication origins and the like. Suitable regulatory sequences are described for example in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

Nucleic acid constructs according to the invention comprise in particular sequences selected from those, specifically mentioned herein or derivatives and homo- logues thereof, as well as the nucleic acid sequences that can be derived from amino acid sequences specifically mentioned herein which are advantageously associated operatively or functionally with one or more regulating signal for controlling, e.g. increasing, gene expression.

In addition to these regulatory sequences, the natural regulation of these sequences can still be present in front of the actual structural genes and optionally can have been altered genetically, so that natural regulation is switched off and the expres- sion of the genes has been increased. The nucleic acid construct can also be of a simpler design, i.e. without any additional regulatory signals being inserted in front of the coding sequence and without removing the natural promoter with its regulation. Instead, the natural regulatory sequence is silenced so that regulation no longer takes place and gene expression is increased.

A preferred nucleic acid construct advantageously also contains one or more of the aforementioned enhancer sequences, functionally associated with the promoter, which permit increased expression of the nucleic acid sequence. Additional advantageous sequences, such as other regulatory elements or terminators, can also be inserted at the 3' end of the DNA sequences. One or more copies of the nucleic acids according to the invention can be contained in the construct. The construct can also contain other markers, such as antibiotic resistances or auxotrophy-complementing genes, optionally for selection on the construct.

Examples of suitable regulatory sequences are contained in promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ιρρ-lac-, lacl ^q" ' T7-, T5-, T3-, gal-, trc-, ara-, rhaP (rhaP _B AD)SP6-, lambda-P _R - or in the lambda-P _L promoter, which find application advan- tageously in Gram-negative bacteria. Other advantageous regulatory sequences are contained for example in the Gram-positive promoters ace, amy and SP02, in the yeast or fungal promoters ADC1 , MFalpha, AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH. Artificial promoters can also be used for regulation.

For expression, the nucleic acid construct is inserted in a host organism advantageously in a vector, for example a plasmid or a phage, which permits optimum expression of the genes in the host. In addition to plasmids and phages, vectors are also to be understood as meaning all other vectors known to a person skilled in the art, e.g. viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally. These vectors represent a further embodiment of the invention.

Suitable plasmids are, for example in E. coli, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1 , pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pi N-l 11 ^{1 3} -B 1 , Agt1 1 or pBdCI; in nocardioform actinomy- cetes pJAM2; in Streptomyces plJ101 , plJ364, plJ702 or plJ361 ; in bacillus pUB1 10, pC194 or pBD214; in Corynebacterium pSA77 or pAJ667; in fungi pALS1 , plL2 or pBB1 16; in yeasts 2alphaM, pAG-1 , YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHIac ⁺ , pBIN19, pAK2004 or pDH51. The aforementioned plasmids represent a small selection of the possible plasmids. Other plasmids are well known to a person skilled in the art and will be found for example in the book Cloning Vectors (Eds. Pouwels P.H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

In a further embodiment of the vector, the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can be inserted advantageously in the form of a linear DNA in the microorganisms and integrated into the genome of the host organism through heterologous or homologous recombination. This linear DNA can comprise a linearized vector such as plasmid or just the nucleic acid construct or the nucleic acid according to the invention.

For optimum expression of heterologous genes in organisms, it is advantageous to alter the nucleic acid sequences in accordance with the specific codon usage employed in the organism. The codon usage can easily be determined on the basis of computer evaluations of other, known genes of the organism in question.

The production of an expression cassette according to the invention is based on fusion of a suitable promoter with a suitable coding nucleotide sequence and a termina- tor signal or polyadenylation signal. Common recombination and cloning techniques are used for this, as described for example in T. Maniatis, E.F. Fritsch and J. Sam- brook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as well as in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

The recombinant nucleic acid construct or gene construct is inserted advantageously in a host-specific vector for expression in a suitable host organism, to permit optimum expression of the genes in the host. Vectors are well known to a person skilled in the art and will be found for example in "Cloning Vectors" (Pouwels P.H. et al., Publ. Elsevier, Amsterdam-New York-Oxford, 1985).

6. Hosts that can be used according to the invention

Depending on the context, the term "microorganism" means the starting micro- organism (wild-type) or a genetically modified microorganism according to the invention, or both.

The term "wild-type" means, according to the invention, the corresponding starting microorganism, and need not necessarily correspond to a naturally occurring organism.

By means of the vectors according to the invention, recombinant microorganisms can be produced, which have been transformed for example with at least one vector according to the invention and can be used for production of the polypeptides according to the invention. Advantageously, the recombinant constructs according to the invention, described above, are inserted in a suitable host system and expressed. Preferably, common cloning and transfection methods that are familiar to a person skilled in the art are used, for example co-precipitation, protoplast fusion, electro- poration, retroviral transfection and the like, in order to secure expression of the stated nucleic acids in the respective expression system. Suitable systems are described for example in Current Protocols in Molecular Biology, F. Ausubel et al., Publ. Wiley Inter- science, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In principle, all prokaryotic organisms can be considered as recombinant host organisms for the nucleic acid according to the invention or the nucleic acid construct. Bacteria are used advantageously as host organisms. Preferably they are selected from native or recombinant bacteria having the ability to produce inclusion bodies of the PHA-, TAG- or WE-type, as in particular the TAG-producing nocardioform actinomy- cetes, in particular of the genus Rhodococcus, Mycobacterium, Nocardia, Gordonia, Skermania and Tsukamurella; as well as TAG-producing Streptomycetes; WE- producing genera Acinetobacter and Alcanivorax; as well as recombinant strains of the genus Escherichia, especially E. coli, Corynebacterium, especially C. glutamicum und Bacillus, especially B. subtilis.

The host organism or host organisms according to the invention then preferably contain at least one of the nucleic acid sequences, nucleic acid constructs or vectors described in this invention, which code for an enzyme activity according to the above definition.

The organisms used in the method according to the invention are grown or bred in a manner familiar to a person skilled in the art, depending on the host organism. As a rule, microorganisms are grown in a liquid medium, which contains a source of car- bon, generally in the form of sugars, a source of nitrogen generally in the form of organic sources of nitrogen such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese and magnesium salts and optionally vitamins, at temperatures between 0°C and 100°C, preferably between 10°C to 60°C with oxygen aeration. The pH of the liquid nutrient medium can be maintained at a fixed value, i.e. regulated or not regulated during growing. Growing can be carried out batch wise, semi-batch wise or continuously. Nutrients can be supplied at the start of fermentation or can be supplied subsequently, either semi-continuously or continuously.

7. Recombinant production of enzymes of the invention

The invention also relates to methods for production of enzymes used in methods according to the invention by cultivating a microorganism which expresses said enzyme, and isolating the desired product from the culture.

The microorganisms as used according to the invention can be cultivated continuously or discontinuously in the batch process or in the fed batch or repeated fed batch process. A review of known methods of cultivation will be found in the textbook by Chmiel (Bioprocesstechnik 1 . Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991 )) or in the textbook by Storhas (Bioreaktoren und periphere Ein- richtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium that is to be used must satisfy the requirements of the par- ticular strains in an appropriate manner. Descriptions of culture media for various mi- croorganisms are given in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D. C, USA, 1981 ).

These media that can be used according to the invention generally comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and/or trace elements.

Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon. Other possible sources of carbon are oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid.

Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds. Examples of sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as corn-steep liquor, soybean flour, soybean pro- tein, yeast extract, meat extract and others. The sources of nitrogen can be used separately or as a mixture.

Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds, for example sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mer- captans and thiols, can be used as sources of sulfur.

Phosphoric acid, potassium dihydrogenphosphate or dipotassium hy- drogenphosphate or the corresponding sodium-containing salts can be used as sources of phosphorus.

Chelating agents can be added to the medium, in order to keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

The fermentation media used according to the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, corn-steep liquor and the like. In addition, suitable precursors can be added to the culture medium. The precise composition of the compounds in the medi- urn is strongly dependent on the particular experiment and must be decided individually for each specific case. Information on media optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Publ. P.M. Rhodes, P.F. Stan- bury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3). Growing media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.

All components of the medium are sterilized, either by heating (20 min at 1.5 bar and 121 °C) or by sterile filtration. The components can be sterilized either together, or if necessary separately. All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed.

The temperature of the culture is normally between 15°C and 45°C, preferably

25°C to 40°C and can be kept constant or can be varied during the experiment. The pH value of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, e.g. fatty acid polyglycol esters, can be used for controlling foaming. To maintain the stability of plas- mids, suitable substances with selective action, e.g. antibiotics, can be added to the medium. Oxygen or oxygen-containing gas mixtures, e.g. the ambient air, are fed into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20°C to 45°C. Culture is continued until a maximum of the desired product has formed. This is normally achieved within 10 hours to 160 hours.

The cells can be disrupted optionally by high-frequency ultrasound, by high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of sever- al of the methods listed.

8. Reaction conditions for biocatalytic methods of the invention

The at least one enzyme which is present during a method of the invention or an individual step of a multistep-method as defined herein above, can be present in living cells naturally or recombinantly producing the enzyme or enzymes, in harvested cells, in dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form. The at least one enzyme may be present in solution or as an enzyme immobilized on a carrier. One or several enzymes may simultaneously be present in soluble and immobilised form.

The methods according to the invention can be performed in common reactors, which are known to those skilled in the art, and in different ranges of scale, e.g. from a laboratory scale (few millilitres to dozens of liters of reaction volume) to an industrial scale (several liters to thousands of cubic meters of reaction volume). If the lipase is used in a form encapsulated by non-living, optionally permeabilized cells, in the form of a more or less purified cell extract or in purified form, a chemical reactor can be used. The chemical reactor usually allows controlling the amount of the at least one enzyme, the amount of the at least one substrate, the pH, the temperature and the circulation of the reaction medium. When the at least one enzyme is present in living cells, the process will be a fermentation. In this case the biocatalytic production will take place in a bioreactor (fermenter), where parameters necessary for suitable living conditions for the living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, and the like) can be controlled. Those skilled in the art are familiar with chemical reactors or bioreactors, e.g. with procedures for up-scaling chemical or biotechnological methods from laboratory scale to industrial scale, or for optimizing process parameters, which are also extensively described in the literature (for biotechnological methods see e.g. Crueger und Crueger, Biotechnologie - Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Munchen, Wien, 1984).

Cells containing the at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French presses, or chemical means, such as hypotonic media, lytic enzymes and detergents present in the medium, or combination of such methods. Examples for detergents are digitonin, n- dodecylmaltoside, octylglycoside, Triton® X-100, Tween ® 20, deoxycholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1 -propansulfonate), Nonidet ® P40 (Ethylphenolpoly(ethyleneglycolether), and the like.

If the at least one enzyme is immobilised, it is attached to an inert carrier. Suitable carrier materials are known in the art and are, e.g., disclosed in EP-A-1 149849, EP- A-1 069 183 and DE-OS 100193773 as well as the literature references cited therein (all of which are specifically enclosed with regard to carrier materials). Examples for suitable carrier materials are clays, clay minerals such as kaolinite, diatomeceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers, such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene. For preparing carrier-bound enzymes the carrier materials usually are used in the form of fine powders, wherein porous forms are preferred. The particle size of the carrier material usually does not exceed 5 mm, in particular 2 mm. In case the at least one enzyme is present in a whole-cell-preparation, said whole-cell-preparation may be present in a free or immobilised form. Suitable carrier materials are e.g. Ca- alginate or Carrageenan. Enzymes as well as cells may directly be linked by glutaral- dehyde. A wide range of immobilisation methods is known in the art (e.g. J. Lalonde and A. Margolin ..Immobilization of Enzymes" in K. Drauz und H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol.111, 991 -1032, Wiley-VCH, Weinheim).

The conversion reaction can be carried out batch wise, semi-batch wise or continuously. Reactants (and optionally nutrients) can be supplied at the start of reaction or can be supplied subsequently, either semi-continuously or continuously.

The reaction of the invention, depending on the particular reaction type ,may be performed in an aqueous or non-aqueous reaction medium. Ester cleavage reaction are preferably performed in the presence of water, in particular in the present of an aqueous-organic solvent system, preferably a 2-phase system. Esterification reactions are predereably performed in the absence of water, more particularly in the presence of an organic solvent which is free or substantially free of water.

An aqueous medium may contain a suitable buffer in order to adjust the pH to a value in the range of 5 to 9, like 6 to 8.

The non-aqueous medium may contain is substantially free of water, i.e. will con- tain less that about 1 wt.-% or 0.5 wt.-% of water.

In particular, biocatalytic methods are performed in an organic non-aqueous medium. As suitable organic solvents there may be mentioned aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic carbohydrates, like benzene, toluene, xy- lenes, chlorobenzene or dichlorobenzene, aliphatic acyclic and ethers, like diethylether, methyl-tert.-butylether, ethyl-tert.-butylether, dipropylether, diisopropylether, dibutyleth- er; or mixtures thereof. Preferably an organic solvent is applied which has the ability to form a biphasic solvent system with water

The concentration of the reactants/substrates may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the initial substrate concentration may be in the 0, 1 to 0,5 M, as for example 10 to 100 mM.

The reaction temperature may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the reaction may be performed at a temperature in a range of from 0 to 70 °C, as for example 20 to 50 or 25 to 40 °C. Examples for reaction temperatures are about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C and about 60°C.

The process may proceed until equilibrium between the substrate and then produces) is achieved, but may be stopped earlier. Usual process times are in the range from 1 minute to 25 hours, in particular 10 min to 6 hours, as for example in the range from 1 hour to 4 hours, in particular 1 .5 hours to 3.5 hours.

8.1 Selective enzymatic esterification of a 3 -unsaturated carboxylic acid, like- homofarnesylic acid

In a preferred embodiment, a mixture of (3E/Z) isomers of the 3-unsaturated carboxylic acid, like in particular of (3E,7E)- and (3Z,7E)-homofarnesylic acid, an aliphatic alcohol and optionally an organic solvent is treated with the lipase enzyme for performing an esterification reaction.

Non-limiting examples of suitable lipases are Candida antarctica lipase (CALB), and the immobilized analog thereof, like Novozym 435 ®.

Non-limiting examples of suitable alcohols are aliphatic CrC ₂ o-alcohols, as for example methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, sec.-butanol, n-pentanol, n-hexanol, n-heptanol and n-octanol.

Non-limiting examples of suitable solvents are in particular aliphatic hydrocar- bons, as for example hexane, cyclohexane, heptane, octane; aromatic hydrocarbons, as for example toluene, xylene; dialkyl ethers, as for example MTBE and diisopropyl ether.

In a preferred embodiment, one equivalent alcohol is used per one equivalent of (3E)- carboxylic acid, in particular (3E,7E)-isomer of homofarnesylic acid, in order to obtain an essentially complete conversion. Applying low amounts of alcohol limits the ester yield.

The reaction is suitably performed in a temperature range of about 0°C and + 80°C. The progress of the reaction may be controlled by means of GC or H PLC analytics.

A separable acid/ester mixture of the carboxylic acid isomers is obtained.

8.2 Selective enzymatic saponification of 3-unsaturated carboxylic acid ester, like homofarnesylic acid esters In another embodiment a separable mixture of isomers is obtained by applying an enzyme catalyzed ester cleavage of an 3E/Z-isomer mixture of the 3-unsturated carboxylic acid, in particular of a mixture of (3E,7E)- and (3Z,7E)-isomers of homofarnesylic acid alkyl esters. In a reaction the free 3E-acid, in particular (3E,7E)- homofarnesylic acid isomer and the non-reacted 3Z ester, in particular the non-reacted (3Z,7E)-homofarnesylic acid ester are obtained.

In a preferred embodiment an isomer mixture of the carboxylic acid alkyl esters, optionally dissolved in an organic solvent, is converted by applying a lipase enzyme in the presence of water.

Non-limiting examples of suitable lipases are Candida antarctica lipase (CALB), and the immobilized analog thereof, like Novozym 435 ®.

Non-limiting examples of suitable alkyl residues of the ester are: methyl, ethyl, n- propyl, i-propyl, n-butyl, i-butyl and sec-butyl

Non-limiting examples of suitable solvents are aliphatic hydrocarbons as, for ex- ample, hexane, cyclohexane, heptane; aromatic hydrocarbons, like toluene and xylene; ethers as, for example, MTBE and diisopropyl ether, THF.

8.3 Preparation of (-)-Ambrox The preparation may be performed starting out from the stereoisomerically pure

(3E/7E) homofarnesylic acid as obtained according to the present invention by applying known methods as depicted in the above Scheme 1.

The disclosure of EP16156410 and WO2010/139719 as stated in Scheme 1 as well as of WO2012/066059 describing the biocatalytic conversion of unsaturated sub- strates by applying cyclase enzymes of different origin is incorporated by reference.

Sclareolide, as for example obtained by the cyclase-catalyzed conversion of

(3E/7E)- homofarnesylic acid of the invention which then is chemically reduced (for example by means of LiAIH ₄ or NaBH ₄ ) to fo rm a mbrox-1 ,4-diol [Mookherjee et al.;

Perfumer and Flavourist (1990), 15: 27]. Ambrox-1 ,4-diol may then be chemically converted by means of different processes to (-)-ambrox. (see for example US

5,274, 134).

The biocatalytic synthesis of compound (-)-ambrox is also described in the literature [Neumann et al.; Biol Chem Hoppe Seyler (1986), 367: 723]. The molecule is obtained from homofarnesol ((3Z, 7E)-4,8,12-trimethyltrideca-3,7,1 1 -trien-1 -ol and the squalene-hopen cyclase (SHC) from Aiicyclobacilius acidocaldarius (formerly Bacillus acidocaldarius) was used as the catalyst.

9. Product isolation The methodology of the present invention can further include a step of recovering an end or intermediate product, optionally in stereoisomerically or enantiomerically substantially pure form. The term "recovering" includes extracting, harvesting, isolating or purifying the compound from culture or reaction media. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like.

Identity and purity of the isolated product may be determined by known techniques, like High Performance Liquid Chromatography (HPLC), gas chromatography (GC), Spektroskopy (like IR, UV, NMR), Colouring methods, TLC, NIRS, enzymatic or microbial assays. ( see for example: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 1 1 27-32; und Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S. 521 -540, S. 540-547, S. 559-566, 575- 581 und S. 581 -587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.)

The following examples only serve to illustrate the invention. The numerous pos- sible variations that are obvious to a person skilled in the art also fall within the scope of the invention.

Experimental Part:

A. Materials

(1 ) Enzymes

Lipase: Novozym 435; commercial product of Novozymes;

immobilized Candida antarctica lipase B Amino acid sequence:

1 MKLLSLTGVA GVLATCVAAT PLVKRLPSGS DPAFSQPKSV LDAGLTCQGA SPSSVSKPIL

61 LVPGTGTTGP QSFDSNWIPL STQLGYTPCW ISPPPFMLND TQVNTEYMVN AITALYAGSG

121 NNKLPVLTWS QGGLVAQWGL TFFPSIRSKV DRLMAFAPDY KGTVLAGPLD ALAVSAPSVW 181 QQTTGSALTT ALRNAGGLTQ IVPTTNLYSA TDEIVQPQVS NSPLDSSYLF NGKNVQAQAV

241 CGPLFVIDHA GSLTSQFSYV VGRSALRSTT GQARSADYGI TDCNPLPAND LTPEQKVAAA

301 ALLAPAAAAI VAGPKQNCEP DLMPYARPFA VGKRTCSGIV TP

Cyclase: Zm-SHC-1 see below

Unless otherwise specified, recombinant proteins are cloned and expressed by standard methods, such as, for example, as described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2 ^nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

(2) Chemicals

Isomer mixtures of homofarnesylic acid are for example obtained by a method as described in European Patent Application, application Number EP 17157950.1 filed on February 24, 2017.

Isomer mixtures of homofarnesylic acid esters have been prepared (e.g. in accordance with the generally known acid-catalyzed esterification of carboxylic acids with alcohols (so-called Fischer esterification - Chemische Berichte 28, 1895, 3252-3258) from isomeric mixtures of homofarnesylic acid.

All other chemical as applied were of laboratory grade.

B. Methods: 1. HPLC parameters for the analysis of homofarnesylic acid compounds

Apparatus : Agilent Series 1 100

Column Chiralpak AD-RH 5μηι 150 ^* 4,6mm von Daicel®

Eluent : -A: Water with 0,1 Vol% H ₃ P0 ₄

-B: Acetonitrile mit 0,1Vol% H ₃ P0 ₄

Detector UV-Detector λ=205 nm, BW=5 nm

Flow rate 1 ,2 ml/min

Injection 5 μί.

Temp. 40°C

Duration 35 min

Pressure approx. 70 bar

GC parameters for the analysis of sclareolide

The conversion of homofamesylie acid into sclareolide can be determined with the following GC system:

Column: 10 m Optima 1

Temperature profile:

0 min: 100°C

5°C/min to 200°C

After 5 min

30°C/min to 320°C thereafter constant

Duration of the method: 30 min

Injector temperature: 280°C Retention times (RT):

Homofarnesylic acid: peak 1 at 1 1 .7 min, peak 2 at 12.1 min ; Sclareolide: approx. 13.5 min

A calibration series, with the aid of which the concentration of unknown samples was determined, is established using authentic material (Sigma, catalog No.: 358002).

2. Lipase activity measurement

Tributyrin Assay according to Beisson, F. et al. Eur. J. Lipid Sci. Technol. 2000, 133-153. B. Examples:

Reference Example 1 : Cloning of the Zm-SHC-1 and expression in E. coli

The gene of the cyclase may be amplified from Zymomonas mobilis with the aid of the oligonucleotides Zm-SHC_fw and Zm-SHC_rev.

In each case 100 ng of primers Zm-SHC_fw and Zm-SHC_rev were mixed in an equimolar ratio. The PCR with genomic DNA from Z. mobilis (ATCC31821 ) was carried out following the manufacturer's instructions using Pwo- polymerase (Roche Applied Science) and the following temperature gradient program: 95°C for 3 min; 30 cycles at 95°C for 30 sec, 50°C for 30 sec and 72°C for 3 min; 72°C for 10 min.; 4°C until used. The PCR product (-2.2 kb) was isolated by agarose gel electrophoresis (1 .2% electrophoresis gel, Invitrogen) and column chromatography (GFX Kit, Amersham Pharmacia) and subsequently sequenced (sequencing primer: Zm-SHC_fw and Zm- SHC_rev). The sequence obtained matches the published sequence. The PCR product was digested with the restriction endonucleases Nde\ and BamYW and ligated into suitably digested vector pDHE19.2 [9]. Sequencing the resulting plasmids gave the nucleic acid sequence shown in SEQ ID NO: 1 . The corresponding amino acid sequence is shown in the following text /(SEQ I D NO:2):

Met Gly lie Asp Arg Met Asn Ser Leu Ser Arg Leu Leu Met Lys Lys

1 5 10 15

lie Phe Gly Ala Glu Lys Thr Ser Tyr Lys Pro Ala Ser Asp Thr lie

20 25 30

lie Gly Thr Asp Thr Leu Lys Arg Pro Asn Arg Arg Pro Glu Pro Thr

35 40 45

Ala Lys Val Asp Lys Thr lie Phe Lys Thr Met Gly Asn Ser Leu Asn

50 55 60

Asn Thr Leu Val Ser Ala Cys Asp Trp Leu lie Gly Gin Gin Lys Pro

65 70 75 80

Asp Gly His Trp Val Gly Ala Val Glu Ser Asn Ala Ser Met Glu Ala

85 90 95

Glu Trp Cys Leu Ala Leu Trp Phe Leu Gly Leu Glu Asp His Pro Leu

100 105 110

Arg Pro Arg Leu Gly Asn Ala Leu Leu Glu Met Gin Arg Glu Asp Gly

115 120 125

Ser Trp Gly Val Tyr Phe Gly Ala Gly Asn Gly Asp lie Asn Ala Thr

130 135 140

Val Glu Ala Tyr Ala Ala Leu Arg Ser Leu Gly Tyr Ser Ala Asp Asn

145 150 155 160

Pro Val Leu Lys Lys Ala Ala Ala Trp lie Ala Glu Lys Gly Gly Leu

165 170 175

Lys Asn lie Arg Val Phe Thr Arg Tyr Trp Leu Ala Leu He Gly Glu

180 185 190

Trp Pro Trp Glu Lys Thr Pro Asn Leu Pro Pro Glu He He Trp Phe

195 200 205

Pro Asp Asn Phe Val Phe Ser He Tyr Asn Phe Ala Gin Trp Ala Arg

210 215 220

Ala Thr Met Val Pro He Ala He Leu Ser Ala Arg Arg Pro Ser Arg

225 230 235 240

Pro Leu Arg Pro Gin Asp Arg Leu Asp Glu Leu Phe Pro Glu Gly Arg

245 250 255

Ala Arg Phe Asp Tyr Glu Leu Pro Lys Lys Glu Gly He Asp Leu Trp

260 265 270

Ser Gin Phe Phe Arg Thr Thr Asp Arg Gly Leu His Trp Val Gin Ser

275 280 285

Asn Leu Leu Lys Arg Asn Ser Leu Arg Glu Ala Ala He Arg His Val

290 295 300 Leu Glu Trp lie lie Arg His Gin Asp Ala Asp Gly Gly Trp Gly Gly 305 310 315 320 lie Gin Pro Pro Trp Val Tyr Gly Leu Met Ala Leu His Gly Glu Gly

325 330 335

Tyr Gin Leu Tyr His Pro Val Met Ala Lys Ala Leu Ser Ala Leu Asp

340 345 350

Asp Pro Gly Trp Arg His Asp Arg Gly Glu Ser Ser Trp lie Gin Ala

355 360 365

Thr Asn Ser Pro Val Trp Asp Thr Met Leu Ala Leu Met Ala Leu Lys 370 375 380

Asp Ala Lys Ala Glu Asp Arg Phe Thr Pro Glu Met Asp Lys Ala Ala 385 390 395 400

Asp Trp Leu Leu Ala Arg Gin Val Lys Val Lys Gly Asp Trp Ser He

405 410 415

Lys Leu Pro Asp Val Glu Pro Gly Gly Trp Ala Phe Glu Tyr Ala Asn

420 425 430

Asp Arg Tyr Pro Asp Thr Asp Asp Thr Ala Val Ala Leu He Ala Leu

435 440 445

Ser Ser Tyr Arg Asp Lys Glu Glu Trp Gin Lys Lys Gly Val Glu Asp 450 455 460

Ala He Thr Arg Gly Val Asn Trp Leu He Ala Met Gin Ser Glu Cys 465 470 475 480

Gly Gly Trp Gly Ala Phe Asp Lys Asp Asn Asn Arg Ser He Leu Ser

485 490 495

Lys He Pro Phe Cys Asp Phe Gly Glu Ser He Asp Pro Pro Ser Val

500 505 510

Asp Val Thr Ala His Val Leu Glu Ala Phe Gly Thr Leu Gly Leu Ser

515 520 525

Arg Asp Met Pro Val He Gin Lys Ala He Asp Tyr Val Arg Ser Glu 530 535 540

Gin Glu Ala Glu Gly Ala Trp Phe Gly Arg Trp Gly Val Asn Tyr He 545 550 555 560

Tyr Gly Thr Gly Ala Val Leu Pro Ala Leu Ala Ala He Gly Glu Asp

565 570 575

Met Thr Gin Pro Tyr He Thr Lys Ala Cys Asp Trp Leu Val Ala His

580 585 590

Gin Gin Glu Asp Gly Gly Trp Gly Glu Ser Cys Ser Ser Tyr Met Glu

595 600 605

He Asp Ser He Gly Lys Gly Pro Thr Thr Pro Ser Gin Thr Ala Trp 610 615 620

Ala Leu Met Gly Leu He Ala Ala Asn Arg Pro Glu Asp Tyr Glu Ala 625 630 635 640

He Ala Lys Gly Cys His Tyr Leu He Asp Arg Gin Glu Gin Asp Gly 645 650 655

Ser Trp Lys Glu Glu Glu Phe Thr Gly Thr Gly Phe Pro Gly Tyr Gly

660 665 670

Val Gly Gin Thr lie Lys Leu Asp Asp Pro Ala Leu Ser Lys Arg Leu

675 680 685

Leu Gin Gly Ala Glu Leu Ser Arg Ala Phe Met Leu Arg Tyr Asp Phe

690 695 700

Tyr Arg Gin Phe Phe Pro lie Met Ala Leu Ser Arg Ala Glu Arg Leu

705 710 715 720

lie Asp Leu Asn Asn

725

The plasmid pDHE-Zm-SHC-1 was transformed into the strain £ coli TG10 pAgro4 pHSG575 [Takeshita et al., Gene 1987, 61 :63-74; Tomoyasu et al., Mol Microbiol 2001 , 40:397-413]. The recombinant £ coli were named £ coli LU 15568.

Reference Example 2: Provision of recombinant homofarnesol cyclase from Z. mobilis

Inoculated from a suitable 2 ml preculture, £. coli LU 15568 was grown for 16 h at 37°C in 20 ml LB- Amp/Spec/Cm (100 g/l ampicillin; 100Mg/l spectinomycin; 20 g/l chloramphenicol), 0.1 mM I PTG, 0.5 g/l rhamnose in 100 ml Erlenmeyer flasks (with baffles), centrifuged at 5000*g/10 min and stored at 4°C. Protein extract was prepared by suspending the cell pellet in 15 ml disruption buffer (0.2 M Tris/HCI, 0.5 M EDTA, pH 8.0), 375 U benzonase (for example Novagen, 25 ΙΙ/μΙ_), 40 μΙ_ PMSF (100 mM, dissolved in /-PropOH), 0.8 g sucrose and approx. 0.5 mg of lysozyme. The reaction mixture was mixed and incubated on ice for 30 min. Thereafter, the mixture was frozen at -20°C.

After the reaction mixture had defrosted, it was made up to approx. 40 ml with distilled water and again incubated on ice for 30 min.

Thereafter, the cells were disrupted 3 times for 3 min using ultrasound (HTU-Soni

130, by G. Heinemann, Schwabisch-Hall, amplitude 80%, 15" pulse/15" pause). After the disruption, the cell debris was removed by centrifugation for 60 min at 4°C and 26 900 *g. The supernatant was discarded and the pellet was resuspended in 100 ml solubilization buffer (50 mM Tris/HCI, 10 mM MgCI2x6H20, 1 % Triton X-100, pH 8.0) and comminuted in a Potter for approx. 5 min. Thereafter, the suspension was maintained on ice for 30 min.

The homogenized extract was recentrifuged for 1 h at 4°C and 26 900 *g, and the pellet was discarded. The extract was employed for the enzyme assays and may be stored over several weeks at -20°C without suffering activity losses. The protein content was in the range of 1 mg/ml. Reference Example 3: Activity determination of the recombinant cyclase from E. co// LU 15568

Homofarnesylic acid ((3£,7£)-4,8, 12-trimethyltrideca-3,7, 1 1 -trienoic acid) was incubated with the protein preparation described in Reference Example 2. Specifically, 0.0412 g of homofarnesylic acid were weighed (20 mM in the reaction mixture; purity 85.1 % composed of Z,Z 0.44%, £,Z 10.13%, £,£ 74.93%), 2.913 ml of water; 0.350 ml of sodium citrate buffer (1 M sodium citrate pH 5.4), 0.560 ml MgCI ₂ (0.5M solution) were pipetted in, and the mixture was warmed for 30 min at 37°C, with stirring. The reaction started with the addition of £ coli LU 15568 homogenate (protein content 35 mg/ml), warmed to 37°C. The reaction mixture was stirred on a magnetic stirrer in an oil bath for 24 h at pH 5.0 at 37°C at maximum stirring speed. The pH was adjusted during the reaction using 0.5M HCI. After incubation for 24 hours, 0.500 ml from the reaction mixture were extracted by vortexing for 30 seconds with 1000 ml of n- heptane/n-propanol 3:2. The organic supernatant after the phase separation was employed in the GC analysis (cf. figure 1 ).

Using the analyses described herein below in greater detail, a conversion rate of 74.5% in total of 82.7% from the £,£ isomer was determined.

Example 1 : Preparation of (3E,7E)-homofarnesylic acid butyl ester

71 g (283 mmol) of a mixture of (3E,7E)-homofarnesylic acid and (3Z,7E)- homofarnesylic acid in a molar ratio of 56 : 44 were dissolved in 360 ml of n-heptane. 21 g (283 mmol) of n-butanol and 470 mg Novozym 435 were added. The mixture was stirred for 48 h at 23°C. The enzyme was separated by filtration. At a temperature of 0°C, 1 15 ml of methanol and 5 ml of water were added. The pH of the mixture was ad- justed to pH = 12 by adding aqueous sodium hydroxide (25%) at a temperature of < 10°C and under agitation.

The stirrer was stopped and the lower phase was separated off. After removal of the solvent, 43.5 g (1 19 mmol) of the homofarnesylic acid butyl ester having a (3E,7E)- content of > 97% were obtained.

Example 2: Influence of different alcohols on selectivity of enzymatic esterifica- tion of homofarnesylic acid in the absence of a solvent:

2 g (8 mmol) of a 57:43 mixture of (3E,7E)- homofarnesylic acid and (3Z,7E)- homofarnesylic acid were dissolved in 15 ml of different alcohols and were stirred at 23°C in the presence of 20 mg Novozym 435. At particular time intervals the composition of the reaction mixture was analyzed by HPLC. The results are summarized in the subsequent Table 1 . Table 1 : HFS Mixture Alcohol ROH Solvent Novozyme Time 3Z,7E 3E,7E 3Z,7E 3E,7E

435 Acid Acid Ester Ester

[mmol] [g] Type mL Type mL mg [ ] Fl% Fl% Fl% Fl%

8 2 EtOH 15 none - 20 0 43 57 - -

5 43 49 < 1 9

8 42 44 < 1 13

24 42 27 < 1 30

32 42 21 1 36

8 2 iPrOH 15 none - 20 0 43 57 - -

5 43 52 < 1 5

8 43 49 < 1 9

24 43 35 < 1 22

32 42 29 < 1 29

8 2 BuOH 15 none - 20 0 43 57 - -

5 43 52 < 1 5

8 43 49 < 1 9

24 43 34 < 1 23

32 42 28 1 29

8 2 tBuOH 15 none - 20 0 43 57 - -

5 43 57 - -

8 43 57 - -

24 43 57 - -

32 43 57 - -

Example 3: The influence of a combination of different alcohols and the solvent heptane on the selectivity of enzymatic esterification of homofarnesylic acid

2 g (8 mmol) of a 57:43 mixture of (3E,7E)- homofarnesylic acid and (3Z,7E)- homofarnesylic acid were dissolved in 15 ml of heptane. 8 mmol of different alcohols and 20 mg Novozym 435 were added and the mixture was stirred at 23°C. At predetermined time intervals the composition of the reaction mixture was analyzed via HPLC. The results are shown in Table 2.

Table 2: HFS Mixture Alcohol ROH Solvent Novozyme Time 3Z,7E 3E,7E 3Z,7E 3E,7E

435 Acid Acid Ester Ester

[mmole] [g] Type mg Type mL mg [h] Fl% Fl% Fl% Fl%

8 2 MeOH 256 Heptane 15 20 0 43 57 - -

7,5 41 22 1 35

24 37 2 6 55

32 33 2 10 55

8 2 EtOH 370 Heptane 15 20 0 43 57 - -

7,5 42 23 1 35

24 40 4 3 53

32 38 3 5 54

8 2 iPrOH 480 Heptane 15 20 0 43 57 - -

7,5 43 44 < 1 13

24 43 27 < 1 30

32 43 20 < 1 38

8 2 BuOH 600 Heptane 15 20 0 43 57 - -

7,5 42 30 1 27

24 41 4 1 53

32 40 3 2 55

8 2 1- 1050 Heptane 15 20 0 43 57 - -

Octanol

7,5 43 22 1 35

24 41 2 3 52

32 41 1 5 54

Example 4: The influence of a combination of butanol and different solvents on the selectivity, of enzymatic esterification of homofarnesylic acid

2 g (8 mmol) of a 57:43 mixture of (3E,7E)-homofarnesylic acid and (3Z,7E)- homofarnesylic acid were dissolved in 15 ml of different solvents. 8 mmol butanol and 20 mg Novozym 435 were added and the mixture was stirred at 23°C. At predetermined time intervals the composition of the reaction mixture was analyzed via HPLC. The results are shown in Table 3.

Table 3: HFS Mixture Alcohol ROH Solvent Novozyme Time 3Z,7E 3E,7E 3Z,7E 3E,7E

435 Acid Acid Ester Ester

[mmole] [g] Type mg Type mL mg [h] Fl% Fl% Fl% Fl%

8 2 BuOH 600 Toluene 15 20 0 43 57 - -

7 42 36 < 1 22

24 42 9 1 48

31 41 5 2 52

8 2 BuOH 600 THF 15 20 0 43 57 - -

7 43 57 0 0

24 43 57 0 0

31 43 55 0 2

8 2 BuOH 600 MTBE 15 20 0 43 57 - -

7 42 42 < 1 16

24 42 16 1 42

31 41 12 1 45

8 2 BuOH 600 CH2CI2 15 20 0 43 57 - -

7 43 56 0 1

24 43 53 0 4

31 43 52 0 5

Example 5: Preparation of free (3E,7E)-homofarnesylic acid via enzymatic ester cleavage

2 g (7.56 mmol) of (3E,7E)- and (3Z,7E)-homofarnesylic acid methyl ester (ratio of (3E,7E):(3Z,7E) = 51 :49) were dissolved in 50 ml of toluene. 10 ml of water and 50 mg of Novozym 435 were added. The mixture was stirred at 23°C. After 6 hours the composition of the reaction mixture was analyzed to be as follows:

36% (3E,7E)-homofarnesylic acid methyl ester,

49% (3Z,7E)-homofarnesylic acid methyl ester,

15% (3E,7E)-homofarnesylic acid and

< 0.1 % (3Z,7E)-homofarnesylic acid .

The enzyme was removed by filtration and the reaction mixture is adjusted to pH > 9 by means of sodium carbonate. The aqueous lower phase was separated. The pH of the aqueous phase was adjusted to a value of < 4 by means of an acid (10 % hydro- chloric acid). Afterwards the phase is extracted with toluene. The obtained toluene phase contains more than 95% of pure (3E,7E)-homofarnesylic acid .

Sequences:

SEQ ID NO: 1 - 326 Nucleic acid/amino acid sequences of various SHC genes

SEQ ID NO: 327-328 PCR primer

SEQ ID NO: 329, 330 Nucleic acid/amino acid sequences of lipase CALB

It is explicitly referred to the references as cited herein.