U.S. patent application number 17/131930 was filed with the patent office on 2021-12-09 for enzymatic cyclization of homofarnesylic acid.
The applicant listed for this patent is BASF SE. Invention is credited to Michael BREUER, Ralf PELZER, Stefan RUDENAUER, Wolfgang SIEGEL.
Application Number | 20210381013 17/131930 |
Document ID | / |
Family ID | 1000005784838 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210381013 |
Kind Code |
A1 |
BREUER; Michael ; et
al. |
December 9, 2021 |
ENZYMATIC CYCLIZATION OF HOMOFARNESYLIC ACID
Abstract
The present invention relates to processes for the preparation
of sclareolide and related compounds by the biocatalytic
cyclization of polyunsaturated carboxylic acid compounds, in
particular of homofarnesylic acid and related compounds; and to a
process for the preparation of ambroxide via the biocatalytic
cyclization of homofarnesylic acid to sclareolide.
Inventors: |
BREUER; Michael; (Darmstadt,
DE) ; SIEGEL; Wolfgang; (Limburgerhof, DE) ;
RUDENAUER; Stefan; (Weinheim, DE) ; PELZER; Ralf;
(Furstenberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF SE |
Ludwigshafen am Rhein |
|
DE |
|
|
Family ID: |
1000005784838 |
Appl. No.: |
17/131930 |
Filed: |
December 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15999299 |
Aug 18, 2018 |
10954538 |
|
|
PCT/EP2017/053795 |
Feb 20, 2017 |
|
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17131930 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 17/04 20130101;
C12Y 504/99017 20130101; C12Y 402/01129 20130101 |
International
Class: |
C12P 17/04 20060101
C12P017/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 19, 2016 |
EP |
16156410.9 |
Claims
1.-15. (canceled)
16. A process for the biocatalytic preparation of a compound of the
general formula II, ##STR00008## wherein R.sup.1, R.sup.2, R.sup.3
and R.sup.4 independently of one another represent H or
C.sub.1-C.sub.4-alkyl, in stereoisomerically pure form or as a
mixture of stereoisomers, wherein the compound is brought into
contact with a protein, which protein is capable of cyclizing a
polyunsaturated carboxylic acid, in particular homofarnesylic
acid.
17. The process as claimed in claim 16, wherein the compound of the
formula I is brought into contact with a protein which has the
enzymatic activity of a squalene-hopene cyclase (SHC).
18. The process as claimed in claim 16, wherein the substrate
employed is a polyunsaturated carboxylic acid of the general
formula I, ##STR00009## wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 have the abovementioned meanings, in particular in
essentially stereoisomer-pure form.
19. The process as per claim 16, wherein homofarnesylic acid, of
the formula Ia, ##STR00010## is employed as the starting material,
in particular in essentially stereoisomerically pure form,
preferably in a proportion of (3E,7E)-homofarnesylic acid of at
least 70 mol %, based on the total amount of homofarnesylic acid
isomers present.
20. The process as claimed in claim 19, wherein sclareolide, of the
formula IIa, ##STR00011## is obtained in stereoisomerically pure
form or as a mixture of stereoisomers.
21. The process as claimed in claim 16, wherein the SHC is selected
from among a) proteins comprising a polypeptide with an amino acid
sequence as per SEQ ID NO: 2, b) by deletion, insertion,
substitution, addition, inversion or a combination of proteins
derived as per a), comprising a polypeptide with a sequence
identity of at least 45%, to the amino acid sequence as per SEQ ID
NO: 2; and c) proteins which are functionally equivalent to a) or
b) and which catalyze the cyclization of homofarnesylic acid to
sclareolide.
22. The process as claimed in claim 16, wherein the biocatalytic
conversion a) is carried out at a pH value of the reaction medium
in the range from approximately 4 to 5.8; and under at least one of
the following further conditions: b) at a substrate concentration
of at least 15 mM; c) at an enzyme concentration of at least 5
mg/ml; d) at a reaction temperature in the range from 32 to
40.degree. C.; e) in a sodium citrate buffer comprising 1 to 20 mM
MgCl.sub.2; and/or f) at a buffer concentration of approximately 10
to 100 mM.
23. The process as claimed in claim 16, wherein the enzymatic
cyclase activity, in particular the activity of the SHC, is present
in a form selected from among: a) a free, optionally partially or
fully purified natural or recombinantly produced cyclase, b)
cyclase as per a) in immobilized form; c) intact cells, comprising
at least one cyclase; d) cell lysates or cell homogenates of cells
as per c).
24. The process as claimed in claim 16, wherein the conversion is
carried out in one-phase aqueous systems or in two-phase
aqueous-organic or solid-liquid systems.
25. The process as claimed in claim 16, wherein the conversion is
carried out at a temperature in the range of 37.degree. C., and a
pH value in the range of from 5 to 5.2.
26. The process as claimed in claim 16, wherein the SHC is isolated
from a microorganism selected among Methylococcus capsalatus,
Rhodopseudomonas palustris, Bradyrhizobium japonicum, Frankia
spec., Streptomyces coelicolor and in particular Zymomonas
mobilis.
27. The process as claimed in claim 16, wherein the SHC is isolated
from an SHC-overexpressing microorganism which is selected among
bacteria of the genus Escherichia, Corynebacterium, Ralstonia,
Clostridium, Pseudomonas, Bacillus, Zymomonas, Rhodobacter,
Streptomyces, Burkholderia, Lactobacillus and Lactococcus.
28. The process as claimed in claim 16, wherein the SHC is isolated
from transgenic SHC-overexpressing bacteria of the species
Escherichia coli, Pseudomonas putida, Burkholderia glumae,
Streptomyces lividans, Streptomyces coelicolor and Zymomonas
mobilis.
29. A process for the preparation of
3a,6,6,9a-tetramethyldodecahydronaphto[2,1-b]furan (ambroxide),
wherein a) homofarnesylic acid is converted into sclareolide by a
process as per claim 16; b) the product of step a) is reduced
chemically to ambroxdiol, and c) ambroxidol from step b) is
cyclized chemically to ambroxide.
30. The process as claimed in claim 29, wherein ambroxide is
(-)-ambroxide
((3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphto[2,1-b]furan
[CAS 6790-58-5]).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
U.S. Ser. No. 15/999,299 which is a national stage application
(under 35 U.S.C. .sctn. 371) of PCT/EP2017/053795, filed Feb. 20,
2017, which claims benefit of European Application No. 16156410.9,
filed Feb. 19, 2016, all of which are incorporated herein by
reference in their entirety.
SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is 074012_0396_01_SL.txt.
The size of the text file is 1,578,598 bytes and the text file was
created on Mar. 5, 2021.
[0003] The present invention relates to processes for the
preparation of sclareolide and related compounds by the
biocatalytic cyclization of polyunsaturated carboxylic acid
compounds, in particular of homofarnesylic acid and related
compounds; and to a process for the preparation of ambroxide via
the biocatalytic cyclization of homofarnesylic acid to
sclareolide.
BACKGROUND OF THE INVENTION
[0004] Compounds with the dodecahydronaphtho[2,1-b]furan skeleton
are of great economic interest as aroma chemicals. In this context,
compound (-)-2 should be mentioned; that is,
(3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphtho-[2,1-b]-furan,
known as the laevorotatory stereoisomer of ambroxan.
[0005] Ambroxan has originally been obtained from sperm whale
ambergris and currently can be prepared mainly via two routes.
Sclareol (3), a constituent of clary sage (Salvia sclarea), is
frequently used as suitable starting material for semisynthetic
material because it already contains the optical information for
compound ((-)-2). Here, the oxidative degradation may be carried
out using chromic acid, permanganate, H.sub.2O.sub.2 or ozone
[Stoll et al.; Helv Chim Acta (1950), 33: 1251]. The resulting
sclareolide (4) is subsequently converted (for example using
LiAlH.sub.4 or NaBH.sub.4) to ambrox-1,4-diol (5) [Mookherjee et
al.; Perfumer and Flavourist (1990), 15: 27]. The preparation of
compound (4) from sclareol (3) can also be effected by
biotransformation with Hyphozyma roseoniger [EP 204009]
##STR00001##
[0006] Finally, ambrox-1,4-diol (5) can be cyclized in a series of
chemical processes to give compound ((-)-2). Research has been
carried out into the preparation of the racemate of ambroxan
(rac-2) via inter alia homofarnesylic acid [U.S. Pat. No. 513,270;
Lucius et al.; Chem Ber (1960), 93: 2663] and
4-(2,6,6-trimethylcyclohex-1-enyl)butan-2-one [Buchi et al.; Helv
Chim Acta (1989), 72: 996].
[0007] In 2002, the market volume of ambroxan was, on average, 20
tonnes per year. This requires a starting base of approximately 33
tonnes of sclareol per year. The production of one tonne of
ambroxan requires 207 tonnes of various individual substances,
which, in turn, bring about the generation of 206 tonnes of waste.
The accumulating substances have different but overall relatively
potent effects on health and environment [Deutsche Bundesstiftung
Umwelt]. Thus, this synthesis consumes a great deal of energy and
requires the use of polluting chemicals.
[0008] The biocatalytic synthesis of compound ((-)-2) has been
described in the literature [Neumann et al.; Biol Chem Hoppe Seyler
(1986), 367: 723]. Here, the molecule is obtained from homofarnesol
(compound (1a), (3Z,7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol).
The catalyst used was the enzyme squalene-hopene cyclase (SHC) from
Alicyclobacillus acidocaldarius (formerly Bacillus acidocaldarius).
Further enzymes for catalyzing the cyclization of homofarnesol to
ambroxan have been described in patent specifications (for example
WO 2012/066059) and in the literature [Neumann et al., loc.
cit.].
[0009] Seitz, M. et al describe in ChemBioChem 2013, 14, 436-439 an
enzymatic process for the preparation of sclareolide from
homofarnesylic acid using the squalene-hopene cyclase from
Zymomonas mobilis (Zm SHCI). However, the product yields obtained
therein are only approximately 7.7 to 22.9%.
[0010] It was an object of the present invention to provide an
improved process for the preparation of ambroxide precursors, in
particular of sclareolide and related compounds, which can be
carried out in a technically more simple and a more economic
fashion than traditional chemical processes (for example reduction
of the number of reaction steps required, and/or more convenient
starting materials). It was a further object to additionally reduce
the arising costs by using readily available starting materials and
by reducing the number of chemical reactions (or steps). In
particular, an improved biocatalytic process for the preparation of
sclareolide should be provided.
SUMMARY OF THE INVENTION
[0011] The above objects were achieved by providing a process for
the preparation of ambroxide precursors, preferably sclareolide, of
the general formula (4), characterized in that homofarnesylic acid
derivatives, in particular homofarnesylic acid of the general
formula (Error! Reference source not found.) is cyclized
biocatalytically, as explained in more detail in the following.
##STR00002##
DESCRIPTION OF THE FIGURES
[0012] FIG. 1A shows the gas chromatography (GC) spectrum of a
homofarnesylic acid cyclization product prepared in accordance with
the invention. FIG. 1B shows the GC of a commercially available
sclareolide preparation.
DETAILED DESCRIPTION OF THE INVENTION
A. General Definitions
[0013] "Homofarnesylic acid" (compound (1b)) is equivalent to
"(3E,7E)-4,8,12-trimethyltrideca-3,7,11-trienoic acid"
[0014] "Sclareolide" (compound (4)) is equivalent to
"(3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyl-1,4,5,5a,7,8,9,9b-octahydrobenzo-
[e]benzofuran-2-one".
[0015] Laevorotatory sclareolide (or compound (-)-4) has the
following formula:
##STR00003##
[0016] "Ambrox", "Ambroxan" and "Amroxide" are used synonymously
herein. They comprise all stereoisomeric forms such as, in
particular, (+)-Ambrox, 3a-epi-(-)Ambrox, 9b-epi-(-) Ambrox and in
particular (-) Ambrox.
[0017] For the purposes of the present invention, "cyclases" are
generally enzymes or enzyme mutants which display in particular the
activity of a homofarnesylic acid cyclase. Suitable enzymes with
the activity of a homofarnesylic acid cyclase are intermolecular
transferases from the subclass of the isomerases; that is to say
proteins with the EC number EC 5.4 (enzyme code as per Eur. J.
Biochem. 1999, 264, 610-650). They are, in particular,
representations of EC 5.4.99.17. Suitable enzymes with the activity
of a homofarnesylic acid cyclase are in particular those cyclases
which also bring about the cyclization of homofarnesylic acid to
sclareolide and/or of squalene to hopene (therefore also
occasionally the name "SHC" squalene-hopene cyclase) and which are
described in detail in the international application
PCT/EP2010/057696, which is expressly referred to here. Mutants
thereof are described for example in WO 2012/066059, which is
expressly referred to here.
[0018] Owing to the reversibility of enzymatic reactions, the
present invention relates to the enzymatic reactions described
herein, in both directions of reaction.
[0019] "Functional mutants" of a "cyclase" comprise the "functional
equivalents" of such enzymes defined herein below.
[0020] The term "biocatalytic process" relates to any process
carried out in the presence of catalytic activity of a "cyclase"
according to the invention or of an enzyme with "cyclase activity",
i.e. processes in the presence of crude, or purified, dissolved,
dispersed or immobilized enzyme, or in the presence of intact
microbial cells which have or express such an enzymatic activity.
Thus, biocatalytic processes comprise enzymatic and microbial
processes.
[0021] The term "stereoisomers" comprises conformational isomers
and in particular configurational isomers, such as enantiomers and
diastereoisomers.
[0022] Generally also encompassed are, in accordance with the
invention, all stereoisomeric forms of the compounds described
herein, such as constitutional isomers and in particular
stereoisomers and mixed stereoisomers, such as, for example,
optical isomers or geometric isomers such as E and Z isomers, and
combinations of these. If a plurality of asymmetric centers are
present in a molecule, then the invention comprises all
combinations of different conformations of these asymmetric
centers, such as, for example, enantiomer pairs.
[0023] The term "stereospecific" means that one of several possible
stereoisomers of a compound with at least one asymmetric center,
prepared in accordance with the invention, is, as the result of the
activity of an enzyme according to the invention, produced in high
"enantiomeric excess" or high "enantiomeric purity", such as, for
example, at least 90% ee, in particular at least 95% ee, or at
least 98% ee, or at least 99% ee. The ee % value is calculated
using the following formula:
ee %=[X.sub.A-X.sub.B]/[X.sub.A+X.sub.B]*100,
wherein X.sub.A and X.sub.B are the molar fraction of the
enantiomers A and B, respectively.
[0024] A "cyclase activity", which has been determined on a
"reference substrate under standard conditions", is, for example,
an enzymatic activity which describes the formation of a cyclic
product from a noncyclic substrate. Examples of standard conditions
are substrate concentrations of from 10 mM to 0.2 M, in particular
15 to 100 mM, such as, for example, approximately 20 to 25 mM; at a
pH 4 to 8, and at temperatures of, for example, from 15 to 30 or 20
to 25.degree. C. Here, the determination can be carried out using
recombinant cyclase-expressing cells, disrupted cyclase-expressing
cells, fractions thereof or enriched or purified cyclase enzyme. A
reference substrate is, in particular, a homofarnesylic acid of the
formula (la); standard conditions are in particular approximately
20 to 25 mM of homofarnesylic acid of the formula (Ia), at 20 to
25.degree. C. and pH 4-6, such as 4.5; as also described in more
detail in the examples.
[0025] The "yield" and/or the "conversion" 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, during which
homofarnesylic acid is converted into sclareolide by means of
cyclases according to the invention. In particular, the conversion
is carried out under precisely defined conditions of, for example,
25, 30, 40, 50 or 60.degree. C. In particular, the yield and/or the
conversion is determined by carrying out the reaction for
converting homofarnesylic acid into sclareolide by means of the
cyclases according to the invention at 30.degree. C. over 16
hours.
[0026] To determine the yield and/or the conversion, one will, in
particular, react a 10 mM homofarnesylic acid solution with a
cyclase solution, the enzyme being present as membrane protein
extract cyclase-expressing cells (isolated for example as described
by [Ochs D. et al.; J. Bacteriol, (1992), 174: 298]) in a protein
content concentration of 0.08 percent by weight.
[0027] A cyclase according to the invention may also be
characterized in that, when homofarnesylic acid is converted to
sclareolide under identical conditions, it shows the 2-, 3-, 4-,
5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-,
19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-, 31-,
32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, 40-, 41-, 42-, 43-, 44-,
45-, 46-, 47- , 48-, 49-, 50-, 51-, 52-, 53-, 54-, 55-, 56-, 57-,
58-, 59-, 60-, 61-, 62-, 63-, 64-, 65-, 66-, 67-, 68-, 69-, 70-,
71-, 72-, 73-, 74-, 75-, 76-, 77-, 78-, 79-, 80-, 81-, 82-, 83-,
84-, 85-, 86-, 87-, 88-, 89-, 90-, 91-, 92-, 93-, 94-, 95-, 96-,
97-, 98-, 99-, 100-, 200-, 500-, 1000-fold or higher yield and/or
conversion in comparison with the squalene-hopene cyclase (SHC)
from Alicyclobacillus acidocaldarius (formerly Bacillus
acidocaldarius). Here, the term "conditions" refers to reaction
conditions such as substrate concentration, enzyme concentration,
reaction period and/or temperature.
[0028] The term "carboxylic acid" comprises both the free acid and
its salt form, such as, for example, its alkali or alkaline-earth
metal salts. This applies analogously to all carboxylic acids
mentioned herein, such as, for example, homofarnesylic acid.
[0029] The term "approximately" denotes a potential change of
.+-.25% of a stated value, especially .+-.15%, .+-.10%, preferably
.+-.5%, .+-.2 or .+-.1%.
[0030] The term "essentially" spans a range of values of
approximately 80% up to and including 100%, such as 85-99.9%,
especially 90 to 99.9%, preferably 95 to 99.9% or 98 to 99.9%, in
particular 99 to 99.9%.
[0031] Unless otherwise indicated, the following general chemical
definitions apply herein:
[0032] "Alkyl" represents saturated, straight-chain or branched, in
particular straight-chain hydrocarbon radicals with 1 to 6, in
particular 1 to 4, carbon atoms, for example methyl, ethyl, propyl,
1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, and
1,1-dimethylethyl as examples of representatives of
C.sub.1-C.sub.4-alkyl; and pentyl, 1-methylbutyl, 2-methylbutyl,
3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl,
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 and 1-ethyl-2-methylpropyl and in particular
methyl, ethyl, n-propyl and n-butyl.
B. Specific Embodiments of the Invention
[0033] The present invention particularly relates to the following
specific embodiments: [0034] 1. A process for the biocatalytic
preparation of a compound of the general formula II,
[0034] ##STR00004## [0035] wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 independently of one another represent H or
C.sub.1-C.sub.4-alkyl, in particular methyl or ethyl, preferably
methyl, [0036] in stereoisomerically pure form or as a mixture of
stereoisomers, [0037] wherein the compound is brought into contact
with a protein, in particular a protein with cyclase activity,
which protein is capable of cyclizing a polyunsaturated carboxylic
acid, in particular homofarnesylic acid. [0038] 2. The process as
per embodiment 1, wherein the compound of the formula I is brought
into contact with a protein which has the enzymatic activity of a
squalene-hopene cyclase (SHC). [0039] 3. The process as per
embodiment 1 or 2, wherein the substrate employed is a
polyunsaturated carboxylic acid of the general formula I,
[0039] ##STR00005## [0040] wherein R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 have the abovementioned meanings, in particular in
essentially stereoisomer-pure form. [0041] 4. The process as per
any of the preceding embodiments, wherein homofarnesylic acid, of
the formula la,
[0041] ##STR00006## [0042] is employed as the starting material, in
particular in essentially stereoisomerically pure form, preferably
in a proportion of (3E,7E)-homofarnesylic acid of at least 70 mol
%, particularly preferably at least 75, 80 or 85 mol %, most
preferably 90, 95 or 99 or 99.9 mol %, based on the total amount of
homofarnesylic acid isomers present. [0043] 5. The process as per
embodiment 4, wherein sclareolide, of the formula Ila,
[0043] ##STR00007## [0044] is obtained in stereoisomerically pure
form or as a mixture of stereoisomers. [0045] 6. The process as per
any of the preceding embodiments, wherein the SHC is selected from
among [0046] a) proteins comprising a polypeptide with an amino
acid sequence as per SEQ ID NO: 2, [0047] b) by deletion,
insertion, substitution, addition, inversion or a combination of
proteins derived as per a), comprising a polypeptide with a
sequence identity of at least 45%, 50%, 55%, 60%, 65%, 70%, in
particular at least 75% or 80%, preferably at least 85%, such as,
for example, at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, to
the amino acid sequence as per SEQ ID NO: 2; and [0048] c) proteins
which are functionally equivalent to a) or b) and which catalyze
the cyclization of homofarnesylic acid to sclareolide. [0049] 7.
The process as per embodiment 6, wherein the functionally
equivalent protein comprises a polypeptide with an amino acid
sequence which is selected from among [0050] a) SEQ ID NO: 3 to 326
and [0051] b) an amino acid sequence which is derived therefrom by
deletion, insertion, substitution, addition, inversion or a
combination and which has a degree of identity of at least 60%,
65%, 70%, particularly at least 75% or 80%, preferably at least
85%, such as, for example, at least 90, 91, 92, 93, 94, 95, 96,
97,98 or 99%. [0052] 8. The process as per any of the preceding
embodiments, wherein the enzymatic cyclase activity, in particular
the activity of the SHC, is present in a form selected from among:
[0053] a) a free, optionally partially or fully purified natural or
recombinantly produced cyclase, [0054] b) cyclase as per a) in
immobilized form; [0055] c) intact cells, comprising at least one
cyclase; [0056] d) cell lysates or cell homogenates of cells as per
c). [0057] 9. The process as per any of the preceding embodiments,
wherein the conversion is carried out in one-phase aqueous systems
or in two-phase aqueous-organic or solid-liquid systems. [0058] 10.
The process as per any of the preceding embodiments, wherein the
conversion is carried out at a temperature in the range from 0 to
60.degree. C., in particular from 10 to 50.degree. C., preferably
from 20 to 40.degree. C. and/or a pH value in the range of from 4
to 8, in particular from 5 to 7. [0059] 11. The process as per any
of the preceding embodiments, wherein the SHC is isolated from a
microorganism selected among Methylococcus capsalatus,
Rhodopseudomonas palustris, Bradyrhizobium japonicum, Frankia
spec., Streptomyces coelicolor and in particular Zymomonas mobilis.
[0060] 12. The process as per any of the preceding embodiments,
wherein the SHC is isolated from an SHC-overexpressing
microorganism which is selected among bacteria of the genus
Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas,
Bacillus, Zymomonas, Rhodobacter, Streptomyces, Burkholderia,
Lactobacillus and Lactococcus. [0061] 13. The process as per any of
the preceding embodiments, wherein the SHC is isolated from
transgenic SHC-overexpressing bacteria of the species Escherichia
coli, Pseudomonas putida, Burkholderia glumae, Streptomyces
lividans, Streptomyces coelicolor and Zymomonas mobilis. [0062] 14.
The process as per any of the preceding embodiments, wherein the
conversion is carried out in batch mode, fed-batch mode or
continuous mode. [0063] 15. The process as per any of the preceding
embodiments, wherein the biocatalytic conversion is carried out
under at least one of the following conditions: [0064] a) at a pH
value of the reaction medium in the range from approximately 4 to
5.9 or 5.8 or 4.5 to 5.8, in particular 4.5 to 5.5 or 5 to 5.5;
[0065] b) at a substrate concentration of at least 15 mM, such as,
for example, up to 100 mM, in particular up to 50 mM, in particular
15 to 30 mM, above all 15 to 25 mM; [0066] c) at an enzyme
concentration of at least 5 mg/ml; in particular 5 to 100,
preferably 10 to 50 or 15 to 40 or 15 to 30 mg/ml [0067] d) at a
reaction temperature in the range from 32 to 40.degree. C., in
particular 35 to 38.degree. C.; [0068] e) in a citrate buffer, in
particular sodium citrate buffer, in particular comprising 1 to 20
mM or 5 to 10 mM MgCl.sub.2 [0069] f) at a buffer concentration of
approximately 10 to 100, in particular 20 to 50 mM. [0070] The
processes according to the invention are carried out preferably
with simultaneous realization of the above conditions a) to b) or
a) to c) or a) to d) or a) to e) or a) to f). Here, any
combinations of parameter ranges, independently of the respective
degree of preference of individual ranges, are part of the present
disclosure. [0071] 16. The process for the preparation of
3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan (ambroxide),
wherein [0072] a) homofarnesylic acid is converted into sclareolide
by a process as per any of claims 1 to 13; [0073] b) the product of
step a) is reduced chemically in a manner known per se to
ambroxdiol, and [0074] c) ambroxidol from step b) is cyclized
chemically in a manner known per se to ambroxide. [0075] 17.
Process as per embodiment 16, wherein ambroxide is (-)-ambroxide
((3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan
[CAS 6790-58-5]).
C. Further Embodiments of the Invention
1. Particularly Suitable Cyclase Sequences
[0076] Cyclases which are useful in accordance with the invention
are SHCs whose SEQ ID NO for the corresponding wild-type sequence,
source organism and Genbank reference number are compiled in the
following table.
TABLE-US-00001 GI No. of S_ID SEQ the reference DB ID NO Organism
sequences s1 seq_ID 2 Zymomonas mobilis AAV90172.1 s20 seq_ID 3
Streptomyces coelicolor CAB39697.1 s911 seq_ID 4 Acetobacter
pasteurianus BAH99456.1 s2 seq_ID 5 Bradyrhizobium sp. ABQ33590.1
s940 seq_ID 6 Zymomonas mobilis EER62728.1 s949 seq_ID 7
Acidithiobacillus caldus EET25937.1 s167 seq_ID 8 Acidithiobacillus
ferrooxidans ACH84004.1 s41 seq_ID 9 Acidobacterium capsulatum
ACO34244.1 s36 seq_ID 10 Acidothermus cellulolyticus ABK53469.1 s83
seq_ID 11 Adiantum capillus-veneris BAF93209.1 s143 seq_ID 12
Ajellomyces capsulatus EDN09769.1 s995 seq_ID 13 Ajellomyces
capsulatus EER40510.1 s163 seq_ID 14 Ajellomyces capsulatus
EEH02950.1 s13 seq_ID 15 Alicyclobacillus acidocaldarius EED08231.1
s14 seq_ID 16 Alicyclobacillus acidocaldarius P33247.4 s1193 seq_ID
17 Alicyclobacillus acidocaldarius AAT70690.1 s21 seq_ID 18
Alicyclobacillus acidoterrestris CAA61950.1 s1189 seq_ID 19
Alicyclobacillus acidoterrestris AAT70691.1 s51 seq_ID 20 Anabaena
variabilis ABA24268.1 s76 seq_ID 21 Anaeromyxobacter sp. ABS28257.1
s159 seq_ID 22 Aspergillus clavatus EAW07713.1 s131 seq_ID 23
Aspergillus flavus EED48353.1 s176 seq_ID 24 Aspergillus fumigatus
EDP50814.1 s126 seq_ID 25 Aspergillus fumigatus EAL84865.1 s178
seq_ID 26 Aspergillus fumigatus EAL86291.2 s121 seq_ID 27
Aspergillus niger CAK43501.1 s115 seq_ID 28 Aspergillus niger
CAK45506.1 s124 seq_ID 29 Aspergillus oryzae BAE63941.1 s119 seq_ID
30 Azotobacter vinelandii EAM07611.1 s223 seq_ID 31 Bacillus
amyloliquefaciens ABS74269.1 s221 seq_ID 32 Bacillus anthracis
AAP27368.1 s976 seq_ID 33 Bacillus cereus EEK66523.1 s225 seq_ID 34
Bacillus cereus EAL12758.1 s972 seq_ID 35 Bacillus cereus
EEL44583.1 s977 seq_ID 36 Bacillus cereus EEK43841.1 s985 seq_ID 37
Bacillus cereus EEK82938.1 s988 seq_ID 38 Bacillus cereus
EEK99528.1 s981 seq_ID 39 Bacillus cereus EEK77935.1 s987 seq_ID 40
Bacillus cereus EEL81079.1 s960 seq_ID 41 Bacillus cereus
EEK88307.1 s979 seq_ID 42 Bacillus cereus EEL63943.1 s974 seq_ID 43
Bacillus cereus EEL59884.1 s956 seq_ID 44 Bacillus cereus
EEL69857.1 s951 seq_ID 45 Bacillus cereus EEL92663.1 s986 seq_ID 46
Bacillus cereus EEL49968.1 s227 seq_ID 47 Bacillus cereus
AAU16998.1 s224 seq_ID 48 Bacillus cereus AAS42477.1 s212 seq_ID 49
Bacillus cereus ACK95843.1 s289 seq_ID 50 Bacillus coahuilensis
205373680 s219 seq_ID 51 Bacillus cytotoxicus ABS22481.1 s230
seq_ID 52 Bacillus licheniformis AAU23777.1 s955 seq_ID 53 Bacillus
mycoides EEL98438.1 s990 seq_ID 54 Bacillus mycoides EEM04821.1
s989 seq_ID 55 Bacillus pseudomycoides EEM16144.1 s247 seq_ID 56
Bacillus pumilus ABV62529.1 s250 seq_ID 57 Bacillus pumilus
EDW21137.1 s249 seq_ID 58 Bacillus sp. EAR64404.1 s218 seq_ID 59
Bacillus sp. EDL66148.1 s241 seq_ID 60 Bacillus subtilis Q796C3.1
s284 seq_ID 61 Bacillus subtilis AAB84441.1 s215 seq_ID 62 Bacillus
thuringiensis ABK86448.1 s984 seq_ID 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 s63 seq_ID 71 Bacterium Ellin514
EEF57225.1 s72 seq_ID 72 Bacterium Ellin514 EEF59508.1 s87 seq_ID
73 Beijerinckia indica ACB96717.1 s69 seq_ID 74 Blastopirellula
marina EAQ81955.1 s543 seq_ID 75 Blastopirellula marina EAQ78122.1
s156 seq_ID 76 Bradyrhizobium japonicum CAA60250.1 s938 seq_ID 77
Acetobacter pasteurianus BAH98349.1 s3 seq_ID 78 Bradyrhizobium sp.
CAL79893.1 s201 seq_ID 79 Brevibacillus brevis BAH44778.1 s148
seq_ID 80 Burkholderia ambifaria EDT05097.1 s158 seq_ID 81
Burkholderia ambifaria EDT37649.1 s149 seq_ID 82 Burkholderia
ambifaria ACB68303.1 s100 seq_ID 83 Burkholderia ambifaria
EDT42454.1 s146 seq_ID 84 Burkholderia cenocepacia EAY66961.1 s139
seq_ID 85 Burkholderia cenocepacia ACA95661.1 s147 seq_ID 86
Burkholderia cenocepacia CAR57099.1 s95 seq_ID 87 Burkholderia
cenocepacia CAR56694.1 s102 seq_ID 88 Burkholderia dolosa
EAY71311.1 s941 seq_ID 89 Burkholderia glumae ACR32572.1 s945
seq_ID 90 Burkholderia glumae ACR30752.1 s132 seq_ID 91
Burkholderia graminis EDT12320.1 s104 seq_ID 92 Burkholderia mallei
ABM48844.1 s140 seq_ID 93 Burkholderia multivorans ABX19650.1 s116
seq_ID 94 Burkholderia multivorans ABX16859.1 s91 seq_ID 95
Burkholderia oklahomensis 167567074 s111 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_ID 100 Burkholderia
sp. ABB06563.1 s98 seq_ID 101 Burkholderia sp. ABB10136.1 s944
seq_ID 102 Burkholderia sp. CCGE1002 EFA54357.1 s89 seq_ID 103
Burkholderia thailandensis 167840988 s113 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 ABO56791.1 s150 seq_ID 108
Burkholderia xenovorans ABE35912.1 s54 seq_ID 109 Candidatus
Koribacter ABF40741.1 s171 seq_ID 110 Candidatus Kuenenia
CAJ71215.1 s79 seq_ID 111 Candidatus Solibacter ABJ82180.1 s99
seq_ID 112 Candidatus Solibacter ABJ82254.1 s917 seq_ID 113
Catenulispora acidiphila ACU75510.1 s65 seq_ID 114 Chthoniobacter
flavus EDY15838.1 s637 seq_ID 115 Chthoniobacter flavus EDY22035.1
s38 seq_ID 116 Crocosphaera watsonii EAM53094.1 s186 seq_ID 117
Cupriavidus taiwanensis CAQ72562.1 s32 seq_ID 118 Cyanothece sp.
ACB53858.1 s40 seq_ID 119 Cyanothece sp. ACK71719.1 s30 seq_ID 120
Cyanothece sp. EDY02410.1 s29 seq_ID 121 Cyanothece sp. ACK66841.1
s47 seq_ID 122 Cyanothece sp. EDX97382.1 s35 seq_ID 123 Cyanothece
sp. EAZ91809.1 s39 seq_ID 124 Cyanothece sp. ACL45896.1 s925 seq_ID
125 Cyanothece sp. PCC 8802 ACV02092.1 s64 seq_ID 126 Desulfovibrio
salexigens EEC62384.1 s74 seq_ID 127 Dryopteris crassirhizoma
BAG68223.1 s59 seq_ID 128 Frankia alni CAJ61140.1 s48 seq_ID 129
Frankia alni CAJ60090.1 s56 seq_ID 130 Frankia sp. ABD10207.1 s60
seq_ID 131 Frankia sp. ABW15063.1 s31 seq_ID 132 Frankia sp.
ABW14125.1 s948 seq_ID 133 Frankia sp. Eulic EFA59873.1 s919 seq_ID
134 Frankia sp. Eulic EFA59089.1 s628 seq_ID 135 Gemmata
obscuriglobus 168700710 s209 seq_ID 136 Geobacillus sp. EED61885.1
s206 seq_ID 137 Geobacillus sp. EDY05760.1 s964 seq_ID 138
Geobacillus sp. Y412MC52 EEN95021.1 s993 seq_ID 139 Geobacillus sp.
Y412MC61 ACX79399.1 s205 seq_ID 140 Geobacillus thermodenitrificans
ABO67242.1 s15 seq_ID 141 Geobacter bemidjiensis ACH40355.1 s8
seq_ID 142 Geobacter lovleyi ACD95949.1 s62 seq_ID 143 Geobacter
metallireducens ABB30662.1 s12 seq_ID 144 Geobacter metallireducens
ABB33038.1 s73 seq_ID 145 Geobacter sp. ACM21577.1 s10 seq_ID 146
Geobacter sp. EDV72707.1 s11 seq_ID 147 Geobacter sp. ACM22003.1
s913 seq_ID 148 Geobacter sp. M18 EET34621.1 s914 seq_ID 149
Geobacter sp. M21 ACT16952.1 s58 seq_ID 150 Geobacter
sulfurreducens AAR36453.1 s7 seq_ID 151 Geobacter sulfurreducens
AAR34018.1 s9 seq_ID 152 Geobacter uraniireducens ABQ25226.1 s46
seq_ID 153 Gloeobacter violaceus BAC91998.1 s67 seq_ID 154
Gluconacetobacter diazotrophicus ACI51585.1 s165 seq_ID 155
Gluconacetobacter diazotrophicus CAP55563.1 s68 seq_ID 156
Gluconobacter oxydans AAW61994.1 s80 seq_ID 157 Granulibacter
bethesdensis ABI63005.1 s937 seq_ID 158 Hyphomicrobium
denitrificans EET65847.1 s932 seq_ID 159 Leptospirillum
ferrodiazotrophum EES53667.1 s24 seq_ID 160 Leptospirillum rubarum
EAY57382.1 s25 seq_ID 161 Leptospirillum sp. EDZ38599.1 s174 seq_ID
162 Magnaporthe grisea EDK02551.1 s153 seq_ID 163 Magnetospirillum
magnetotacticum 46203107 s49 seq_ID 164 Methylacidiphilum
infernorum ACD82457.1 s169 seq_ID 165 Methylobacterium ACK83067.1
chloromethanicum s75 seq_ID 166 Methylobacterium ACK86232.1
chloromethanicum s946 seq_ID 167 Methylobacterium extorquens
CAX24364.1 s141 seq_ID 168 Methylobacterium nodulans ACL61886.1
s152 seq_ID 169 Methylobacterium populi ACB79998.1 s162 seq_ID 170
Methylobacterium radiotolerans ACB27373.1 s180 seq_ID 171
Methylobacterium sp. ACA20611.1 s175 seq_ID 172 Methylocella
silvestris ACK52150.1 s181 seq_ID 173 Methylococcus capsulatus
CAA71098.1 s55 seq_ID 174 Microcystis aeruginosa CAO86472.1 s101
seq_ID 175 Neosartorya fischeri EAW20752.1 s129 seq_ID 176
Nitrobacter hamburgensis ABE63461.1 s161 seq_ID 177 Nitrobacter sp.
EAQ34404.1 s160 seq_ID 178 Nitrobacter winogradskyi ABA05523.1 s157
seq_ID 179 Nitrococcus mobilis EAR22397.1 s164 seq_ID 180
Nitrosococcus oceani ABA57818.1 s170 seq_ID 181 Nitrosomonas
europaea CAD85079.1 s173 seq_ID 182 Nitrosomonas eutropha
ABI59752.1 s943 seq_ID 183 Nitrosomonas sp. AL212 EET32702.1 s142
seq_ID 184 Nitrosospira multiformis ABB75845.1 s52 seq_ID 185
Nostoc punctiforme ACC84529.1 s45 seq_ID 186 Nostoc sp. BAB72732.1
s122 seq_ID 187 Oligotropha carboxidovorans ACI93782.1 s233 seq_ID
188 Paenibacillus sp. EDS49994.1 s991 seq_ID 189 Paenibacillus sp.
JDR-2 ACS99948.1 s950 seq_ID 190 Paenibacillus sp. oral taxon 786
EES74793.1 s1280 seq_ID 191 Paramecium tetraurelia 145542269 s71
seq_ID 192 Pelobacter carbinolicus ABA87701.1 s5 seq_ID 193
Pelobacter carbinolicus ABA87615.1 s66 seq_ID 194 Pelobacter
propionicus ABK98395.1 s16 seq_ID 195 Pelobacter propionicus
ABK98811.1 s136 seq_ID 196 Penicillium chrysogenum CAP99707.1 s936
seq_ID 197 Planctomyces limnophilus EEO67214.1 s1158 seq_ID 198
Planctomyces limnophilus EEO68341.1 s526 seq_ID 199 Planctomyces
maris EDL58855.1 s992 seq_ID 200 Polypodiodes niponica BAI48071.1
s942 seq_ID 201 Polypodiodes niponica BAI48070.1 s1202 seq_ID 202
Populus trichocarpa EEF12098.1 s168 seq_ID 203 Ralstonia eutropha
AAZ64302.1 s190 seq_ID 204 Ralstonia eutropha CAJ96989.1 s81 seq_ID
205 Ralstonia metallidurans ABF11015.1 s110 seq_ID 206 Ralstonia
metallidurans ABF11268.1 s123 seq_ID 207 Rhizobium sp. P55348.1
s657 seq_ID 208 Rhodopirellula baltica CAD74517.1 s4 seq_ID 209
Rhodopseudomonas palustris ABJ08391.1 s130 seq_ID 210
Rhodopseudomonas palustris CAA71101.1 s155 seq_ID 211
Rhodopseudomonas palustris ABD06434.1 s97 seq_ID 212
Rhodopseudomonas palustris ABD87279.1 s135 seq_ID 213
Rhodopseudomonas palustris ACF02757.1 s84 seq_ID 214 Rhodospirillum
rubrum ABC20867.1 s1279 seq_ID 215 Rubrobacter xylanophilus
ABG05671.1 s915 seq_ID 216 Saccharomonospora viridis ACU97316.1 s42
seq_ID 217 Saccharopolyspora erythraea CAM03596.1 s82 seq_ID 218
Schizosaccharomyces japonicus EEB08219.1 s923 seq_ID 219
Sphaerobacter thermophilus ACZ39437.1 s924 seq_ID 220 Streptomyces
albus 239983547 s23 seq_ID 221 Streptomyces avermitilis BAC69361.1
s44 seq_ID 222 Acaryochloris marina ABW29816.1 s921 seq_ID 223
Streptomyces filamentosus 239945642 s934 seq_ID 224 Streptomyces
flavogriseus EEW70811.1 s920 seq_ID 225 Streptomyces ghanaensis
239927462 s922 seq_ID 226 Streptomyces griseoflavus 256812310 s28
seq_ID 227 Streptomyces griseus BAG17791.1 s926 seq_ID 228
Streptomyces hygroscopicus 256775136 s916 seq_ID 229 Streptomyces
lividans 256783789 s33 seq_ID 230 Streptomyces peucetius ACA52082.1
s27 seq_ID 231 Streptomyces pristinaespiralis EDY61772.1 s933
seq_ID 232 Streptomyces scabiei CBG68454.1 s37 seq_ID 233
Streptomyces sp. EDX25760.1 s34 seq_ID 234 Streptomyces sp.
EDY46371.1 s931 seq_ID 235 Streptomyces sp. AA4 256668250 s918
seq_ID 236 Streptomyces sp. C 256770952 s929 seq_ID 237
Streptomyces sp. Mg1 254385931 s928 seq_ID 238 Streptomyces sp.
SPB74 254379682 s930 seq_ID 239 Streptomyces sp. SPB78 256680470
s26 seq_ID 240 Streptomyces sviceus EDY55942.1 s927 seq_ID 241
Streptomyces viridochromogenes 256805984 s61 seq_ID 242
Synechococcus sp. EDX84551.1 s935 seq_ID 243 Synechococcus sp. PCC
7335 254422098 s53 seq_ID 244 Synechocystis sp. BAA17978.1
s22 seq_ID 245 Syntrophobacter fumaroxidans ABK18414.1 s6 seq_ID
246 Syntrophobacter fumaroxidans ABK17672.1 s912 seq_ID 247
Teredinibacter turnerae ACR13362.1 s57 seq_ID 248
Thermosynechococcus elongatus BAC09861.1 s43 seq_ID 249
Trichodesmium erythraeum ABG50159.1 s1178 seq_ID 250 Uncultured
organism ACA58560.1 s1176 seq_ID 251 Uncultured organism ABL07557.1
s1165 seq_ID 252 Uncultured organism ACA58559.1 s1166 seq_ID 253
Uncultured organism ACA58558.1 s1168 seq_ID 254 Uncultured organism
ABL07560.1 s1169 seq_ID 255 Uncultured organism ABL07565.1 s1170
seq_ID 256 Uncultured organism ABL07566.1 s1167 seq_ID 257
Uncultured organism ACA58545.1 s1171 seq_ID 258 Uncultured organism
ACA58535.1 s1180 seq_ID 259 Uncultured organism ACA58549.1 s1179
seq_ID 260 Uncultured organism ACA58554.1 s1181 seq_ID 261
Uncultured organism ACA58555.1 s1182 seq_ID 262 Uncultured organism
ACA58556.1 s1235 seq_ID 263 Uncultured organism ACA58530.1 s1188
seq_ID 264 Uncultured organism ACA58534.1 s1237 seq_ID 265
Uncultured organism ACA58552.1 s1223 seq_ID 266 Uncultured organism
ABL07558.1 s1200 seq_ID 267 Uncultured organism ABL07542.1 s1236
seq_ID 268 Uncultured organism ACA58539.1 s1238 seq_ID 269
Uncultured organism ACA58537.1 s1233 seq_ID 270 Uncultured organism
ACA58543.1 s1173 seq_ID 271 Uncultured organism ABL07553.1 s1241
seq_ID 272 Uncultured organism ABL07540.1 s1242 seq_ID 273
Uncultured organism ABL07544.1 s1225 seq_ID 274 Uncultured organism
ACA58557.1 s1183 seq_ID 275 Uncultured organism ACA58520.1 s1197
seq_ID 276 Uncultured organism ACA58524.1 s1185 seq_ID 277
Uncultured organism ACA58522.1 s1190 seq_ID 278 Uncultured organism
ACA58525.1 s1187 seq_ID 279 Uncultured organism ACA58523.1 s1184
seq_ID 280 Uncultured organism ACA58521.1 s1204 seq_ID 281
Uncultured organism ACA58547.1 s1221 seq_ID 282 Uncultured organism
ACA58544.1 s1198 seq_ID 283 Uncultured organism ACA58546.1 s1226
seq_ID 284 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 s1207 seq_ID 290 Uncultured organism
ABL07564.1 s1212 seq_ID 291 Uncultured organism ABL07563.1 s1208
seq_ID 292 Uncultured organism ABL07562.1 s1209 seq_ID 293
Uncultured organism ABL07559.1 s1214 seq_ID 294 Uncultured organism
ABL07556.1 s1216 seq_ID 295 Uncultured organism ACA58528.1 s1219
seq_ID 296 Uncultured organism ACA58536.1 s1192 seq_ID 297
Uncultured organism ABL07533.1 s1195 seq_ID 298 Uncultured organism
ABL07536.1 s1174 seq_ID 299 Uncultured organism ABL07545.1 s1186
seq_ID 300 Uncultured organism ABL07548.1 s1196 seq_ID 301
Uncultured organism ACA58561.1 s1172 seq_ID 302 Uncultured organism
ABL07555.1 s1194 seq_ID 303 Uncultured organism ABL07541.1 s1211
seq_ID 304 Uncultured organism ABL07554.1 s1220 seq_ID 305
Uncultured organism ABL07547.1 s1203 seq_ID 306 Uncultured organism
ABL07550.1 s1199 seq_ID 307 Uncultured organism ABL07551.1 s1228
seq_ID 308 Uncultured organism ACA58509.1 s1201 seq_ID 309
Uncultured organism ACA58514.1 s1205 seq_ID 310 Uncultured organism
ABL07543.1 s1206 seq_ID 311 Uncultured organism ABL07534.1 s1177
seq_ID 312 Uncultured organism ABL07546.1 s1210 seq_ID 313
Uncultured organism ABL07535.1 s1175 seq_ID 314 Uncultured organism
ABL07552.1 s1191 seq_ID 315 Uncultured organism ABL07549.1 s1222
seq_ID 316 Uncultured organism ACA58553.1 s1244 seq_ID 317
Uncultured organism ABL07539.1 s1213 seq_ID 318 Uncultured organism
ACA58532.1 s1239 seq_ID 319 Uncultured organism ACA58548.1 s1215
seq_ID 320 Uncultured organism ABL07561.1 s1240 seq_ID 321
Uncultured organism ACA58533.1 s1234 seq_ID 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 the cyclase which is
herein also referred to as Zm-SHC-1.
2. Further Proteins/Enzyme Mutants According to the Invention
[0077] The present invention is not limited to the proteins with
cyclase activity which are specifically disclosed herein, but,
rather, also extends to functional equivalents thereof.
[0078] "Functional equivalents" or analogs of the specifically
disclosed enzymes, in particular of SEQ ID NO: 2 to 6, are, within
the scope of the present invention, polypeptides which differ from
them and which still retain the desired biological activity, such
as, in particular, cyclase activity.
[0079] Thus, for example, "functional equivalents" are understood
as meaning enzymes and mutants which, in a used test for "cyclase
activity" within the meaning of the invention (i.e. with a
reference substrate under standard conditions) have an at least 1%,
in particular at least approximately 5 to 10%, such as, for
example, at least 10% or at least 20%, such as, for example, at
least 50% or 75% or 90%, higher or lower activity of an enzyme
comprising an amino acid sequence specifically defined herein (in
particular SEQ ID NO: 2 to 6).
[0080] The activity data for functional equivalents will, unless
otherwise specified, refer herein to activity determinations
carried out by means of a reference substrate under standard
conditions as defined herein.
[0081] The "cyclase activity" within the meaning of the invention
can be detected with the aid of various known tests. Without being
limited thereto, a test using a reference substrate such as, for
example, homofarnesylic acid, under standard conditions as
described hereinabove and explained in the experimental part, shall
be mentioned.
[0082] Furthermore, functional equivalents are stable for example
between pH 4 to 11 and advantageously have a pH optimum in a range
of from pH 5 to 10, such as, in particular 6.5 to 9.5 or 7 to 8 or
approximately at 7.5, and a temperature optimum in the range of
from 15.degree. C. to 80.degree. C. or 20.degree. C. to 70.degree.
C., such as, for example approximately 30 to 60.degree. C. or
approximately 35 to 45.degree. C., such as at 40.degree. C.
[0083] In accordance with the invention, "functional equivalents"
are in particular also understood as meaning "mutants" which are
derived from SEQ ID NO:2 to 326, in particular from SEQ ID NO: 2 to
6, and which display, in at least one sequence position of the
abovementioned amino acid sequences, an amino acid other than the
specifically mentioned amino acid but still have one of the
abovementioned biological activities.
[0084] "Functional equivalents" comprise the mutants obtainable by
one or more, such as, for example, 1 to 50, 2 to 30, 2 to 15, 4 to
12 or 5 to 10 mutations such as amino acid additions,
substitutions, deletions and/or inversions, where the
abovementioned modifications may occur in any sequence position as
long as they lead to a mutant with the property profile according
to the invention. Functional equivalence exists in particular also
when the reactivity patterns between mutant and unmodified
polypeptide agree in terms of quality, i.e. for example identical
substrates are converted at different rates.
[0085] Nonlimiting examples of suitable amino acid substitutions
are compiled in the following table:
TABLE-US-00002 Original residue Examples of the substitution Ala
Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro
His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu;
Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile;
Leu
[0086] "Functional equivalents" in the above sense are also
"precursors" of the polypeptides described, and also "functional
derivatives" and "salts" of the polypeptides.
[0087] "Precursors" here are natural or synthetic precursors of the
polypeptides with or without the desired biological activity.
[0088] The term "salts" is understood as meaning not only salts of
carboxyl groups, but also acid addition salts of amino groups of
the protein molecules according to the invention. Salts of carboxyl
groups may be prepared in a manner known per se and comprise
inorganic salts such as, for example, sodium, calcium, ammonium,
iron and zinc salts and salts with organic bases such as, for
example, amines such as triethanolamine, arginine, lysine,
piperidine and the like. Acid addition salts such as, for example,
salts with mineral acids such as hydrochloric acid or sulfuric acid
and salts with organic acids such as acetic acid and oxalic acid
are likewise subject matter of the invention.
[0089] Likewise, "functional derivatives" of polypeptides according
to the invention can be prepared on functional amino acid side
groups or at their N- or C-terminal ends with the aid of 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, prepared by
reaction with acyl groups; or O-acyl derivatives of free hydroxyl
groups, prepared by reaction with acyl groups.
[0090] Naturally, "functional equivalents" also comprise
polypeptides which can be obtained from other organisms, and
naturally occurring variants. By means of sequence comparison, for
example, areas of homologous sequence regions can be established,
and equivalent enzymes can be determined based on the specific
information of the invention.
[0091] "Functional equivalents" likewise comprise fragments,
preferably individual domains or sequence motifs, of the
polypeptides according to the invention, which have for example the
desired biological function.
[0092] "Functional equivalents" are furthermore fusion proteins
which have one of the aforementioned polypeptide sequences or
functional equivalents derived therefrom and at least one further,
functionally different, heterologous sequence in functional N- or
C-terminal linkage (i.e. without mutual substantial functional
impairment of the fusion protein moieties). Nonlimiting examples of
heterologous sequences of this kind are, for example, signal
peptides, histidine anchors or enzymes.
[0093] "Functional equivalents" which are also comprised in
accordance with the invention are homologs to the specifically
disclosed proteins. These have at least 60%, preferably at least
75%, in particular at least 85%, such as, for example, 90, 91, 92,
93, 94, 95, 96, 97, 98 or 99%, homology (or identity) to one of the
specifically disclosed amino acid sequences, calculated by the
algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA)
85(8), 1988, 2444-2448. A homology or identity, expressed as a
percentage, of a homologous polypeptide according to the invention
means in particular an identity, expressed as a percentage, of the
amino acid residues based on the total length of one of the amino
acid sequences described specifically herein.
[0094] The identity data, expressed as a percentage, may also be
determined with the aid of BLAST alignments, algorithm blastp
(protein-protein BLAST), or by applying the Clustal settings
specified herein below.
[0095] If a protein glycosylation is possible, "functional
equivalents" according to the invention comprise proteins of the
abovementioned type in deglycosylated or glycosylated form, and
modified forms which are available by altering the glycosylation
pattern.
[0096] Homologs of the proteins or polypeptides according to the
invention may be generated by mutagenesis, for example by point
mutation, extension or truncation of the protein.
[0097] Homologs of the proteins according to the invention may be
identified by screening combinatorial libraries of mutants such as,
for example, truncated mutants. For example, a variegated library
of protein variants can be generated by combinatorial mutagenesis
at the nucleic acid level, such as, for example, by enzymatically
ligating the mixture of synthetic oligonucleotides. A multiplicity
of methods exist which can be used for generating libraries of
potential homologs from a degenerate oligonucleotide sequence. The
chemical synthesis of a degenerate gene sequence may be carried out
in an automatic DNA synthesizer, and the synthetic gene can then be
ligated into a suitable expression vector. The use of a degenerate
set of genes makes it possible to provide, in a mixture, all those
sequences which code for the desired set of potential protein
sequences. Processes for synthesizing the degenerate
oligonucleotides are known to the skilled worker (for example
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. 11:477).
[0098] A plurality of techniques for screening gene products of
combinatorial libraries which have been generated by point
mutations or truncation or for screening cDNA libraries for gene
products with a selected property are known in the art. These
techniques may be adapted to the rapid screening of the gene
libraries which have been generated by combinatorial mutagenesis of
homologs according to the invention. The most frequently used
techniques for screening large gene libraries, as the basis for
high-throughput analysis, comprise cloning the gene library into
replicable expression vectors, transforming the suitable cells with
the resulting vector library and expressing the combinatorial genes
under conditions under which the detection of the desired activity
facilitates the isolation of the vector which codes for the gene
whose product has been detected. Recursive Ensemble Mutagenesis
(REM), a technique which increases the frequency of functional
mutants in the libraries, may be used in combination with the
screening tests for identifying homologs (Arkin and Yourvan (1992)
PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering
6(3):327-331).
3. Nucleic Acids and Constructs
3.1 Nucleic Acids
[0099] Nucleic acids which code for the enzyme with cyclase
activity as described above are also subject matter of the
invention.
[0100] The present invention also relates to nucleic acids with a
certain degree of identity to the specific sequences described
herein.
[0101] "Identity" between two nucleic acids is understood as
meaning the identity of the nucleotides over in each case the
entire length of the nucleic acid, in particular the identity which
is calculated by comparison with the aid of the Vector NTI Suite
7.1 Software from Informax (USA) using the Clustal method (Higgins
D G, Sharp P M. Fast and sensitive multiple sequence alignments on
a microcomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1),
setting the following parameters:
Multiple Alignment Parameters:
TABLE-US-00003 [0102] 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 weighting 0
Pairwise Alignment Parameter:
TABLE-US-00004 [0103] FAST algorithm on K-tuple size 1 Gap penalty
3 Window size 5 Number of best diagonals 5
[0104] Alternatively, the identity may also be determined according
to the method of 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, according to the
website: http://www.ebi.ac.uk/Tools/clustalw/index.html# and using
the following parameters:
TABLE-US-00005 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
[0105] All the nucleic acid sequences mentioned herein (single- and
double-stranded DNA and RNA sequences, such as, for example, cDNA
and mRNA) can be prepared in manner known per se by chemical
synthesis from the nucleotide building blocks such as, for example,
by fragment condensation of individual overlapping, complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides can be effected for example in a
manner known per se by the phosphoamidite method (Voet, Voet,
2.sup.nd ed., Wiley Press New York, pages 896-897). The adding-on
of synthetic oligonucleotides and filling-in of gaps with the aid
of the Klenow fragment of the DNA polymerase and ligation reactions
as well as general cloning methods are described in Sambrook et al.
(1989), Molecular Cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0106] Subject matter of the invention are also nucleic acid
sequences (single- and double-stranded DNA and RNA sequences, such
as, for example, cDNA and mRNA) which code for one of the above
polypeptides and their functional equivalents, which are obtainable
for example using artificial nucleotide analogs.
[0107] The invention relates both to isolated nucleic acid
molecules, which code for polypeptides or proteins according to the
invention or for biological active segments thereof, and nucleic
acid fragments which can be used for example for use as
hybridization probes or as primers for identifying or amplifying
coding nucleic acids according to the invention.
[0108] The nucleic acid molecules according to the invention may
additionally contain untranslated sequences at the 3'- and/or 5'
end of the coding gene region.
[0109] The invention furthermore comprises the nucleic acid
molecules which are complementary to the specifically described
nucleotide sequences, or a segment thereof.
[0110] The nucleotide sequences according to the invention make it
possible to generate probes and primers which may be used for
identifying and/or cloning homologous sequences in other types of
cells and organisms. Such probes or primers usually comprise a
nucleotide sequence region which, under "stringent" conditions (see
hereinbelow), hybridizes to at least approximately 12, preferably
at least approximately 25, such as, for example, approximately 40,
50 or 75, contiguous nucleotides of a sense strand of a nucleic
acid sequence according to the invention or of a corresponding
antisense strand.
[0111] An "isolated" nucleic acid molecule is separated from the
other nucleic acid molecules which are present in the natural
source of the nucleic acid and may in addition be essentially free
from other cellular material or culture media, if prepared by
recombinant techniques, or free from chemical precursors or other
chemicals if chemically synthesized.
[0112] A nucleic acid molecule according to the invention can be
isolated by means of standard techniques of molecular biology and
the sequence information provided according to the invention. For
example, cDNA can be isolated from a suitable cDNA library by using
one of the specifically disclosed complete sequences or a segment
thereof as hybridization probe and standard hybridization
techniques (as described, for example, in Sambrook, J., Fritsch, E.
F. and Maniatis, T. Molecular Cloning: A Laboratory Manual.
2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a
nucleic acid molecule comprising one of the disclosed sequences or
a segment thereof can be isolated by polymerase chain reaction,
with the oligonucleotide primers which have been generated on the
basis of this sequence being used. The nucleic acid amplified thus
can be cloned into a suitable vector and characterized by DNA
sequence analysis. The oligonucleotides according to the invention
can furthermore be prepared by standard synthesis methods, for
example using an automatic DNA synthesizer.
[0113] Nucleic acid sequences according to the invention or
derivatives thereof, homologs or parts of these sequences can be
isolated from other bacteria for example using customary
hybridization methods or the PCR technology, for example by genomic
libraries or cDNA libraries. These DNA sequences hybridize under
standard conditions to the sequences according to the
invention.
[0114] "Hybridize" is understood as meaning the ability of a poly-
or oligonucleotide to bind to an almost complementary sequence
under standard conditions, while nonspecific binding between
noncomplementary partners does not occur under these conditions. To
this end, the sequences may be 90-100% complementary. The property
of complementary sequences of being able to specifically bind to
one another is exploited for example in the Northern or Southern
blot technique or in primer binding in PCR or RT-PCR.
[0115] For the hydridization, short oligonucleotides of the
conservative regions are advantageously used in. However, longer
fragments of the nucleic acids according to the invention or the
complete sequences may also be used for the hybridization. These
standard conditions vary depending on the nucleic acid
(oligonucleotide, longer fragment or complete sequence), or
depending on which type of nucleic acid, DNA or RNA, is being used
for the hybridization. Thus, for example, the melting temperatures
for DNA: DNA hybrids are by approximately 10.degree. C. lower than
those of DNA: RNA-hybrids of the same length.
[0116] Depending on the nucleic acid, standard conditions are
understood as meaning, for example, temperatures of between 42 and
58.degree. C. in an aqueous buffer solution with a concentration of
between 0.1 to 5.times.SSC (1.times.SSC=0.15 M NaCl, 15 mM sodium
citrate, pH 7.2) or additionally in the presence of 50% formamide
such as, for example, 42.degree. C. in 5.times.SSC, 50% formamide.
Advantageously, the hybridization conditions for DNA: DNA hybrids
are 0.1.times.SSC and temperatures of between approximately
20.degree. C. to 45.degree. C., preferably between approximately
30.degree. C. to 45.degree. C. For DNA: RNA hybrids, the
hybridization conditions are advantageously 0.1.times.SSC and
temperatures of between approximately 30.degree. C. to 55.degree.
C., preferably between approximately 45.degree. C. to 55.degree. C.
These stated temperatures for the hybridization are examples of
calculated melting point values for a nucleic acid with a length of
approximately 100 nucleotides and a G+C content of 50% in the
absence of formamide. The experimental conditions for the DNA
hybridization are described in relevant textbooks of genetics, such
as, for example, Sambrook et al., "Molecular Cloning", Cold Spring
Harbor Laboratory, 1989, and can be calculated using formulae known
by a person skilled in the art, for example as a function of the
length of the nucleic acids, the type of the hybrids or the G+C
content. Further information on hybridization can be obtained by a
person skilled in the art 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, IRL Press at Oxford University
Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A
Practical Approach, IRL Press at Oxford University Press,
Oxford.
[0117] The "hybridization" may take place in particular under
stringent conditions. Such hybridization conditions are described,
for example, by Sambrook, J., Fritsch, E. F., Maniatis, T., in:
Molecular Cloning (A Laboratory Manual), 2.sup.nd ed., 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.
[0118] "Stringent" hybridization conditions are taken to mean in
particular: incubation at 42.degree. C. overnight in a solution
consisting of 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate and 20 g/ml denatured,
sheared salmon sperm DNA, followed by a step of washing the filters
with 0.1.times.SSC at 65.degree. C.
[0119] Subject matter of the invention are also derivatives of the
specifically disclosed or derivable nucleic acid sequences.
[0120] Thus, for example, further cyclase-mutant-encoding nucleic
acid sequences according to the invention may be derived for
example from SEQ ID NO:1 or from the coding sequences to SEQ ID NO:
2 to 326, in particular SEQ ID NO: 2 to 6, by an F486 mutation or
F486-analogous mutation and differ therefrom by addition,
substitution, insertion or deletion of individual or several
nucleotides, but continue to code for polypeptides with the desired
property profile.
[0121] Also comprised in accordance with the invention are those
nucleic acid sequences which comprise so-called silent mutations or
which are modified in comparison with a specifically mentioned
sequence in accordance with the codon usage of a specific source or
host organism, as are naturally occurring variants such as, for
example, splice variants or allelic variants, thereof.
[0122] Subject matter are likewise sequences obtainable by
conservative nucleotide substitutions (i.e. the amino acid in
question is replaced by an amino acid with the same charge, size,
polarity and/or solubility).
[0123] Subject matter of the invention are also the molecules which
are derived from the specifically disclosed nucleic acids by means
of sequence polymorphisms. These genetic polymorphisms may exist
between individuals within a population owing to natural variation.
These natural variations usually bring about a variance of from 1
to 5% in the nucleotide sequence of a gene.
[0124] Derivatives of the cyclases-encoding nucleic acid sequences
according to the invention derived from sequence SEQ ID NO: 1 or
from one of the coding sequences to SEQ ID NO: 2 to 326, in
particular SEQ ID NO: 2 to 6, are understood as meaning, for
example, allelic variants which have at least 60% homology at the
derived amino acid level, preferably at least 80% homology,
especially preferably at least 90% homology over the entire
sequence region (in respect of homology at the amino acid level,
reference may be made to what has been said above in connection
with the polypeptides). Advantageously, the homologies can be
higher over part-regions of the sequences.
[0125] Furthermore, derivatives are understood as meaning homologs
of the nucleic acid sequences according to the invention, for
example fungal or bacterial homologs, truncated sequences,
single-stranded DNA or RNA of the coding and noncoding DNA
sequences.
[0126] Furthermore, derivatives are understood as meaning for
example fusions with promoters. The promoters, which are arranged
upstream of the specified nucleotide sequences, may have been
changed by at least one nucleotide substitution, at least one
insertion, inversion and/or deletion, without, however, adversely
affecting the functionality/activity of the promoters. Moreover,
the efficacy of the promoters may be enhanced by modifying their
sequence, or the promoters may be exchanged fully for more
effective promoters, also from organisms from different
species.
3.2 Generation of Functional Mutants
[0127] Furthermore, a person skilled in the art is familiar with
processes for generating functional mutants of enzymes according to
the invention.
[0128] Depending on the technique used, a person skilled in the art
can introduce entirely random or else more targeted mutations into
genes or else noncoding nucleic acid sections (which are, for
example, important for regulating expression) and subsequently
construct the gene libraries. The methods of molecular biology
which are required for this purpose are known to a person skilled
in the art and described, for example, in Sambrook and Russell,
Molecular Cloning. 3.sup.rd ed., Cold Spring Harbor Laboratory
Press 2001.
[0129] Methods of modifying genes and thus of modifying the
proteins encoded by them have long been known to a person skilled
in the art, such as, for example, [0130] site-specific mutagenesis,
where individual or multiple nucleotides of a gene are replaced in
a targeted manner (Trower M K (Ed.) 1996; In vitro mutagenesis
protocols. Humana Press, New Jersey), [0131] saturation
mutagenesis, where a codon for any amino acid may be replaced or
added at any gene locus (Kegler-Ebo D M, Docktor C M, DiMaio D
(1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M,
Valcarel R, Stunnenberg H G (1994) Nucleic Acids Res 22:541; Barik
S (1995) Mol. Biotechnol. 3:1), [0132] error-prone polymerase chain
reaction (error-prone PCR), where nucleotide sequences are mutated
by erroneously working DNA polymerases (Eckert K A, Kunkel T A
(1990) Nucleic Acids Res. 18:3739); [0133] the SeSaM method
(sequence saturation method), where preferential substitutions are
prevented by the polymerase. Schenk et al., Biospektrum, vol. 3,
2006, 277-279 [0134] the passaging of genes in mutator strains, in
which, for example, an increased mutation rate of nucleotide
sequences takes place on account of defective DNA repair mechanisms
(Greener A, Callahan M, Jerpseth B (1996) An efficient random
mutagenesis technique using an E. coli mutator strain. In: Trower M
K (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey),
or [0135] DNA shuffling, where a pool of closely related genes is
formed and digested and the fragments are used as templates for a
polymerase chain reaction, in which mosaic genes of full length are
finally produced by repeated strand separation and reannealing
(Stemmer W P C (1994) Nature 370:389; Stemmer W P C (1994) Proc.
Natl. Acad. Sci. USA 91:10747).
[0136] Using "directed evolution" (described, inter alia, in Reetz
M T and Jaeger K-E (1999), Topics Curr. Chem. 200:31; Zhao H, Moore
J C, Volkov A A, Arnold F H (1999), Methods for optimizing
industrial enzymes by directed evolution, In: Demain A L, Davies J
E (Ed.) Manual of industrial microbiology and biotechnology.
American Society for Microbiology), a person skilled in the art can
also generate functional mutants in a selective manner and also on
a large scale. Here, in a first step, gene libraries of the
respective proteins are initially produced, it being possible to
employ, for example, the methods indicated hereinabove. The gene
libraries are expressed in a suitable manner, for example by
bacteria or by phage display systems.
[0137] The relevant genes of host organisms that express functional
mutants with properties which largely correspond to the desired
properties may be subjected to a further round of mutation. The
steps of mutation and selection or screening may be repeated
iteratively until the functional mutants present possess the
desired properties in an adequate measure. As a result of this
iterative procedure, a limited number of mutations, such as, for
example, 1, 2, 3, 4 or 5 mutations, may be performed stepwise, and
assessed and selected for their effect on the respective enzyme
property. Then, the selected mutant may be subjected to a further
mutation step in the same manner. The number of individual mutants
to be studied may be significantly decreased thereby.
[0138] The results according to the invention also provide
important information with respect to structure and sequence of the
respective enzymes, which are required for generating, in a
targeted fashion, further enzymes with desired modified properties.
In particular, it is possible to define so-called "hot spots", i.e.
sequence segments which are potentially suitable for modifying an
enzyme property via the introduction of targeted mutations.
[0139] Likewise, information is derivable in respect of amino acid
sequence positions in whose surroundings mutations may be carried
out which will presumably have little effect on the enzymatic
activity and which may be referred to as potential "silent
mutations".
3.3 Constructs
[0140] A subject matter of the invention are furthermore, in
particular recombinant, expression constructs comprising, under the
genetic control of regulatory nucleic acid sequences, a nucleic
acid sequence which codes for a polypeptide according to the
invention; and, in particular recombinant, vectors comprising at
least one of these expression constructs.
[0141] According to the invention, an "expression unit" is
understood as meaning a nucleic acid which has expression activity
and which comprises a promoter as herein defined and which, after
functional linkage to a nucleic acid to be expressed or to a gene,
will regulate the expression, in other words the transcription and
the translation, of this nucleic acid or this gene. This is why it
is also referred to in this context as a "regulatory nucleic acid
sequence". In addition to the promoter, further regulatory elements
such as, for example, enhancers may be present.
[0142] According to the invention, an "expression cassette" or
"expression construct" is understood as meaning an expression unit
which is functionally linked to the nucleic acid to be expressed or
the gene to be expressed. In contrast to an expression unit, an
expression cassette, therefore, does not only comprise nucleic acid
sequences which regulate transcription and translation, but also
those nucleic acid sequences which are to be expressed as a protein
as a result of transcription and translation.
[0143] Within the context of the invention, the terms "expression"
or "overexpression" describe the production or increase of the
intracellular activity of one or more enzymes in a microorganism
which are encoded by the corresponding DNA. To this end, it is
possible, for example, to introduce a gene into an organism, to
replace an existing gene by a different gene, to increase the copy
number of the gene(s), to use a strong promoter or to use a gene
which codes for a corresponding enzyme with a high activity, and
these measures can optionally be combined.
[0144] Preferably, such constructs according to the invention
comprise a promoter 5'-upstream and a terminator sequence
3'-downstream of the respective coding sequence and, optionally,
further customary regulatory elements, in each case operably linked
to the coding sequence.
[0145] According to the invention, a "promoter", a "nucleic acid
with promoter activity" or a "promoter sequence" is understood as
meaning a nucleic acid which, in functional linkage with a nucleic
acid to be transcribed, regulates the transcription of this nucleic
acid.
[0146] In this context, a "functional" or "operable" linkage is
understood as meaning, for example, the sequential arrangement of
one of the nucleic acids with promoter activity and a nucleic acid
sequence to be transcribed and optionally further regulatory
elements such as, for example, nucleic acid sequences which ensure
the transcription of nucleic acids and, for example, a terminator
in such a way that each of the regulatory elements can fulfill its
function upon transcription of the nucleic acid sequence. A direct
linkage in the chemical sense is not necessarily required for this
purpose. Genetic control sequences such as, for example, enhancer
sequences can also exert their function on the target sequence from
positions which are located at a greater distance, or indeed from
other DNA molecules. Preferred arrangements are those in which the
nucleic acid sequence to be transcribed is positioned behind (i.e.
at the 3' end) of the promoter sequence so that the two sequences
are covalently bonded to each other. In this context, the distance
between the promoter sequence and the nucleic acid sequence to be
expressed transgenically may be less than 200 base pairs or less
than 100 base pairs or less than 50 base pairs.
[0147] In addition to promoters and terminator, the following may
be mentioned as examples of other regulatory elements: 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, Calif. (1990).
[0148] Nucleic acid constructs according to the invention comprise
in particular a sequence coding for a cyclase, for example SEQ ID
NO: 1, or coding for a cyclase as per SEQ ID NO. 2 to 326 or
derivatives and homologs thereof, and the nucleic acid sequences
which can be derived therefrom and which have been linked
operatively or functionally with one or more regulatory signals,
advantageously for controlling, for example increasing, gene
expression.
[0149] In addition to these regulatory sequences, the natural
regulation of these sequences may still be present before the
actual structural genes and optionally may have been genetically
modified so that the natural regulation has been switched off and
expression of the genes has been enhanced. The nucleic acid
construct may, however, also be of simpler construction, i.e. no
additional regulatory signals have been inserted before the coding
sequence and the natural promoter, with its regulation, has not
been removed. Instead, the natural regulatory sequence is mutated
such that regulation no longer takes place and the gene expression
is increased.
[0150] A preferred nucleic acid construct advantageously also
comprises one or more of the already mentioned "enhancer" sequences
in functional linkage with the promoter, which sequences make
possible an enhanced expression of the nucleic acid sequence.
Additional advantageous sequences may also be inserted at the
3'-end of the DNA sequences, such as further regulatory elements or
terminators. One or more copies of the nucleic acids according to
the invention may be present in a construct. In the construct,
other markers, such as genes which complement auxotrophisms or
antibiotic resistances, may also optionally be present so as to
select for the construct.
[0151] Examples of suitable regulatory sequences are present in
promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac,
lacl.sup.q, T7, T5, T3, gal, trc, ara, rhaP (rhaP.sub.BAD)SP6,
lambda-P.sub.R or in the lambda-P.sub.L promoter, and these are
advantageously employed in Gram-negative bacteria. Further
advantageous regulatory sequences are present for example in the
Gram-positive promoters amy and SPO2, in the yeast or fungal
promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
Artificial promoters may also be used for regulation.
[0152] For expression in a host organism, the nucleic acid
construct is inserted advantageously into a vector such as, for
example, a plasmid or a phage, which makes possible optimal
expression of the genes in the host. Vectors are also understood as
meaning, in addition to plasmids and phages, all the other vectors
which are known to the skilled worker, that is to say for example
viruses such as SV40, CMV, baculovirus and adenovirus, transposons,
IS elements, phasmids, cosmids and linear or circular DNA. These
vectors are capable of being replicated autonomously in the host
organism or else chromosomally. These vectors are a further
development of the invention.
[0153] 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,
pIN-III.sup.113-B1, .lamda.gt11 or pBdCI, in Streptomyces pIJ101,
pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in
Corynebacterium pSA77 or pAJ667, in fungi pALS1, plL2 or pBB116, in
yeasts 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants
pLGV23, pGHlac.sup.+, pBIN19, pAK2004 or pDH51. The abovementioned
plasmids are a small selection of the plasmids which are possible.
Further plasmids are well known to the skilled worker and can be
found for example in the book Cloning Vectors (Eds. Pouwels P. H.
et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 90401
8).
[0154] In a further development of the vector, the vector which
comprises the nucleic acid construct according to the invention or
the nucleic acid according to the invention can advantageously also
be introduced into the microorganisms in the form of a linear DNA
and integrated into the host organism's genome via heterologous or
homologous recombination. This linear DNA can consist of a
linearized vector such as a plasmid or only of the nucleic acid
construct or the nucleic acid according to the invention.
[0155] For optimal expression of heterologous genes in organisms,
it is advantageous to modify the nucleic acid sequences to match
the specific "codon usage" used in the organism. The "codon usage"
can be determined readily by computer evaluations of other, known
genes of the organism in question.
[0156] An expression cassette according to the invention is
generated by fusing a suitable promoter to a suitable coding
nucleotide sequence and a terminator or polyadenylation signal.
Customary recombination and cloning techniques are used for this
purpose, as are described, for example, in T. Maniatis, E. F.
Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and
in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with
Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Greene Publishing Assoc. and Wiley Interscience
(1987).
[0157] For expression in a suitable host organism, the recombinant
nucleic acid construct or gene construct is advantageously inserted
into a host-specific vector which makes possible optimal expression
of the genes in the host. Vectors are well known to the skilled
worker and can be found for example in "Cloning Vectors" (Pouwels
P. H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
4. Microorganisms
[0158] Depending on the context, the term "microorganism" may refer
to the wild-type microorganism or to a genetically modified,
recombinant microorganism, or to both.
[0159] With the aid of the vectors according to the invention it is
possible to generate recombinant microorganisms which are
transformed for example with at least one vector according to the
invention and which can be employed for the production of the
polypeptides according to the invention. Advantageously, the
above-described recombinant constructs according to the invention
are introduced into a suitable host system and expressed therein.
In this context, customary cloning and transfection methods which
are known to the skilled worker, such as, for example,
coprecipitation, protoplast fusion, electroporation, retroviral
transfection and the like, are preferably used so as to allow
expression of the abovementioned nucleic acids in the expression
system in question. Suitable systems are described for example in
Current Protocols in Molecular Biology, F. Ausubel et al., Ed.,
Wiley Interscience, New York 1997, or Sambrook et al. Molecular
Cloning: A Laboratory Manual. 2.sup.nd Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0160] Suitable recombinant host organisms for the nucleic acid
according to the invention or the nucleic acid construct are, in
principle, all prokaryotic or eukaryotic organisms. Microorganisms
such as bacteria, fungi or yeasts are advantageously used as host
organisms. Gram-positive or Gram-negative bacteria, preferably
bacteria from the families Enterobacteriaceae, Pseudomonadaceae,
Rhizobiaceae, Streptomycetaceae or Nocardiaceae, especially
preferably bacteria from the genera Escherichia, Pseudomonas,
Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium,
Clostridium or Rhodococcus, are advantageously used. Very
especially preferred is the genus and species Escherichia coli.
Further advantageous bacteria can additionally be found in the
group of the alpha-proteobacteria, beta-proteobacteria or
gamma-proteobacteria.
[0161] In this context, the host organism(s) according to the
invention contain(s) preferably at least one of the nucleic acid
sequences, nucleic acid constructs or vectors which are described
in the present invention and which code for an enzyme with
phenylethanol dehydrogenase activity as defined hereinabove.
[0162] Depending on the host organism, the organisms used in the
process according to the invention are grown or cultured in a
manner with which the skilled worker is familiar. As a rule,
microorganisms are grown in a liquid medium which comprises a
carbon source, usually in the form of sugars, a nitrogen source,
usually in the form of organic nitrogen sources such as yeast
extract or salts such as ammonium sulfate, trace elements such as
iron salts, manganese salts, magnesium salts and optionally
vitamins, at temperatures of between 0.degree. C. and 100.degree.
C., preferably between 10.degree. C. to 60.degree. C., while
passing in oxygen gas. The pH of the liquid medium may be
maintained at a fixed value, that is to say may be regulated during
culturing, or not. Culturing may take place batchwise,
semibatchwise or continuously. Nutrients may be provided at the
beginning of the fermentation or fed in semicontinuously or
continuously.
5. Recombinant Production of Enzymes According to the Invention
[0163] The invention furthermore relates to processes for the
recombinant production of polypeptides according to the invention
or functional biologically active fragments thereof, wherein a
polypeptide-producing microorganism is cultured, the expression of
the polypeptides is optionally induced, and the polypeptides are
isolated from the culture. If desired, the polypeptides can also be
produced on an industrial scale in this manner.
[0164] The microorganisms produced according to the invention may
be cultured continuously or discontinuously by the batch method or
the fed-batch method or the repeated fed-batch method. An overview
of known cultivation methods can be found in the textbook by Chmiel
(Bioproze.beta.technik 1. Einfuhrung in die Bioverfahrenstechnik
[Bioprocess technology 1. Introduction to bioprocess technology]
(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by
Storhas (Bioreaktoren and periphere Einrichtungen [Bioreactors and
peripheral equipment] (Vieweg Verlag, Braunschweig/Wiesbaden,
1994)).
[0165] The culture medium to be used must suitably meet the
requirements of the respective strains. Descriptions of culture
media for various microorganisms are given in the manual "Manual of
Methods for General Bacteriology" of the American Society for
Bacteriology (Washington D.C., USA, 1981).
[0166] These media which can be used in accordance with the
invention usually comprise one or more carbon sources, nitrogen
sources, inorganic salts, vitamins and/or trace elements.
[0167] Preferred carbon sources are sugars, such as mono-, di- or
polysaccharides. Very good carbon sources 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 of sugar refining. It can also be
advantageous to add mixtures of different carbon sources. Other
possible carbon sources are oils and fats, for example soya oil,
sunflower oil, peanut oil, and coconut fat, fatty acids such as,
for example, palmitic acid, stearic acid or linoleic acid,
alcohols, for example glycerol, methanol or ethanol, and organic
acids, for example acetic acid or lactic acid.
[0168] Nitrogen sources are usually organic or inorganic nitrogen
compounds or materials that comprise these compounds. Examples of
nitrogen sources comprise ammonia gas or ammonium salts, such as
ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium
carbonate or ammonium nitrate, nitrates, urea, amino acids or
complex nitrogen sources, such as corn steep liquor, soya flour,
soya protein, yeast extract, meat extract and others. The nitrogen
sources may be used individually or as a mixture.
[0169] Inorganic salt compounds that can be present in the media
comprise the chloride, phosphorus or sulfate salts of calcium,
magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc,
copper and iron.
[0170] Inorganic sulfur-comprising compounds, for example sulfates,
sulfites, dithionites, tetrathionates, thiosulfates, sulfides as
well as organic sulfur compounds, such as mercaptans and thiols,
may be used as the sulfur source.
[0171] Phosphoric acid, potassium dihydrogen phosphate or
dipotassium hydrogen phosphate or the corresponding
sodium-comprising salts may be used as the phosphorus source.
[0172] Chelating agents may 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.
[0173] The fermentation media used in accordance with the invention
usually also comprise other growth factors such as vitamins or
growth promoters, which include for example biotin, riboflavin,
thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
Growth factors and salts often originate from the components of
complex media, such as yeast extract, molasses, corn steep liquor
and the like. Moreover, suitable precursors can be added to the
culture medium. The exact composition of the compounds in the
medium depends greatly on the respective experiment and is decided
for each specific case individually. Information on media
optimization can be found in the textbook "Applied Microbiol.
Physiology, A Practical Approach" (Ed. P. M. Rhodes, P. F.
Stanbury, IRL Press (1997), p. 53-73, ISBN 0 19 963577 3). Growth
media can also be obtained from commercial suppliers, such as
Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the
like.
[0174] All media components are sterilized, either by heat (20 min
at 1.5 bar and 121.degree. C.) or by filter sterilization. The
components may be sterilized either together or separately if
necessary. All media components may be present at the beginning of
a culture or can be added either continuously or batchwise.
[0175] The culture temperature is normally between 15.degree. C.
and 45.degree. C., preferably 25.degree. C. to 40.degree. C. and
can be varied or kept constant during the experiment. The pH of the
medium should be in the range of from 5 to 8.5, preferably around
7.0. The pH for growing can be controlled during growing by adding
basic compounds such as sodium hydroxide, potassium hydroxide,
ammonia or ammonia water, or acidic compounds such as phosphoric
acid or sulfuric acid. Antifoams, for example fatty acid polyglycol
esters, may be used for controlling foaming. To maintain the
stability of plasmids, suitable selectively acting substances, such
as, for example, antibiotics, may be added to the medium. To
maintain aerobic conditions, oxygen or oxygen-comprising gas
mixtures, such as, for example, ambient air, are passed into the
culture. The temperature of the culture is normally in the range of
from 20.degree. C. to 45.degree. C. The culture is continued until
a maximum of the desired product has formed. This target is
normally reached within 10 hours to 160 hours.
[0176] The fermentation liquor is subsequently processed further.
Depending on the requirements, the biomass may be removed from the
fermentation liquor completely or partially by separation
techniques, for example centrifugation, filtration, decanting or a
combination of these methods, or may be left in it completely.
[0177] If the polypeptides are not secreted into the culture
medium, the cells can also be disrupted and the product can be
obtained from the lysate by known protein isolation methods. The
cells can optionally be disrupted with high-frequency ultrasound,
high pressure, for example in a French press, by osmolysis, by the
action of detergents, lytic enzymes or organic solvents, by means
of homogenizers, or by a combination of several of the
aforementioned methods.
[0178] The polypeptides may be purified by known chromatographic
techniques, such as molecular sieve chromatography (gel
filtration), such as Q-Sepharose chromatography, ion-exchange
chromatography and hydrophobic chromatography, and with other usual
techniques such as ultrafiltration, crystallization, salting out,
dialysis and native gel electrophoresis. Suitable methods are
described, for example, in Cooper, T. G., Biochemische
Arbeitsmethoden [Biochemical methods], Verlag Walter de Gruyter,
Berlin, New York, or in Scopes, R., Protein Purification, Springer
Verlag, New York, Heidelberg, Berlin.
[0179] For isolating the recombinant protein, it may be
advantageous to use vector systems or oligonucleotides which extend
the cDNA by defined nucleotide sequences and therefore code for
modified polypeptides or fusion proteins, which for example serve
for easier purification. Suitable modifications of this type are,
for example, so-called "tags", which function as anchors, for
example the modification known as hexa-histidine anchor, or
epitopes that can be recognized as antigens of antibodies
(described, for example, in Harlow, E. and Lane, D., 1988,
Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press).
These anchors can serve for attaching the proteins to a solid
carrier, for example a polymer matrix, which may, for example, be
used as packing in a chromatography column, or may be used on a
microtiter plate or on some other carrier.
[0180] At the same time, these anchors may also be used for
recognition of the proteins. For recognition of the proteins, it is
furthermore also possible to use usual markers, such as fluorescent
dyes, enzyme markers, which form a detectable reaction product
after reaction with a substrate, or radioactive markers, alone or
in combination with the anchors for derivatization of the
proteins.
[0181] To express mutants according to the invention, one may refer
to the description of the expression of the wild-type enzyme EbN1
and the expression systems which are useful therefor, in
WO2005/108590 and WO2006/094945, which are expressly referred to
herewith.
6. Enzyme Immobilization
[0182] The enzymes used according to the invention can be used in
free or immobilized form in the processes described herein. An
immobilized enzyme is to be understood as an enzyme that is fixed
to an inert carrier. Suitable carrier materials and the enzymes
immobilized thereon are known from EP-A-1149849, EP-A-1069183 and
DE-A 100193773 and from the references cited therein. In this
respect, the disclosure of these documents is incorporated herein
in its entirety by reference. The suitable carrier materials
include, for example, clays, clay minerals such as kaolinite,
diatomaceous earth, perlite, silica, aluminum oxide, 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 making the supported enzymes,
the carrier materials are usually employed in finely-divided,
particulate form, with porous forms being preferred. The particle
size of the carrier material is usually no more than 5 mm, in
particular no more than 2 mm (particle-size distribution curve).
Similarly, when using the dehydrogenase as a whole-cell catalyst, a
free or immobilized form can be selected. Carrier materials are,
for example, Ca alginate and carrageenan. Enzymes as well as cells
may also be crosslinked directly with glutaraldehyde (cross-linking
to CLEAs). Corresponding and other immobilization techniques are
described, for example, in J. Lalonde and A. Margolin
"Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme
Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH,
Weinheim. Further information on biotransformations and bioreactors
for carrying out processes according to the invention are also
given, for example, in Rehm et al. (Ed.) Biotechnology, 2nd Edn.,
Vol. 3, Chapter 17, VCH, Weinheim.
7. Enzymatic Cyclization of Polyunsaturated Carboxylic Acids
7.1 General Aspects
[0183] The cyclization process according to the invention is
carried out in particular in the presence of an enzyme, where the
enzyme is encoded by a nucleic acid sequence according to SEQ ID
NO: 1 or a functional equivalent thereof, wherein the nucleic acid
sequence is a constituent of a gene construct or vector. Such gene
constructs or vectors are described in detail in international
application PCT/EP2010/057696 on pages 16 to 20, which is expressly
referred to here.
[0184] The host cell, which contains a gene construct or a vector,
in which the nucleic acid sequence that codes for the enzyme with
the desired activity is present is also called a transgenic
organism. The generation of such transgenic organisms is known in
principle and is discussed for example in international application
PCT/EP2010/057696 on page 20, to which reference is expressly made
here.
[0185] Cells from the group comprising bacteria, cyanobacteria,
fungi and yeasts are preferably selected as transgenic organisms.
The cell is preferably selected from fungi of the genus Pichia or
bacteria of the genera Escherichia, Corynebacterium, Ralstonia,
Clostridium, Pseudomonas, Bacillus, Zymomonas, Rhodobacter,
Streptomyces, Burkholderia, Lactobacillus or Lactococcus. The cell
is especially preferably selected from bacteria of the species
Escherichia coli, Pseudomonas putida, Burkholderia glumae,
Streptomyces lividans, Streptomyces coelicolor or Zymomonas
mobilis.
[0186] Preference is given to a process according to the invention
which is characterized in that the enzyme with the activity of a
homofarnesylic acid cyclase is encoded by a gene which has been
isolated from a microorganism, selected from among Zymomonas
mobilis, Methylococcus capsulatus, Rhodopseudomonas palustris,
Bradyrhizobium japonicum, Frankia spec, Streptomyces coelicolor and
Acetobacter pasteurianus. Particular mention is given to the
relevant genes from Zymomonas mobilis, Streptomyces coelicolor,
Bradyrhizobium japonicum and Acetobacter pasteurianus.
[0187] Preference is furthermore given to a process according to
the invention which is characterized in that the enzyme with the
cyclase activity has been generated by a microorganism which
overproduces the enzyme and which has been selected from the group
of microorganisms consisting of the genera Escherichia,
Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus,
Zymomonas, Rhodobacter, Streptomyces, Burkholderia, Lactobacillus
and Lactococcus.
[0188] Specific mention is made of a process according to the
invention which is characterized in that the enzyme with the
cyclase activity has been produced by transgenic microorganisms of
the species Escherichia coli, Pseudomonas putida, Burkholderia
glumae, Corynebacterium glutamicum, Saccharomyces cerevisiae,
Pichia pastoris, Streptomyces lividans, Streptomyces coelicolor,
Bacillus subtilis or Zymomonas mobilis, which overproduce the
enzyme with the cyclase activity.
[0189] Further embodiments for carrying out the biocatalytic
cyclization process according to the invention.
[0190] The process according to the invention is characterized in
that the enzyme is present in at least one of the following forms:
[0191] a) free, optionally purified or partially purified
polypeptide; [0192] b) immobilized polypeptide; [0193] c)
polypeptide, isolated from cells, as per a) or b); [0194] d) intact
cell, optionally quiescent or growing cells comprising at least one
such polypeptide; [0195] e) lysate or homogenate of the cells as
per d).
[0196] A further embodiment of the process according to the
invention is characterized in that the cells are microorganisms,
preferably transgenic microorganisms expressing at least one
heterologous nucleic acid molecule encoding for a polypeptide with
the cyclase activity.
[0197] A preferred embodiment of the process according to the
invention comprises at least the following steps a), b) and d):
[0198] a) to isolate or to recombinantly generate a microorganism
producing an enzyme with cyclase activity from a natural source,
[0199] b) to multiply this microorganism, [0200] c) optionally to
isolate the enzyme with cyclase activity from the microorganism or
to prepare a protein fraction comprising this enzyme, and [0201] d)
to transfer the microorganism of step b) or the enzyme of step c)
into a medium which comprises substrate, for example homofarnesylic
acid of the general formula (la).
[0202] In the process according to the invention, a substrate is
brought into contact and/or incubated with the enzyme having
cyclase activity in a medium in such a way that the substrate, for
example homofarnesylic acid is reacted in the presence of the
enzyme to give sclareolide. The medium is preferably an aqueous
reaction medium.
[0203] The pH of the aqueous reaction medium in which the process
according to the invention is carried out by preference is
advantageously maintained between pH 4 and 12, preferably between
pH 4.5 and 9, especially preferably between pH 5 and 8.
[0204] The aqueous reaction media are preferably buffered solutions
which, as a rule, have a pH of preferably from 5 to 8. A buffer
which may be used can be a citrate, phosphate, TRIS
(tris(hydroxymethyl)aminomethane) or MES buffer
(2-(N-morpholino)ethanesulfonic acid). The reaction medium may
additionally also comprise further additives such as, for example,
detergents (for example taurodeoxycholate).
[0205] The substrate, for example homofarnesylic acid is preferably
introduced into the enzymatic reaction at a concentration of 2-200
mM, especially preferably 5-25 mM, and can be resupplied
continuously or batchwise.
[0206] As a rule, the enzymatic cyclization takes place at a
reaction temperature below the deactivation temperature of the
enzyme used and above -10.degree. C. Preferably, the process
according to the invention is carried out at a temperature of
between 0.degree. C. and 95.degree. C., especially preferably at a
temperature of between 15.degree. C. and 60.degree. C., in
particular between 20 and 40.degree. C., for example at about 25 to
30.degree. C.
[0207] Especially preferred is a process according to the invention
in which the reaction of homofarnesylic acid to sclareolide is
carried out at a temperature in the range of from 20 to 40.degree.
C. and/or a pH in the range of from 4 to 8.
[0208] Besides these one-phase aqueous systems, in another variant
of the invention, two-phase systems are also used. Here, as well as
an aqueous phase, organic non-water-miscible reaction media are
used as the second phase. As a result, the reaction products
accumulate in the organic phase. After the reaction, the product in
the organic phase can readily be separated from the aqueous phase
that comprises the biocatalyst.
[0209] Preferred is a process according to the invention
characterized in that the conversion of homofarnesylic acid is
carried out in one-phase aqueous systems or two-phase systems, or
that the conversion of sparingly soluble homofarnesylic acid salts
is carried out in two-phase aqueous/solid systems.
[0210] The reaction product can be extracted using organic solvents
and optionally distilled for purification.
[0211] Examples of suitable organic solvents are aliphatic
hydrocarbons, preferably having 5 to 8 carbon atoms, such as
pentane, cyclopentane, hexane, cyclohexane, heptane, octane or
cyclooctane, halogenated aliphatic hydrocarbons, preferably having
one or two carbon atoms, such as dichloromethane, chloroform,
carbon tetrachloride, dichloroethane or tetrachloroethane, aromatic
hydrocarbons, such as benzene, toluene, the xylenes, chlorobenzene
or dichlorobenzene, aliphatic acyclic and cyclic ethers or
alcohols, preferably having 4 to 8 carbon atoms, such as ethanol,
isopropanol, diethyl ether, methyl tert-butyl ether, ethyl
tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether,
tetrahydrofuran or esters such as ethyl acetate or n-butyl acetate
or ketones such as methyl isobutyl ketone or dioxane, or mixtures
of these. The abovementioned heptane, methyl tert-butyl ether,
diisopropyl ether, tetrahydrofuran, ethyl acetate are especially
preferably used.
[0212] The cyclases used in accordance with the invention can be
used in the process according to the invention as free or
immobilized enzyme, as already described above.
[0213] For the process according to the invention, it is possible
to use quiescent or growing, free or immobilized cells which
comprise nucleic acids, nucleic acid constructs or vectors which
code for the cyclase. Disrupted cells, such as cell lysates or cell
homogenates, may also be used. Disrupted cells are understood as
meaning, for example, cells which have been permeabilized by a
treatment for example with solvents, or cells that have been
disrupted by enzymatic treatment, by mechanical treatment (for
example French press or ultrasound) or by some other method. The
crude extracts thus obtained are advantageously suitable for the
process according to the invention. Purified or partially purified
enzymes may also be used for the process.
[0214] If free organisms or enzymes are used for the process
according to the invention, they are expediently separated prior to
the extraction, for example by filtration or centrifugation.
[0215] The process according to the invention can be operated
batchwise, semibatchwise or continuously.
7.2 Preferred Conversion of Homofarnesylic Acid into
Sclareolide
[0216] In particular, the invention relates to a process for the
preparation of sclareolide in which [0217] a) homofarnesylic acid
is brought into contact and/or incubated with the homofarnesol
ambroxan cyclase and [0218] b) sclareolide is isolated.
[0219] In one embodiment of the invention, homofarnesylic acid is
brought into contact and/or incubated with the cyclase in a medium
such that a conversion of homofarnesylic acid into sclareolide in
the presence of cyclase takes place. Preferably, the medium is an
aqueous reaction medium. The aqueous reaction media are preferably
buffered solutions which, as a rule, have a pH of preferably from 5
to 8. A citrate, phosphate, TRIS (tris(hydroxymethyl)aminomethane),
MES (2-(N-morpholino)ethanesulfonic acid) buffer may be used as the
buffer. Furthermore, the reaction medium may additionally comprise
other additions, such as, for example, detergents (for example
taurodeoxycholate).
[0220] The substrate is preferably employed in the enzymatic
conversion at a concentration of 5-100 mM, especially preferably of
15-25 mM, and may be supplied continuously or batchwise.
[0221] As a rule, the enzymatic cyclization is carried out at a
reaction temperature below the deactivation temperature of the
cyclase employed, and above -10.degree. C. It is especially
preferably in the range of from 0 to 100.degree. C., in particular
from 15 to 60.degree. C. and specifically from 20 to 40.degree. C.,
for example at approximately 30.degree. C.
[0222] The reaction product sclareolide may be extracted with
organic solvents, selected from the group of those mentioned
hereinbelow, and, for purification, optionally be distilled.
[0223] Besides these one-phase aqueous systems, two-phase systems
are also employed in a further variant of the invention. Here,
ionic liquids are used as the second phase, but preferably organic
reaction media which are immiscible with water are applied as the
second phase. The reaction products thereby accumulate in the
organic phase. After the reaction, ambroxan, present in the organic
phase, can be separated readily from the aqueous phase, which
contains the biocatalyst.
[0224] Nonaqueous reaction media are understood as meaning reaction
media which comprise less than 1% by weight, preferably less than
0.5% by weight, of water, based on the total weight of the liquid
reaction medium. The conversion can be carried out in particular in
an organic solvent.
[0225] Examples of suitable organic solvents are aliphatic
hydrocarbons, preferably having 5 to 8 carbon atoms, such as
pentane, cyclopentane, hexane, cyclohexane, heptane, octane or
cyclooctane, halogenated aliphatic hydrocarbons, preferably having
one or two carbon atoms, such as dichloromethane, chloroform,
carbon tetrachloride, dichloroethane or tetrachloroethane, aromatic
hydrocarbons, such as benzene, toluene, the xylenes, chlorobenzene
or dichlorobenzene, aliphatic acyclic and cyclic ethers or
alcohols, preferably having 4 to 8 carbon atoms, such as ethanol,
isopropanol, diethyl ether, methyl tert-butyl ether, ethyl
tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether,
tetrahydrofuran or esters such as ethyl acetate or n-butyl acetate
or ketones such as methyl isobutyl ketone or dioxane, or mixtures
of these. The abovementioned heptane, methyl tert-butyl ether,
diisopropyl ether, tetrahydrofuran, ethyl acetate are especially
preferably used.
[0226] The conversion of homofarnesylic acid to sclareolide may be
performed not only in one-phase aqueous systems, but also in
two-phase systems. In the case of two-phase systems, those
mentioned hereinabove are employed. It is preferred to use the
abovementioned organic solvents which are immiscible with water as
the second phase. Thereby, the reaction products accumulate in the
organic phase. After the reaction, ambroxan, which is in the
inorganic phase, can be separated readily from the aqueous phase
which comprises the biocatalyst.
[0227] A further subject matter of the present invention is a
process for the biocatalytic preparation of sclareolide,
characterized in that the enzyme is a polypeptide which is encoded
by a nucleic acid molecule comprising at least one nucleic acid
molecule selected from the group consisting of: [0228] a) nucleic
acid molecule which codes for a polypeptide comprising the sequence
shown in SEQ ID NO 2; [0229] b) nucleic acid molecule which
comprises at least one polynucleotide of the sequence shown in SEQ
ID NO 1; [0230] c) nucleic acid molecule which codes for a
polypeptide whose sequence has an identity of at least 45% to the
sequences SEQ ID NO 2; [0231] d) nucleic acid molecule according to
(a) to (c) which codes for a functionally equivalent polypeptide or
fragment of the sequence according to SEQ ID NO 2; [0232] e)
nucleic acid molecule, coding for a functionally equivalent
polypeptide with the activity of a homofarnesylic acid cyclase,
which is obtained by amplifying a nucleic acid molecule from a cDNA
library or from genomic DNA by means of the primers according to
SEQ ID NO: No. 327 and 328, or the nucleic acid molecule,
chemically synthesized by de-novo synthesis; [0233] f) nucleic acid
molecule, coding for a functionally equivalent polypeptide with the
activity of a homofarnesylic acid cyclase, which hybridizes under
stringent conditions with a nucleic acid molecule according to (a)
to (c); [0234] g) nucleic acid molecule, coding for a functionally
equivalent polypeptide with the activity of a homofarnesylic acid
cyclase, which can be isolated from a DNA library using a nucleic
acid molecule according to (a) to (c) or their subfragments of at
least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500
nt, as a probe under stringent hybridization conditions; and [0235]
h) nucleic acid molecule, coding for a functionally equivalent
polypeptide with the activity of a homofarnesylic acid cyclase,
wherein the sequence of the polypeptide has an identity of at least
30% to the sequences SEQ ID NO 2; [0236] i) nucleic acid molecule,
coding for a functionally equivalent polypeptide with the activity
of a homofarnesylic acid cyclase, wherein the polypeptide is
encoded by a nucleic acid molecule selected from the group of
nucleic acids stated in a) to h) [0237] j) nucleic acid molecule,
coding for a functionally equivalent polypeptide with the activity
of a homofarnesylic acid cyclase, wherein the polypeptide has an
analogous or similar binding site as a polypeptide encoded by a
nucleic acid molecule selected from the group of those described in
a) to h).
[0238] For the purposes of the invention, an analogous or similar
binding site is a conserved domain or motif of the amino acid
sequence with a homology of 80%, especially preferably 85%, 86%,
87%, 88%, 89%, 90%, in particular 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% or 100%, which ensures the binding of the same
substrate, in particular homofarnesylic acid.
[0239] Preferably, the nucleic acid molecule c) has an identity of
at least 50%, 60%, 65%, 70%, 75%, 80%, especially preferably 85%,
86%, 87%, 88%, 89%, 90%, in particular 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99%, to SEQ ID NO: 1.
[0240] Likewise, a functionally equivalent polypeptide has an
identity of at least 50%, 60%, 65%, 70%, 75%, 80%, especially
preferably 85%, 86%, 87%, 88%, 89%, 90%, in particular 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99%, to SEQ ID NO: 2. Instead of
the term "identity", the term "homologous" or "homology" may also
be used synonymously.
[0241] The invention will now be described with reference to the
following nonlimiting examples:
Experimental Part
[0242] Unless specific information has been given in the examples
which follow, the general information hereinbelow applies.
A. GENERAL INFORMATION
[0243] All materials and microorganisms employed are commercially
available products.
[0244] 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.sup.nd Edition, Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989.
B. EXAMPLES
Example 1
Cloning of the Zm-SHC and Expression in E. coli
[0245] The gene of the cyclase may be amplified from Zymomonas
mobilis with the aid of the oligonucleotides Zm-SHC_fw and
Zm-SHC_rev.
TABLE-US-00006 Primer: Primer No. sequence (5'->3') Position
Zm-SHC_fw gcgctgtttcatatgggtattgaca N-term (SEQ ID NO: 327) primer
Zm-SHC_rev gcgcttaccctggatcctcgaaaat C-term (SEQ ID NO: 328)
primer
[0246] 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.degree. C. for 3 min; 30
cycles at 95.degree. C. for 30 sec., 50.degree. C. for 30 sec and
72.degree. C. for 3 min; 72.degree. C. for 10 min.; 4.degree. C.
until used. The PCR product (.about.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.
[0247] The PCR product was digested with the restriction
endonucleases NdeI and BamHI 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 ID
NO:2):
TABLE-US-00007 Met Gly Ile Asp Arg Met Asn Ser Leu Ser Arg Leu Leu
Met Lys Lys 1 5 10 15 Ile Phe Gly Ala Glu Lys Thr Ser Tyr Lys Pro
Ala Ser Asp Thr Ile 20 25 30 Ile Gly Thr Asp Thr Leu Lys Arg Pro
Asn Arg Arg Pro Glu Pro Thr 35 40 45 Ala Lys Val Asp Lys Thr Ile
Phe Lys Thr Met Gly Asn Ser Leu Asn 50 55 60 Asn Thr Leu Val Ser
Ala Cys Asp Trp Leu Ile Gly Gln Gln 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 Gln Arg Glu Asp Gly
115 120 125 Ser Trp Gly Val Tyr Phe Gly Ala Gly Asn Gly Asp Ile 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
Ile Ala Glu Lys Gly Gly Leu 165 170 175 Lys Asn Ile Arg Val Phe Thr
Arg Tyr Trp Leu Ala Leu Ile Gly Glu 180 185 190 Trp Pro Trp Glu Lys
Thr Pro Asn Leu Pro Pro Glu Ile Ile Trp Phe 195 200 205 Pro Asp Asn
Phe Val Phe Ser Ile Tyr Asn Phe Ala Gln Trp Ala Arg 210 215 220 Ala
Thr Met Val Pro Ile Ala Ile Leu Ser Ala Arg Arg Pro Ser Arg 225 230
235 240 Pro Leu Arg Pro Gln 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 Ile
Asp Leu Trp 260 265 270 Ser Gln Phe Phe Arg Thr Thr Asp Arg Gly Leu
His Trp Val Gln Ser 275 280 285 Asn Leu Leu Lys Arg Asn Ser Leu Arg
Glu Ala Ala Ile Arg His Val 290 295 300 Leu Glu Trp Ile Ile Arg His
Gln Asp Ala Asp Gly Gly Trp Gly Gly 305 310 315 320 Ile Gln Pro Pro
Trp Val Tyr Gly Leu Met Ala Leu His Gly Glu Gly 325 330 335 Tyr Gln
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 Ile Gln 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 Gln Val Lys Val
Lys Gly Asp Trp Ser Ile 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 Ile Ala Leu 435 440 445 Ser Ser Tyr Arg
Asp Lys Glu Glu Trp Gln Lys Lys Gly Val Glu Asp 450 455 460 Ala Ile
Thr Arg Gly Val Asn Trp Leu Ile Ala Met Gln Ser Glu Cys 465 470 475
480 Gly Gly Trp Gly Ala Phe Asp Lys Asp Asn Asn Arg Ser Ile Leu Ser
485 490 495 Lys Ile Pro Phe Cys Asp Phe Gly Glu Ser Ile 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 Ile Gln Lys Ala Ile
Asp Tyr Val Arg Ser Glu 530 535 540 Gln Glu Ala Glu Gly Ala Trp Phe
Gly Arg Trp Gly Val Asn Tyr Ile 545 550 555 560 Tyr Gly Thr Gly Ala
Val Leu Pro Ala Leu Ala Ala Ile Gly Glu Asp 565 570 575 Met Thr Gln
Pro Tyr Ile Thr Lys Ala Cys Asp Trp Leu Val Ala His 580 585 590 Gln
Gln Glu Asp Gly Gly Trp Gly Glu Ser Cys Ser Ser Tyr Met Glu 595 600
605 Ile Asp Ser Ile Gly Lys Gly Pro Thr Thr Pro Ser Gln Thr Ala Trp
610 615 620 Ala Leu Met Gly Leu Ile Ala Ala Asn Arg Pro Glu Asp Tyr
Glu Ala 625 630 635 640 Ile Ala Lys Gly Cys His Tyr Leu Ile Asp Arg
Gln Glu Gln 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 Gln Thr Ile Lys Leu
Asp Asp Pro Ala Leu Ser Lys Arg Leu 675 680 685 Leu Gln Gly Ala Glu
Leu Ser Arg Ala Phe Met Leu Arg Tyr Asp Phe 690 695 700 Tyr Arg Gln
Phe Phe Pro Ile Met Ala Leu Ser Arg Ala Glu Arg Leu 705 710 715 720
Ile Asp Leu Asn Asn 725
[0248] The plasmid pDHE-Zm-SHC-1 was transformed into the strain E.
coli TG10 pAgro4 pHSG575 [Takeshita et al., Gene 1987, 61:63-74;
Tomoyasu et al., Mol Microbiol 2001, 40:397-413]. The recombinant
E. coli were named E. coli LU15568.
Example 2
Provision of Recombinant Homofarnesol Cyclase from Z. mobilis
[0249] Inoculated from a suitable 2 ml preculture, E. coli LU15568
was grown for 16 h at 37.degree. C. in 20 ml LB-Amp/Spec/Cm (100
.mu.g/I ampicillin; 100 .mu.g/I spectinomycin; 20 .mu.g/I
chloramphenicol), 0.1 mM IPTG, 0.5 g/I rhamnose in 100 ml
Erlenmeyer flasks (with baffles), centrifuged at 5000*g/10 min and
stored at 4.degree. C. Protein extract was prepared by suspending
the cell pellet in 15 ml disruption buffer (0.2 M Tris/HCl, 0.5 M
EDTA, pH 8.0), 375 U benzonase (for example Novagen, 25 U/.mu.L),
40 .mu.L PMSF (100 mM, dissolved in i-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.degree. C.
[0250] 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.
[0251] 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.degree.
C. and 26 900*g. The supernatant was discarded and the pellet was
resuspended in 100 ml solubilization buffer (50 mM Tris/HCl, 10 mM
MgCl2.times.6H2O, 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.
[0252] The homogenized extract was recentrifuged for 1 h at
4.degree. 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.degree. C. without suffering activity losses.
The protein content was in the range of 1 mg/ml.
Example 3
Activity Determination of the Recombinant Cyclase from E. coli
LU15568
[0253] Homofarnesylic acid (1b,
(3E,7E)-4,8,12-trimethyltrideca-3,7,11-trienoic acid)) was
incubated with the protein preparation described in 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%, E,Z
10.13%, E,E 74.93%), 2.913 ml of water; 0.350 ml of sodium citrate
buffer (1M sodium citrate pH 5.4), 0.560 ml MgCl.sub.2 (0.5M
solution) were pipetted in, and the mixture was warmed for 30 min
at 37.degree. C., with stirring. The reaction started with the
addition of E. coli LU15568 homogenate (protein content 35 mg/ml),
warmed to 37.degree. C. The reaction mixture was stirred on a
magnetic stirrer in an oil bath for 24 h at pH 5.0 at 37.degree. C.
at maximum stirring speed. The pH was adjusted during the reaction
using 0.5M HCl. 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. FIG.
1).
[0254] Using the analyses described hereinbelow in greater detail,
a conversion rate of 74.5% in total of 82.7% from the E,E isomer
was determined.
[0255] The conversion of homofarnesylic acid (1b) into sclareolide
(3) can be determined with the following GC system: [0256] Column:
10 m Optima 1 [0257] Temperature profile: [0258] 0 min: 100.degree.
C. [0259] 5.degree. C./min to 200.degree. C. [0260] After 5 min:
30.degree. C./min to 320.degree. C. [0261] thereafter constant
[0262] Duration of the method: 30 min [0263] Injector temperature:
280.degree. C. [0264] Retention times (RT): [0265] Homofarnesylic
acid: peak 1 at 11.7 min, peak 2 at 12.1 min; [0266] Sclareolide:
approx. 13.5 min [0267] A calibration series, with the aid of which
the concentration of unknown samples was determined, is established
using authentic material (Sigma, catalog No.: 358002).
[0268] Reference is made expressly to the disclosure of the
publications cited herein.
Sequences:
[0269] SEQ ID NO: 1-326 Nucleic acid/amino acid sequences of
various SHC genes [0270] SEQ ID NO: 327-328 PCR primer
[0271] What follows now is a list of SHC enzyme sequences which are
particularly useful in accordance with the invention:
Enzyme Sequences
TABLE-US-00008 [0272]>seq_ID 4
MNMASRFSLKKILRSGSDTQGTNVNTLIQSGTSDIVRQKPAPQEPADLSAL
KAMGNSLTHTLSSACEWLMKQQKPDGHWVGSVGSNASMEAEWCLALWFLGL
EDHPLRPRLGKALLEMQRPDGSWGTYYGAGSGDINATVESYAALRSLGYAE
DDPAVSKAAAWIISKGGLKNVRVFTRYWLALIGEWPWEKTPNLPPEIIWFP
DNFVFSIYNFAQWARATMMPLAILSARRPSRPLRPQDRLDALFPGGRANFD
YELPTKEGRDVIADFFRLADKGLHWLQSSFLKRAPSREAAIKYVLEWIIWH
QDADGGWGGIQPPWVYGLMALHGEGYQFHHPVMAKALDALNDPGWRHDKGD
ASWIQATNSPVWDTMLSLMALHDANAEERFTPEMDKALDWLLSRQVRVKGD
WSVKLPNTEPGGWAFEYANDRYPDTDDTAVALIAIASCRNRPEWQAKGVEE
AIGRGVRWLVAMQSSCGGWGAFDKDNNKSILAKIPFCDFGEALDPPSVDVT
AHVLEAFGLLGLPRDLPCIQRGLAYIRKEQDPTGPWFGRWGVNYLYGTGAV
LPALAALGEDMTQPYISKACDWLINCQQENGGWGESCASYMEVSSIGHGAT
TPSQTAWALMGLIAANRPQDYEAIAKGCRYLIDLQEEDGSWNEEEFTGTGF
PGYGVGQTIKLDDPAISKRLMQGAELSRAFMLRYDLYRQLFPIIALSRASR LIKLGN
>seq_ID 2 MGIDRMNSLSRLLMKKIFGAEKTSYKPASDTIIGTDTLKRPNRRPEPTAKV
DKTIFKTMGNSLNNTLVSACDWLIGQQKPDGHWVGAVESNASMEAEWCLAL
WFLGLEDHPLRPRLGNALLEMQREDGSWGVYFGAGNGDINATVEAYAALRS
LGYSADNPVLKKAAAWIAEKGGLKNIRVFTRYVVLALIGEWPWEKTPNLPP
EIIWFPDNFVFSIYNFAQWARATMVPIAILSARRPSRPLRPQDRLDELFPE
GRARFDYELPKKEGIDLWSQFFRTTDRGLHWVQSNLLKRNSLREAAIRHVL
EWIIRHQDADGGWGGIQPPWVYGLMALHGEGYQLYHPVMAKALSALDDPGW
RHDRGESSWIQATNSPVWDTMLALMALKDAKAEDRFTPEMDKAADWLLARQ
VKVKGDWSIKLPDVEPGGWAFEYANDRYPDTDDTAVALIALSSYRDKEEWQ
KKGVEDAITRGVNWLIAMQSECGGWGAFDKDNNRSILSKIPFCDFGESIDP
PSVDVTAHVLEAFGTLGLSRDMPVIQKAIDYVRSEQEAEGAWFGRWGVNYI
YGTGAVLPALAAIGEDMTQPYITKACDWLVAHQQEDGGWGESCSSYMEIDS
IGKGPTTPSQTAWALMGLIAANRPEDYEAIAKGCHYLIDRQEQDGSWKEEE
FTGTGFPGYGVGQTIKLDDPALSKRLLQGAELSRAFMLRYDFYRQFFPIMA LSRAERLIDLNN
>seq_ID 5 MTVTSSASARATRDPGNYQTALQSTVRAAADWLIANQKPDGHWVGRAESNA
CMEAQWCLALWFMGLEDHPLRKRLGQSLLDSQRPDGAWQVYFGAPNGDINA
TVEAYAALRSLGFRDDEPAVRRAREWIEAKGGLRNIRVFTRYVVLALIGEW
PWEKTPNIPPEVIWFPLWFPFSIYNFAQWARATLMPIAVLSARRPSRPLPP
ENRLDALFPHGRKAFDYELPVKAGAGGWDRFFRGADKVLHKLQNLGNRLNL
GLFRPAATSRVLEWMIRHQDFDGAWGGIQPPWIYGLMALYAEGYPLNHPVL
AKGLDALNDPGWRVDVGDATYIQATNSPVWDTILTLLAFDDAGVLGDYPEA
VDKAVDWVLQRQVRVPGDWSMKLPHVKPGGWAFEYANNYYPDTDDTAVALI
ALAPLRHDPKWKAKGIDEAIQLGVDWLIGMQSQGGGWGAFDKDNNQKILTK
IPFCDYGEALDPPSVDVTAHIIEAFGKLGISRNHPSMVQALDYIRREQEPS
GPWFGRWGVNYVYGTGAVLPALAAIGEDMTQPYIGRACDWLVAHQQADGGW
GESCASYMDVSAVGRGTTTASQTAWALMALLAANRPQDKDAIERGCMWLVE
RQSAGTWDEPEFTGTGFPGYGVGQTIKLNDPALSQRLMQGPELSRAFMLRY
GMYRHYFPLMALGRALRPQSHS >seq_ID 6
MTVSTSSAFHHSSLSDDVEPIIQKATRALLEKQHQDGHWVFELEADATIPA
EYILLKHYLGEPEDLEIEAKIGRYLRRIQGEHGGWSLFYGGDLDLSATVKA
YFALKMIGDSPDAPHMLRARNEILARGGAMRANVFTRIQLALFGAMSWEHV
PQMPVELMLMPEWFPVHINKMAYVVARTVLVPLLVLQALKPVARNRRGILV
DELFVPDVLPTLQESGDPIWRRFFSALDKVLHKVEPYVVPKNMRAKAIHSC
VHFVTERLNGEDGLGAIYPAIANSVMMYDALGYPENHPERAIARRAVEKLM
VLDGTEDQGDKEVYCQPCLSPIWDTALVAHAMLEVGGDEAEKSAISALSWL
KPQQILDVKGDWAWRRPDLRPGGWAFQYRNDYYPDVDDTAVVTMAMDRAAK
LSDLHDDFEESKARAMEWTIGMQSDNGGWGAFDANNSYTYLNNIPFADHGA
LLDPPTVDVSARCVSMMAQAGISITDPKMKAAVDYLLKEQEEDGSWFGRWG
VNYIYGTWSALCALNVAALPHDHLAIQKAVAWLKNIQNEDGGWGENCDSYA
LDYSGYEPMDSTASQTAWALLGLMAVGEANSEAVTKGINWLAQNQDEEGLW
KEDYYSGGGFPRVFYLRYHGYSKYFPLWALARYRNLKKANQPIVHYGM
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210381013A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210381013A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References