Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02348962 2001-04-27
WO 00/26355 PCT/BP99/07952
-1-
Constructing production strains for the preparation of substituted phenols by
specifically inactivating genes of eu~enol and ferulic acid catabolism
The present invention relates to the construction of production strains and to
a
process for preparing substituted methoxyphenols, in particular vanillin.
DE-A 4 227 076 (process for preparing substituted methoxyphenols, and
microorganism which is suitable for this purpose) describes the preparation of
substituted methoxyphenols using a novel Pseudomonas sp.. The starting
material in
this context is eugenol and the products are ferulic acid, vanillic acid,
coniferyl
alcohol and coniferyl aldehyde.
An extensive review of the biotransformations which were possible using
ferulic
acid, which was written by Rosazza et al. (Biocatalytic transformation of
ferulic acid:
an abundant aromatic natural product; J. Ind. Microbiol. 15:457-471), also
appeared
in 1995.
The genes and enzymes for synthesizing coniferyl alcohol, coniferyl aldehyde,
ferulic
acid, vanillic and vanillin acid from Pseudomonas sp. were described in EP-A
0 845 532.
The enzymes for converting traps-ferulic acid into traps-feruloyl-SCoA ester
and
subsequently into vanillin, and also the gene for cleaving the ester, were
described by
the Institute of Food Research, Norwich, GB, in WO 97/35999. In 1998, the
content
of the patent also appeared in the form of scientific publications (Gasson et
al. 1998.
Metabolism of ferulic acid to vanillin. J. Biol. Chem. 273:4163-4170; Narbad
and
Gasson 1998. Metabolism of ferulic acid via vanillin using a novel CoA-
dependent
pathway in a newly isolated strain of Pseudomonas fluorescens. Microbiology
144:1397 - 1405).
if a AJi
CA 02348962 2001-04-27
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DE-A 195 32 317 describes the use of Amycolatopsis sp. for obtaining vanillin
from
ferulic acid fermentatively in high yields.
The known processes suffer from the disadvantage that they either achieve only
very
low yields of vanillin or make use of relatively expensive starting compounds.
While
the last-mentioned process (DE-A 195 32 317) does achieve high yields, the use
of
Pseudomonas sp. HR199 and Amycolatopsis sp. HR167 for biotransforming eugenol
into vanillin requires a fermentation which is carried out in two steps,
consequently
leading to substantial expense and consumption of time.
to
The object of the present invention is therefore to construct organisms which
are able
to convert the relatively inexpensive raw material eugenol into vanillin in a
one-step
process.
This object is achieved by means of constructing production strains of
unicellular or
multicellular organisms, which strains are characterized in that enzymes of
eugenol
and/or ferulic acid catabolism are inactivated such that the intermediates
coniferyl
alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid
accumulate.
.... 2o The production strain may be unicellular or multicellular.
Accordingly, the invention
can relate to microorganisms, plants or animals. Furthermore, use can also be
made
of extracts which are obtained from the production strain. According to the
invention,
preference is given to using unicellular organisms. These latter organisms can
be
microorganisms or animal or plant cells. According to the invention,
particular
preference is given to using fungi and bacteria. The highest preference is
given to
bacterial species. Those bacteria which may in particular be used, after their
eugenol
and/or ferulic acid catabolism has/have been altered, are species of
Rhodococcus,
Pseudomonas and Escherichia.
In the simplest case, known, conventional microbiological methods can be used
for
isolating the organisms which may be employed in accordance with the
invention.
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Thus, the enzymic activity of the proteins involved in eugenol and/or ferulic
acid
catabolism can be altered by using enzyme inhibitors. Furthermore, the enzymic
activity of the proteins involved in eugenol and/or ferulic acid catabolism
can be
altered by mutating the genes which encode these proteins. Such mutations can
be
generated in a random manner by means of classical methods, for example by
using
UV irradiation or mutation-inducing chemicals.
Recombinant DNA methods, such as deletions, insertions and/or nucleotide
exchanges, are likewise suitable for isolating the novel organisms. Thus, the
genes of
1o the organisms can, for example, be inactivated using other DNA elements (S2
elements). Suitable vectors can likewise be used for replacing the intact
genes with
gene structures which are altered and/or inactivated. In this context, the
genes which
are to be inactivated, and the DNA elements which are employed for the
inactivation,
can be obtained by means of classical cloning techniques or by means of
polymerase
chain reactions (PCR).
For example, in one possible embodiment of the invention, eugenol catabolism
and
ferulic acid catabolism can be altered by inserting S2 elements, or
introducing
deletions, into appropriate genes. In this context, the abovementioned
recombinant
2o DNA methods can be used to inactivate the functions of the genes, which
encode
dehydrogenases, synthetases, hydratase-adolases, thiolases or demethylases,
such that
production of the relevant enzymes is blocked. Preferably, the genes are those
which
encode coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases,
feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases,
vanillin dehdrogenases or vanillic acid demethylases. Very particular
preference is
given to genes which encode the amino acid sequences specified in EP-A 0845532
and/or nucleotide sequences which encode their allelic variations.
The invention accordingly also relates to gene structures for preparing
transformed
organisms and mutants.
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Preference is given to employing gene structures in which the nucleotide
sequences
encoding dehydrogenases, synthetases, hydratase-aldolases, thiolases or
demethylases
are inactivated for isolating the organisms and mutants. Particular preference
is given
to gene structures in which the nucleotide sequences encoding coniferyl
alcohol
dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases,
enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehydrogenases or
vanillic acid demethylases are inactivated. Very particular preference is
given to gene
structures which exhibit the structures given in Figures la to lr having the
nucleotide
sequences which are depicted in Figures 2a to 2r and/or nucleotide sequences
to encoding their allelic variations. In this context, particular preference
is given to
nucleotide sequences 1 to 18.
The invention also encompasses the part sequences of these gene structures as
well as
functional equivalents. Functional equivalents are to be understood as meaning
those
derivatives of the DNA in which individual nucleobases have been exchanged
(wobble exchanges) without the function being altered. Amino acids may also be
exchanged at the protein level without this resulting in an alteration in
function.
One or more DNA sequences can be inserted upstream and/or downstream of the
2o gene structures. By cloning the gene structures, it is possible to obtain
plasmids or
vectors which are suitable for the transformation and/or transfection of an
organism
and/or for conjugative transfer into an organism.
The invention furthermore relates to plasmids and/or vectors for preparing the
organisms and mutants which are transformed in accordance with the invention.
These organisms and mutants consequently harbour the gene structures which
have
been described. The present invention accordingly also relates to organisms
which
harbour the said plasmids and/or vectors.
The nature of the plasmids and/or vectors depends on what they are being used
for. In
order, for example, to be able to replace the intact genes of eugenol and/or
ferulic
CA 02348962 2001-04-27
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acid catabolism in pseudomonads with the genes which have been inactivated
with
omega elements, there is a need for vectors which, on the one hand, can be
transferred into pseudomonads (conjugatively transferable plasmids) but which,
on
the other hand, cannot be replicated in these organisms and are consequently
unstable
in pseudomonads (so-called suicide plasmids). DNA segments which are
transferred
into pseudomonads with the aid of such a plasmid system can only be retained
if they
become integrated by homologous recombination into the genome of the bacterial
cell.
1o The described gene structures, vectors and plasmids may be used for
preparing
different transformed organisms or mutants. The said gene structures can be
used for
replacing intact nucleic acid sequences with altered and/or inactivated gene
structures. In the cells, which can be obtained by transformation or
transfection or
conjugation, the intact gene is replaced, by homologous recombination, with
the
altered and/or inactivated gene structure, as a consequence of which the
resulting
cells now only possess the altered and/or inactivated gene structure in their
genome.
In this way, preferably genes can be altered and/or inactivated, in accordance
with the
invention, such that the relevant organisms are able to produce coniferyl
alcohol,
coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid.
.. 20
Mutants of the strain Pseudomonas sp. HR199 (DSM 7063), which was described in
detail in DE-A 4 227 076 and EP-A 0845532, are examples of production strains
which have been constructed in this way in accordance with the invention, with
the
corresponding gene structures ensuing, inter alia, from Figures la to lr, in
combination with Figures 2a to 2r:
1. Pseudomonas sp. HR199calAS2Km, which contains the S2Km-inactivated
calA gene in place of the intact calA gene encoding coniferyl alcohol
dehydrogenase (Fig. la; Fig. 2a).
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2. Pseudomonas sp. HR199calASZGm, which contains the 52Gm-inactivated
calA gene in place of the intact calA gene encoding coniferyl alcohol
dehydrogenase (Fig. lb; Fig. 2b).
3. Pseudomonas sp. HR199ca1A0, which contains the deletion-inactivated calA
gene in place of the intact calA gene encoding coniferyl
alcohol
dehydrogenase (Fig. lc; Fig. 2c).
4. Pseudomonas sp. HR199calBSZKm, which contains the S2Km-
inactivated
calB gene in place of the intact calB gene encoding
coniferyl aldehyde
", dehydrogenase (Fig. ld; Fig. 2d)
5. Pseudomonas sp. HR199calBS2Gm, which contains the S2Gm-
inactivated
calB gene in place of the intact calB gene encoding
coniferyl aldehyde
dehydrogenase (Fig. le; Fig. 2e).
6. Pseudomonas sp. HR199ca1B0, which contains the deletion-
inactivated
calB
gene in place of the intact calB gene encoding coniferyl
aldehyde
dehydrogenase (Fig.lf; Fig. 2f).
7. Pseudomonas sp. HR199fcsS2Km, which contains the S2Km-
inactivated
fcs
gene in place of the intact fcs gene encoding feruloyl-CoA
synthetase (Fig.lg;
Fig. 2g).
8. Pseudomonas sp. HR199fcsS2Gm, which contains the S2Gm-
inactivated
fcs
gene in place of the intact fcs gene encoding feruloyl-CoA
synthetase (Fig.lh;
Fig. 2h).
9. Pseudomonas sp. HR199fcs0, which contains the deletion-inactivated fcs
gene in place of the intact fcs gene encoding feruloyl-CoA synthetase (Fig.li;
Fig. 2i).
10. Pseudomonas sp. HR199echS2Km, which contains the S2Km-inactivated ech
gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase
(Fig.lj; Fig. 2j).
11. Pseudomonas sp. HR199echS2Gm, which contains the S2Gm-inactivated ech
gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase
3o (Fig.lk; Fig. 2k).
CA 02348962 2001-04-27
7 _
12. Pseudomonas sp. HR199ech0, which contains the deletion-inactivated ech
gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase
(Fig.ll; Fig. 21).
13. Pseudomonas sp. HR199aat52Km, which contains the S2Km-inactivated aat
gene in place of the intact aat gene ecnoding beta-ketothiolase (Fig. lm;
Fig. 2m).
14. Pseudomonas sp. HR199aatSZGm, which contains the S2Gm-inactivated aat
gene in place of the intact aat gene encoding beta-ketothiolase (Fig.ln;
..~. Fig. 2n).
l0 15. Pseudomonas sp. HR199aat0, which contains the deletion-inactivated aat
gene in place of the intact aat gene encoding beta-ketothiolase (Fig.lo; 20).
16. Pseudomonas sp. HR199vdhSZKm, which contains the S2Km-inactivated vdh
gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lp;
Fig. 2p).
17. Pseudomonas sp. HR199vdhS2Gm, which contains the SZGm-inactivated vdh
gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lq;
Fig. 2q).
18. Pseudomonas sp. HR199vdh0, which contains the deletion-inactivated vdh
gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lr;
Fig. 2r).
19. Pseudomonas sp. HR199vdhBS2Km, which contains the S2Km-inactivated
vdhB gene in place of the intact vdhB gene encoding vanillin dehydrogenase
II.
20. Pseudomonas sp. HR199vdhBS2,Gm, which contains the S2Gm-inactivated
vdhB gene in place of the intact vdhB gene encoding vanillin dehydrogenase
II.
21. Pseudomonas sp. HR199vdhB~, which contains the deletion-inactivated vdhB
gene in place of the intact vdhB gene encoding vanillin dehydrogenase II.
22. Pseudomonas sp. HR199adhS2Km, which contains the S2Km-inactivated adh
3o gene in place of the intact adh gene encoding alcohol dehydrogenase.
CA 02348962 2001-04-27
_ g _
23. Pseudomonas sp. HR199adh52Gm, which contains the S2Gm-inactivated adh
gene in place of the intact adh gene encoding alcohol dehydrogenase.
24. Pseudomonas sp. HR199adh0 which contains the deletion-inactivated adh
gene in place of the intact adh gene encoding alcohol dehydrogenase.
25. Pseudomonas sp. HR199vanAS2Km, which contains the SZKm-inactivated
vanA gene in place of the intact vanA gene encoding the a-subunit of vanillic
acid demethylase.
26. Pseudomonas sp. HR 199vanA52Gm, which contains the S2Gm-inactivated
~.. vanA gene in place of the intact vanA gene encoding the a-subunit of
vanillic
1o acid demethylase.
27. Pseudomonas sp. HR 199vanA~, which contains the deletion-inactivated vanA
gene in place of the intact vanA gene encoding the a-subunit of vanillic acid
demethylase.
28. Pseudomonas sp. HR199vanBSZKm, which contains the S2Km-inactivated
vanB gene in place of the intact vanB gene encoding the (3-subunit of vanillic
acid demethylase.
29. Pseudomonas sp. HR199vanBS2Gm, which contains the S2Gm-inactivated
vanB gene in place of the intact vanB gene encoding the (3-subunit of vanillic
acid demethylase.
30. Pseudomonas sp. HR199vanB0, which contains the deletion-inactivated vanB
gene in place of the intact vanB gene encoding the (3-subunit of vanillic acid
demethylase.
The invention additionally relates to a process for the biotechnological
preparation of
organic compounds. In particular, this process can be used to prepare
alcohols,
aldehydes and organic acids. The latter are preferably coniferyl alcohol,
coniferyl
aldehyde, ferulic acid, vanillin and vanillic acid.
The above-described organisms are employed in the novel process. The organisms
3o which are very particularly preferred include bacteria, in particular the
Pseudomonas
CA 02348962 2001-04-27
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species. Specifically, the abovementioned Pseudomonas species can preferably
be
employed for the following processes:
1. Pseudomonas sp. HR199calAS2Km, Pseudomonas sp. HR199calAS2Gm and
Pseudomonas sp. HR199ca1A0 for preparing coniferyl alcohol from eugenol.
2. Pseudomonas sp. HR 199calBSZKm, Pseudomonas sp. HR 199ca1B52Gm and
Pseudomonas sp. HR199ca1B0 for preparing coniferyl aldehyde from eugenol
m~~ or coniferyl alcohol.
to
3. Pseudomonas sp. HR199fcsSZKm, Pseudomonas sp. HR199fcs52Gm, Pseu-
domonas sp. HR 199fcs0, Pseudomonas sp. HR 199echS2Km, Pseudomonas
sp. HR199echSZGm and Pseudomonas sp. HR199ech~ for preparing ferulic
acid from eugenol or coniferyl alcohol or coniferyl aldehyde.
IS
4. Pseudomonas sp. HR 199vdhSZKm, Pseudomonas sp. HR 199vdhSZGm, Pseu-
domonas sp. HR 199vdhd, Pseudomonas sp. HR 199vdhS2GmvdhBS2Km,
Pseudomonas sp. HR 199vdhS2KmvdhBS2Gm, Pseudomonas sp. HR 199vdh0
vdhBS2Gm and Pseudomonas sp. HR199vdhwdhBS2Km for preparing
2o vanillin from eugenol or coniferyl alcohol or coniferyl aldehyde or ferulic
acid.
5. Pseudomonas sp. HR199vanAS2,Km, Pseudomonas sp. HR199vanAS2Gm,
Pseudomonas sp. HR 199vanA~, Pseudomonas sp. HR 199vanBS2Km,
25 Pseudomonas sp. HR199vanBS2Gm and Pseudomonas sp. HR199vanB0 for
preparing vanillic acid from eugenol or coniferyl alcohol or coniferyl
aldehyde or ferulic acid or vanillin.
Eugenol is the preferred substrate. However, it is also possible to add
further
3o substrates or even to replace the eugenol with another substrate.
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Suitable nutrient media for the organisms which are employed in accordance
with the
invention are synthetic, semisynthetic or complex culture media. These media
may
comprise carbon-containing and nitrogen-containing compounds, inorganic salts,
where appropriate trace elements, and vitamins.
Carbon-containing compounds which may be suitable are carbohydrates,
hydrocarbons or organic standard chemicals. Examples of compounds which may
preferably be used are sugars, alcohols or sugar alcohols, organic acids or
complex
mixtures.
to
The sugar is preferably glucose. The organic acids which may preferably be
employed are citric or acetic acid. Examples of the complex mixtures are malt
extract, yeast extract, casein or casein hydrolysate.
Inorganic compounds are suitable nitrogen-containing substrates. Examples of
these
are nitrates and ammonium salts. Organic nitrogen sources can also be used.
These
sources include yeast extract, Soya bean meal, casein, casein hydrolysate and
corn
steep liquor.
2o Examples of the inorganic salts which may be employed are sulphates,
nitrates,
chlorides, carbonates and phosphates. The metals which the said salts contain
are
preferably sodium, potassium, magnesium, manganese, calcium, zinc and iron.
The temperature for the culture is preferably in the range from 5 to
100°C. The range
from 15 to 60°C is particularly preferred, with 22 to 37°C being
most preferred.
The pH of the medium is preferably 2 to 12. The range from 4 to 8 is
particularly
preferred.
3o In principle, any bioreactor known to the skilled person can be employed
for carrying
out the novel process. Preferential consideration is given to any appliance
which is
CA 02348962 2001-04-27
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suitable for submerged processes. This means that vessels which do or do not
possess
a mechanical mixing device may be employed in accordance with the invention.
Examples of the latter are shaking apparatuses, and bubble column reactors or
loop
reactors. The former preferably include all the known appliances which are
fitted
with stirrers of any design.
The novel process can be carned out continuously or batchwise. The
fermentation
time required for achieving a maximum quantity of product depends on the
specific
nature of the organism employed. However, in principle, the fermentation times
are
between 2 and 200 hours.
The invention is explained in more detail below while refernng to examples:
Mutants of the eugenol-utilizing strain Pseudomonas sp. HR199 (DSM 7063) were
generated in a targeted manner by specifically inactivating genes of eugenol
catabolism by means of inserting omega elements or introducing deletions. The
omega elements employed were DNA segments which encoded resistances to the
antibiotics kanamycin (S2Km) and gentamycin (S2Gm). These resistance genes
were
isolated from Tn5 and the plasmid pBBRIMCS-5 using standard methods. The genes
. 2o calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB, which encode
coniferyl
alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, feruloyl-CoA
synthetase,
enoyl-CoA hydratase-aldolase, beta-ketothiolase, vanillin dehdrogenase,
alcohol
dehydrogenase, vanillin dehdrogenase II and vanillic acid demethylase, were
isolated
from genomic DNA of the strain Pseudomonas sp. HR199 using standard methods
and cloned into pBluescript SK . By means of digesting with suitable
restriction
endonucleases, DNA segments were removed from these genes (deletion) or
substituted with S2 elements (insertion), resulting in the respective gene
being
inactivated. The genes which had been mutated in this manner were recloned
into
conjugatively transferable vectors and subsequently introduced into the strain
3o Pseudomonas sp. HR199. Suitable selection was used to obtain
transconjugants
which had replaced the respective functional wild-type gene with the newly
CA 02348962 2001-04-27
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introduced inactivated gene. The insertion and deletion mutants which were
obtained
in this way now only possessed the respective inactivated gene. This procedure
was
used to obtain both mutants possessing only one defective gene and multiple
mutants,
in which several genes had been inactivated in this manner. These mutants were
employed for biotransforming
a) eugenol into coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin
and/or
vanillic acid;
b) coniferyl alcohol into coniferyl aldehyde, ferulic acid, vanillin and/or
vanillic acid;
c) coniferyl aldehyde into ferulic acid, vanillin and/or vanillic acid;
to d) ferulic acid into vanillin and/or vanillic acid, and
e) vanillin into vanillic acid.
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Materials and Methods
Conditions for growing the bacteria.
Strains of Escherichia coli were propagated at 37°C in Luria-Bertani
(LB) or M9
mineral medium (J. Sambrook, E. F. Fritsch and T. Maniatis. 1989. Molecular
cloning: a laboratory manual. 2nd Edition., Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, New York). Strains of Pseudomonas sp. were propagated at
30°C in Nutrient Broth (NB, 0.8%, wt/vol) or in mineral medium (MM) (H.
G.
Schlegel, et al. 1961. Arch. Mikrobiol. 38:209-222) or HR mineral medium (HR-
1o MM) (J. Rabenhorst, 1996. Appl. Microbiol. Biotechnol. 46:470-474.).
Ferulic acid,
vanillin, vanillic acid and protocatechuic acid were dissolved in dimethyl
sulphoxide
and added to the respective medium to give a final concentration of 0.1%
(wt/vol).
Eugenol was either added directly to the medium to give a final concentration
of
0.1% (vol/vol) or applied to filter paper (circular filter 595, Schleicher &
Schuell,
Dassel, Germany) in the lids of MM agar plates. When transconjugants and
mutants
of Pseudomonas sp. were being propagated, tetracycline, kanamycin and
gentamycin
were employed in final concentrations of 25 pg/ml, 100 p.~ml and 7.5 p.g/ml,
respectively.
2o Qualitative and quantitative detection of metabolic intermediates in
culture
supernatants.
Culture supernatants were analysed by high pressure liquid chromatography
(Knauer
HPLC) either directly or after dilution with doubly distilled H20. The
chromatography was carried out on Nucleosil 100 C18 (7 Vim, 250 x 4 mm). 0.1%
(vol/vol) formic acid and acetonitrile was used as the solvent. The course of
the
gradient employed for eluting the substances was as follows:
00:00 - 06:30 -~ 26% acetonitrile
06:30 - 08:00 -~ 100% acetonitrile
08:00 - 12:00 -~ 100% acetonitrile
12:00 - 13:00 -~ 26% acetonitrile
13:00 - 18:00 -~ 26% acetonitrile
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Purification of vanillin dehydrogenase II.
The purification was carried out at 4°C.
Crude extract.
Pseudomonas sp. HR199 cells which had been propagated on eugenol were washed
in 10 mM sodium phosphate buffer, pH 6.0, then resuspended in the same buffer
and
disrupted by being passed twice through a French press (Amicon, Silver Spring,
Maryland, USA) at a pressure of 1000 psi. The cell homogenate was subjected to
an
1o ultracentrifugation (1 h, 100,000 x g, 4°C), resulting in the
soluble fraction of crude
extract being obtained as the supernatant.
Anion exchange chromatography on DEAE Sephacel.
The soluble fraction of the crude extract was dialysed overnight against 10 mM
sodium phosphate buffer, pH 6Ø The dialysate was loaded onto a DEAE-Sephacel
column (2.6 cm x 35 cm, bed volume[BV]: 186 ml) which had been equilibrated
with 10 mM sodium phosphate buffer, pH 6.0, and which had a flow rate of
0.8 ml/min. The column was rinsed with two BV of 10 mM sodium phosphate
buffer, pH 6Ø The vanillin dehydrogenase II (VDH II) was eluted with a
linear salt
2o gradient of from 0 to 400 mM NaCI in 10 mM sodium phosphate buffer, pH 6.0
(750
ml). 10 ml fractions were collected. Fractions having a high VDH II activity
were
combined to form the DEAE pool.
Determining the vanillin dehydrogenase activity.
The VDH activity was determined at 30°C using an optical enzymic
test. The
reaction mixture, whose volume was 1 ml, contained 0.1 mmol of potassium
phosphate (pH 7.1), 0.125 ~.mol of vanillin, 0.5 p.mol of NAD, 1.2 ~Cmol of
pyruvate
(Na salt), lactate dehydrogenase (1 U; from pig heart) and enzyme solution.
The
oxidation of vanillin was monitored at ~, = 340 nm (~~anil~in = 11.6
cm2/~mol). The
3o enzyme activity was given in units (U), with 1 U corresponding to the
quantity of
enzyme which converts 1 p.mol of vanillin per minute. The protein
concentrations in
CA 02348962 2001-04-27
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the samples were determined using the method of Lowry et al. (O. H. Lowry, N.
J.
Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem. 193:265-275).
Determining the coniferyl alcohol dehydrogenase activity.
The CADH activity was determined at 30°C using an optical enzymic
test in
accordance with Jaeger et al. (E. L. Jaeger, Eggeling and H. Sahm. 1981.
Current
Microbiology. 6:333-336). The reaction mixture, whose volume was 1 ml,
contained
0.2 mmol of tris/HCl (pH 9.0), 0.4 ~mol of coniferyl alcohol, 2 ~Cmol of NAD,
0.1 mmol of semicarbazide and enzyme solution. The reduction of NAD was
to monitored at ~. = 340 nm (~ = 6.3 cm2/~mol). The enzyme activity was given
units
(U), with 1 U corresponding to the quantity of enzyme which converts 1 ~,mol
of
substrate per minute. The protein concentrations in the samples were
determined by
the method of Lowry et al. (O. H. Lowry, N. J. Rosebrough, A. L. Farr and R.
J. Randall. 1951. J. Biol. Chem. 193:265-275).
Determining the coniferyl aldehyde dehydrogenase activity.
The CALDH activity was determined at 30°C using an optical enzymic
test. The
reaction mixture, whose volume was 1 ml, contained 0.1 mmol of tris/HCl (pH
8.8),
0.08 ~mol of coniferyl aldehyde, 2.7 ~,mol of NAD and enzyme solution. The
oxidation of coniferyl aldehyde to ferulic acid was monitored at ~. = 400 nm
(E =
34 cm2/p,mol). The enzymic activity was given in units (U) with 1 U
corresponding
to the quantity of enzyme which converts 1 ~mol of substrate per minute. The
protein
concentrations in the samples were determined by the method of Lowry et al.
(O. H.
Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem.
193:265-275).
Determining the feruloyl-CoA synthetase (ferulic acid thiokinase) activity.
The FCS activity was determined at 30°C using an optical enzymic test
which was a
modification of that of Zenk et al. (Zenk et al. 1980. Anal. Biochem. 101:182-
187).
3o The reaction mixture, whose volume was 1 ml, contained 0.09 mmol of
potassium
phosphate (pH 7.0), 2.1 ~,mol of MgCl2, 0.7 ~mol of ferulic acid, 2 ~mol of
ATP,
CA 02348962 2001-04-27
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0.4 ~mol of coenzyme A and enzyme solution. The formation of the CoA ester
from
ferulic acid was monitored at ~, = 345 nm (~ = 10 cm2/~Cmol). The enzymic
activity
was given in units (U), with 1 U corresponding to the quantity of enzyme which
converts 1 ~.mol of substrate per minute. The protein concentrations in the
samples
were determined using the method of Lowry et al. (O. H. Lowry, N. J.
Rosebrough,
A. L. Farr and R. J. Randall. 1951. J. Biol. Chem. 193:265-275).
Electrophoretic methods.
Protein-containing extracts were fractionated under native conditions in 7.4%
(wt/vol) polyacrylamide gels using the method of Stegemann et al. (Stegemann
et al.
1973. Z. Naturforsch. 28c:722-732) and under denaturing conditions in
11.5°Io
(wt/vol) polyacrylamide gels using the method of Laemmli (Laemmli, U. K. 1970.
Nature (London) 227:680-685). Serva Blue R was used for non-specific protein
staining. For specifically staining the coniferyl alcohol dehydrogenase,
coniferyl
aldehyde dehydrogenase and vanillin dehydrogenase, the gels were rebuffered
for
min in 100 mM KP buffer (pH 7.0) and subequently incubated at 30°C in
the
same buffer to which 0.08% (wtlvol) NAD, 0.04% (wtlvol) p-nitro blue
tetrazolium
chloride, 0.003% (wt/vol) phenazine methosulphate and 1 mM of the respective
substrate had been added until corresponding colour bands became visible.
- 20
Transfer of proteins from polyacrylamide gels to PVDF membranes.
Proteins were transferred from SDS-polyacrylamide gels to PVDF membranes
(Waters-Millipore, Bedford, Mass., USA) using a Semidry Fastblot appliance
(B32/33, Biometra, Gottingen, Germany) in accordance with the manufacturer's
instructions.
Determining N-terminal amino acid sequences.
N-terminal amino acid sequences were determined using a Protein Peptide
Sequencer
(Type 477 A, Applied Biosystems, Foster City, USA) and a PTH analyser in
3o accordance with the manufacturer's instructions.
CA 02348962 2001-04-27
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Isolating and manipulating DNA
Genomic DNA was isolated using the method of Marmur (J. Marmur, 1961. J. Mol.
Biol. 3:208-218). Other plasmid DNA and/or DNA restriction fragments was/were
isolated and analysed using standard methods (J. E. Sambrook, F. Fritsch and
T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition., Cold
Spring
Harbor Laboratory Press, Cold Spring Habor, New York).
Transferring DNA.
Competent Escherichia coli cells were prepared and transformed using the
method of
1o Hanahan (D. Hanahan, 1983. J. Mol. Biol. 166:557-580). Conjugative plasmid
transfer between plasmid-harbouring Escherichia coli S 17-1 strains (donor)
and
Pseudomonas sp.strains (recipient) was performed on NB agar plates in
accordance
with the method of Friedrich et al. (B. Friedrich et al. 1981. J. Bacteriol.
147:198-
205), or by means of a "minicomplementation method" on MM agar plates
containing 0.5% (wt/vol) gluconate as the C source and 25 p,g of
tetracycline/ml or
100 ~.g of kanamycin/ml. In this case, cells of the recipient were applied in
one
direction as an inoculation streak. After 5 min, cells of the donor strains
were then
applied as inoculation streaks, with these streaks crossing the recipient
inoculation
streak. After incubating at 30°C for 48 h, the transconjugants grew
directly
2o downstream of the crossing site whereas neither the donor strain nor the
recipient
strain was able to grow.
Hybridization experiments.
DNA restriction fragments were fractionated electrophoretically in a 0.8%
(wt/vol)
agarose gel in 50 mM tris- 50 mM boric acid- 1.25 mM EDTA buffer (pH 8.5) (J.
E.
Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory
manual.
2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York.).
The transfer of the denatured DNA out of the gel onto a positively charged
nylon
membrane (pore size: 0.45 ~,m, Pall Filtrationstechnik, Dreieich, Germany),
the
3o subsequent hybridization with biotinylated or digoxigenin-labelled DNA
probes, and
the preparation of these DNA probes, were all performed using standard methods
CA 02348962 2001-04-27
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(J. E. Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a
laboratory
manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York).
DNA sequencing.
Nucleotide sequences were determined "non-radioactively" in accordance with
the
Sanger et al. (Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 74:5463-5467)
dideoxy
chain termination method using a "LI-COR" DNA Sequencer Model 4000L"
~y (LI-COR Inc., Biotechnology Division, Lincoln, NE, USA) and using a "thermo
1o sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-
dGTP"
(Amersham Life Science, Amersham International plc., Little Chalfont,
Buckinghamshire, England), in each case in accordance with the manufacturer's
instructions.
Synthetic oligonucleotides were used to carry out sequencing in accordance
with the
"primer-hopping strategy" of Strauss et al. (E. C. Strauss et al. 1986. Anal.
Biochem.
154:353-360).
Chemicals, biochemicals and enzymes.
2o Restriction enzymes, T4 DNA ligase, lambda DNA and enzymes and substrates
for
the optical enzymic tests were obtained from C.F. Boehringer & Sohne
(Mannheim,
Germany) or from GIBCO/BRL (Eggenstein, Germany). [y-32P]ATP was from
Amersham/Buchler (Braunschweig, Germany). Oligonucleotides were obtained from
MWG-Biotech GmbH (Ebersberg, Germany). Type NA agarose was obtained from
Pharmacia-LKB (Uppsala, Sweden). All other chemicals were from Haarmann &
Reimer (Holzminden, Germany), E. Merck AG (Darmstadt, Germany), Fluka
Chemie (Bucks, Switzerland), Serva Feinbiochemica (Heidelberg, Germany) or
Sigma Chemie (Deisenhofen, Germany).
CA 02348962 2001-04-27
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Examples
Example 1
Constructing omega elements which mediate resistances to kanamycin (S2 Km)
or gentamycin (S2Gm).
For constructing the S2,Km element, the 2099 by BgII fragment of Transposons
Tn5
(E. A. Auerswald, G. Ludwig and H. Schaller. 1981. Cold Spring Harb. Symp.
Quant. Biol. 45:107-113; E. Beck, G. Ludwig, E. A. Auerswald, B. Reiss and H.
Schaller. 1982. Genes 19:327-336; P. Mazodier, P. Cossart, E. Giraud and F.
Gasser.
1985. Nucleic Acids Res. 13:195-205) was isolated on a preparative scale. The
fragment was shortened down to approx. 990 by by treating it with Bal 31
nuclease.
This fragment, which now only comprised the kanamycin resistance gene
(encoding
an aminoglycoside-3'-O-phosphotransferase), was then ligated to SmaI-cut
pSKsym
DNA (pBluescript SK derivative which contains a symmetrically constructed
multiple cloning site [SaII, HindI>Z, EcoRI, SmaI, EcoRI, HindIlI, SaII]). It
was
possible to reisolate the S2Km element from the resulting plasmid as a SmaI
fragment, an EcoRI fragment, a HindIlZ fragment or a SaII fragment.
2o For constructing the S2Gm element, the 983 by EaeI fragment of the plasmid
pBRIMCS-5 (M. E. Kovach, P. H. Elzer, D. S. Hill, G. T. Robertson, M. A.
Farris,
R. M. Roop and K. M. Peterson. 1995. Genes 166:175-176) was isolated on a
preparative scale and then treated with mung bean nuclease (progressive
digestion of
single-stranded DNA molecule ends). This fragment, which now only comprised
the
gentamycin resistance gene (encoding a gentamycin-3-acetyltransferase), was
then
ligated to SmaI-cleaved pSKsym DNA (see above). It was possible to reisolate
the
52Gm element from the resulting plasmid as a SmaI fragment, an EcoRI fragment,
a
HindIll fragment or a SaII fragment.
CA 02348962 2001-04-27
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Example 2
Cloning the genes from Pseudomonas sp. HR199 (DSM7063) which were to be
inactivated by inserting S2 elements or by means of deletions.
The fcs, ech, vdh and aat genes were cloned separately proceeding from the E.
coli
S 17-1 strains DSM 10439 and DSM 10440 and using the plasmids pE207 and pE5-1
(see EP-A 0845532). The given fragments were isolated on a preparative scale
from
these plasmids and treated as described below:
to For cloning the fcs gene, the 2350 by SalIlEcoRI fragment from plasmid
pE207 and
the 3700 by EcoRIlSaII fragment from plasmid pE5-1 were cloned together in
pBluescript SK such that the two fragments were joined together by way of the
EcoRI ends. The 6050 by SuII fragment was isolated on a preparative scale from
the
resulting hybrid plasmid and shortened down to approx. 2480 by by being
treated
with Bal 31 nuclease. PstI linkers were subsequently ligated to the ends of
the
fragment and, after digestion with PstI, the fragment was cloned into
pBluescript SK
(pSKfcs). After transformation of E. coli XL1 blue, clones were obtained which
expressed the fcs gene and exhibited an FCS activity of 0.2 U/mg of protein.
2o For cloning the ech gene, the 3800 by Hind>ZIlEcoRI fragment from plasmid
pE207
was isolated on a preparative scale and shortened down to approx. 1470 by by
treating it with Bal 31 nuclease. EcoRI linkers were then ligated to the ends
of the
fragment and, after digestion with EcoRI, the fragment was cloned into
pBluescript
SK (pSKech).
For cloning the vdh gene, the 2350 by SalIlEcoRI fragment from plasmid pE207
was
isolated on a preparative scale. After cloning into pBluescript SK , the
fragment was
truncated at one end by approx. 1530 by using an exonuclease III/mung bean
nuclease system. An EcoRI linker was then ligated to the end of the fragment
and,
3o after digestion with EcoRI, the fragment was cloned into pBluescript SK
(pSKvdh).
CA 02348962 2001-04-27
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Following transformation of E. coli XL1 blue, clones were obtained which
expressed
the VDH gene and exhibited a VDH activity of 0.01 U/mg of protein.
For cloning the aat gene, the 3700 by EcoRIlSaII fragment from plasmid pE5-1
was
isolated on a preparative scale and shortened down to approx. 1590 by by
treating it
with Bal 31 nuclease. EcoRI linkers were then ligated to the ends of the
fragment
and, after digestion with EcoRI, the fragment was cloned into pBluescript SK
(pSKaat).
1o Example 3
Inactivating the above-described genes by inserting S2 elements or by deleting
constituent regions of these genes.
Plasmid pSKfcs, which contained the fcs gene, was digested with BssHII,
resulting in
~5 a 1290 by fragment being excised from the fcs gene. Following religation,
the
deletion derivative of the fcs gene (fcs0) (see Figs. li and 2i) was obtained
in cloned
form in pBluescript SK (pSKfcsO). In addition, after the fragment had been
excised,
the omega elements S2Km and S2Gm were ligated in in its stead. This resulted
in the
S2-inactivated derivatives of the fcs gene (fcsS2Km, see Figs. lg and 2g) and
20 (fcsS2Gm, see Fig. lh and 2h) being obtained in cloned form in pBluescript
SK
(pSKfcsS2Km and pSKfcsS2Gm). It was not possible to detect any FCS activity in
crude extracts of the resulting E. coli clones, whose hybrid plasmids
possessed an fcs
gene which was inactivated by deletion or by SZ element insertion.
25 Plasmid pSKech, which contained the ech gene, was digested with NruI,
resulting in
a 53 by fragment and a 430 by fragment being excised from the ech gene. After
religation, the deletion derivative of the ech gene (echo, see Fig. 11 and 21)
was
obtained in cloned form in pBluescript SK (pSKechO). In addition, after the
fragments had been excised, the omega elements S2Km and S2Gm were ligated in
in
3o their stead. This resulted in the SZ-inactivated derivatives of the ech
gene (echS2Km
CA 02348962 2001-04-27
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and echS2Gm) being obtained in cloned form in pBluescript SK (pSKechSZKm and
pSKechS2Gm).
Plasmid pSKvdh, which contained the vdh gene, was digested with BssHII,
resulting
in a 210 by fragment being excised from the vdh gene. After religation, the
deletion
derivative of the vdh gene (vdh0, see Figs. to and 20) was obtained in cloned
form in
pBluescript SK (pSKvdhO). In addition, after the fragment had been excised,
the
omega elements S2Km and S2Gm were ligated in in its stead. This resulted in
the S2-
inactivated derivatives of the vdh gene (vdhS2,Km and vdhSZGm) being obtained
in
to cloned form in pBluescript SK (pSKvdh52Km, see Figs. lm and 2m) and
(pSKvdhS2Gm, see Figs. In and 2n). It was not possible to detect any VDH
activity
in crude extracts of the resulting E. coli clones, whose hybrid plasmids
possessed a
vdh gene which was inactivated by deletion or by SZ element insertion.
Plasmid pSKaat, which contained the aat gene, was digested with BssHII,
resulting
in a 59 by fragment being excised from the aat gene. After religation, the
deletion
derivative of the aat gene (aat0, see Figs. lr and 2r) was obtained in cloned
form in
pBluescript SK (pSKaatO). In addition, after the fragment had been excised,
the
omega elements SZKm and S2Gm were ligated in in its stead. This resulted in
the SZ-
inactivated derivatives of the aat gene (aatS2Km, see Figs. lp and 2p) and
(aatS2Gm,
see Figs. lq and 2q) being obtained in cloned form in pBluescript SK
(pSKaatS2Km
and pSKaat52Gm).
CA 02348962 2001-04-27
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Example 4
Subcloning the S2 element-inactivated genes into the conjugatively
transferable
"suicide plasmid" pSUP202.
In order to be able to replace the intact genes in Pseudomonas sp. HR199 with
the S2-
element inactivated genes, there is a need for a vector which can, on the one
hand, be
transferred into pseudomonads (conjugatively transferable plasmids) but which,
on
the other hand, cannot replicate in these bacteria and is consequently
unstable in
~, pseudomonads ("suicide plasmid"). DNA segments which are transferred into
1o pseudomonads using such a plasmid system can only be retained if they are
integrated by means of homologous recombination (RecA-dependent recombination)
into the genome of the bacterial cell. In the present case, the "suicide
plasmid"
pSUP202 (Simon et al. 1983. In: A. Piihler. Molecular genetics of the bacteria-
plant
interaction. Springer Verlag, Berlin, Heidelberg, New York, pp. 98-106) was
used.
Following digestion with PstI, the inactivated genes fcsS2Km and fcsS2Gm were
isolated from plasmids pSKfcs52,Km and pSKfcsS2Gm and ligated to PstI-cleaved
pSUP202 DNA. The ligation mixtures were transformed into E. coli S 17-1.
Selection
took place on tetracycline-containing LB medium which also contained kanamycin
or
"- 20 gentamycin, respectively. Kanamycin-resistant transformants whose hybrid
plasmid
(pSUPfcsS2Km) contained the inactivated gene fcsS2Km were obtained. The
corresponding hybrid plasmid (pSUPfcsSZGm) of the gentamycin-resistant
transformants contained the inactivated gene fcsS2Gm.
Following EcoRI digestion, the inactivated genes echS2Km and echS2Gm were
isolated from plasmids pSKechSZKm and pSKechS2Gm and ligated to EcoRI-cleaved
pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1.
Selection
took place on tetracycline-containing LB medium which also contained kanamycin
or
gentamycin, respectively. Kanamycin-resistant transformants whose hybrid
plasmid
(pSUPechS2Km) contained the inactivated gene echS2Km were obtained. The
CA 02348962 2001-04-27
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corresponding hybrid plasmid (pSUPechS2Gm) of the gentamycin-resistant
transformants contained the inactivated gene echS2Gm.
Following EcoRI digestion, the inactivated genes vdhS2Km and vdhSZGm were
isolated from plasmids pSKvdhS2Km and pSKvdhS2Gm and ligated to EcoRI-cleaved
pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1.
Selection
took place on tetracycline-containing LB medium which also contained kanamycin
or
gentamycin, respectively. Kanamycin-resistant transformants whose hybrid
plasmid
(pSUPvdhS2Km) contained the inactivated gene vdhS2,Km were obtained. The
1o corresponding hybrid plasmid (pSUPvdhS2Gm) of the gentamycin-resistant
transformants contained the inactivated gene vdhS2Gm.
Following EcoRI digestion, the inactivated genes aatS2Km and aatS2Gm were
isolated from plasmids pSKaatS2Km and pSKaatS2Gm and ligated to EcoRI-cleaved
pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1.
Selection
took place on tetracycline-containing LB medium which also contained kanamycin
or
gentamycin, respectively. Kanamycin-resistant transformants whose hybrid
plasmid
(pSUPaatSZKm) contained the inactivated gene aat52Km were obtained. The
corresponding hybrid plasmid (pSUPaatS2Gm) of the gentamycin-resistant
2o transformants contained the inactivated gene aatSZGm.
Example 5
Subcloning the deletion-inactivated genes into the conjugatively transferable
"suicide plasmid" PHE55, which possesses the "sacB selection system".
In order to be able to replace the intact genes in Pseudomonas sp. 1-IR199
with the
deletion-inactivated genes, there is a need for a vector which possesses the
properties
which have already been described in the case of pSUP202. Since no possibility
(no
antibiotic resistance) exists of selecting for successful replacement of the
genes in
3o Pseudomonas sp. I-IR199 in the case of deletion-inactivated genes, in
contrast to the
S2 element-inactivated genes, another selection system had to be used. In the
"sacB
CA 02348962 2001-04-27
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selection system", the replacing, deletion-inactivated gene is cloned in a
plasmid
which possesses the sacB gene in addition to an antibiotic resistance gene.
Following
the conjugative transfer of this hybrid plasmid into a pseudomonad, the
plasmid is
integrated by means of homologous recombination at the site in the genome at
which
the intact gene is located (first crossover). This results in a
"heterogenotic" strain
which possesses both an intact gene and a deletion-inactivated gene, with
these genes
being separated from each other by the pHE55 DNA. These strains exhibit the
resistance which is encoded by the vector and also possess an active sacB
gene. The
intention then is that the pHE55 DNA, together with the intact gene, should
then be
to separated out of the genomic DNA by means of a second homologous
recombination
event (second crossover). This recombination event results in a strain which
now
only possesses the inactivated gene. In addition, the pHE55-coded antibiotic
resistance and the sacB gene are both lost. If strains are streaked on sucrose-
containing media, the growth of strains which express the sacB gene is
inhibited
since the gene product converts sucrose into a polymer which is accumulated in
the
periplasm of the cells. The growth of cells which no longer carry the sacB
gene as a
result of the second recombination event having taken place is consequently
not
inhibited. In order to have a possibility of selecting phenotypically for the
integration
of the deletion-inactivated gene, this gene is not exchanged for an intact
gene;
rva 2o instead, use is made of a strain in which the gene to be replaced is
already "labelled"
by the insertion of an S2 element. When successful replacement takes place,
the
resulting strain loses the antibiotic resistance which is encoded by the S2
element.
Following digestion with PstI, the inactivated gene fcs~ was isolated from
plasmid
pSKfcsO and ligated to PstI-cleaved pHE55 DNA. The ligation mixture was
transformed into E. coli S 17-1. Selection took place on tetracycline-
containing LB
medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEfcsO)
contained the inactivated gene fcs0, were obtained.
Following digestion with EcoRI, the inactivated gene echo was isolated from
plasmid pSKechO and treated with mung bean nuclease (generation of blunt
ends).
CA 02348962 2001-04-27
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The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated pHE55
DNA. The ligation mixture was transformed into E. coli S17-1. Selection took
place
on tetracycline-containing LB medium. Tetracycline-resistant transformants,
whose
hybrid plasmid (pHEechO) contained the inactivated gene echo, were obtained
Following digestion with EcoRI, the inactivated gene vdh0 was isolated from
plasmid pSKvdhO and treated with mung bean nuclease. The fragment was ligated
to
BamHI-cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture
was transformed into E. coli S17-1. Selection took place on tetracycline-
containing
LB medium. Tetracycline-resistant transformants, whose hybrid plasmid
(pHEvdhO)
contained the inactivated gene vdh~, were obtained.
Following digestion with EcoRI, the inactivated gene aat0 was isolated from
plasmid
pSKaatO and treated with mung bean nuclease. The fragment was ligated to BamHI-
is cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was
transformed into E. coli S 17-1. Selection took place on tetracycline-
containing LB
medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEaatO)
contained the inactivated gene aat0, were obtained.
CA 02348962 2001-04-27
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Example 6
Generating mutants of the strain Pseudomonas sp. HR199 in which genes of
eugenol catabolism have been specifically inactivated by inserting an S2-
element.
The strain Pseudomonas sp. HR199 was employed as the recipient in conjugation
experiments in which strains of E. coli S 17-1 harbouring the hybrid plasmids
of
pSUP202 which are listed below were used as donors. The transconjugants were
selected on gluconate-containing mineral medium which contained the antibiotic
corresponding to the ~ element. It was possible to distinguish between
"homogenotic" (replacement of the intact gene with the S2 element insertion-
inactivated gene by means of a double crossover) and "heterogenotic"
(integration of
the hybrid plasmid into the genome by means of a single crossover)
transconjugants
on the basis of the pSUP202-encoded tetracycline resistance.
The mutants Pseudomonas sp. HR199 fcsS2Km and Pseudomonas sp. HR199
fcs52Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli S17-
1
(pSUPfcsSZKm) and E. coli S 17-1 (pSUPfcsS2Gm), respectively. The replacement
of
the intact fcs gene with the S2Km-inactivated or S2Gm-inactivated gene
(fcsS2Km and
fcsSZGm, respectively) was verified by means of DNA sequencing.
The mutants Pseudomonas sp. HR 199 echS2Km and Pseudomonas sp. HR 199
echS2Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli
S17-1 (pSUPechSZKm) and E. coli S17-1 (pSUPech52Gm), respectively. The
replacement of the intact ech gene with the SZKm-inactivated or S2Gm-
inactivated
gene (ech52Km and echS2Gm, respectively) was verified by means of DNA
sequencing.
The mutants Pseudomonas sp. HR 199 vdh52Km and Pseudomonas sp. HR 199
vdhS2Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli
3o S17-1 (pSUPvdhS2Km) and E. coli S17-I (pSUPvdhSZGm), respectively. The
CA 02348962 2001-04-27
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replacement of the-intact vdh gene with the SZKm-inactivated or S2,Gm-
inactivated
gene (vdhS2Km and vdhSZGm, respectively) was verified by means of DNA
sequencing.
The mutants Pseudomonas sp. HR199 aatS2Km and Pseudomonas sp. HR199
aatS2Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli
S17-1 (pSUPaatSZKm) and E. coli S17-1 (pSUPaatSZGm), respectively. The
replacement of the intact aat gene with the S2Km-inactivated or S2Gm-
inactivated
gene (aatS2Km and aatS2Gm, respectively) was verified by means of DNA
to sequencing.
The mutant Pseudomonas sp. HR 199 fcsS2KmvdhS2Gm was obtained after
conjugating Pseudomonas sp. HR199 fcsS2Km with E. coli S17-1 (pSUPvdhS2Gm).
The replacement of the intact vdh gene with the 52Gm-inactivated gene
(vdhS2Gm)
was verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR199 vdh52KmaatS2Gm was obtained after
conjugating Pseudomonas sp. HR199 vdhS2Km with E. coli S17-1 (pSUPaatS2Gm).
The replacement of the intact aat gene with the S2Gm-inactivated gene
(aatS2Gm)
2o was verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR 199 vdhS2KmechS2Gm was obtained after
conjugating Pseudomonas sp. HR199 vdhS2Km with E. coli S17-1 (pSUPechS2Gm).
The replacement of the intact ech gene with the S2Gm-inactivated gene
(echSZGm)
was verified by means of DNA sequencing.
CA 02348962 2001-04-27
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Example 7
Generating of mutants of the strain Pseudomonas sp. HR199 in which genes of
eugenol catabolism have been specifically inactivated by deleting a
constituent
region.
The strains Pseudomonas sp. I-18199 fcs52Km, Pseudomonas sp. HR199 echS2Km,
Pseudomonas sp. HR 199 vdhS2Km and Pseudomonas sp. HR 199 aatS2Km were
employed as recipients in conjugation experiments in which strains of E. coli
S17-1
_,-" harbouring the hybrid plasmids of pHE55 which are listed below were used
as
to donors. The "heterogenotic" transconjugants were selected on gluconate-
containing
mineral medium which also contained the antibiotic corresponding to the 52
element
in addition to tetracycline (pHE55-encoded resistance). After streaking out on
sucrose-containing mineral medium, transconjugants were obtained which had
eliminated the vector DNA by means of a second recombination event (second
crossover). By streaking out on mineral medium which was without antibiotic or
which contained the antibiotic corresponding to the S2 element, it was
possible to
identify the mutants in which the S2 element-inactivated gene had been
replaced with
the deletion-inactivated gene (no antibiotic resistance).
'~'~' 2o The mutant Pseudomonas sp. HR 199 fcs0 was obtained after conjugating
Pseudomonas sp. HR 199 fcsS2,Km with E. coli S 17-1 (pl-lEfcsO). The
replacement of
the S2Km inactivated gene (fcs52Km) with the deletion-inactivated gene (fcs0)
was
verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR 199 echo was obtained after conjugating
Pseudomonas sp. HR199 echSZKm with E. coli S17-1 (pHEechO). The replacement
of the S2Km-inactivated gene (echS2Km) with the deletion-inactivated gene
(echo)
was verified by means of DNA sequencing.
3o The mutant Pseudomonas sp. 1-18199 vdh0 was obtained after conjugating
Pseudomonas sp. T1R199 vdhS2Km with E. coli S17-1 (pHEvdh~). The replacement
CA 02348962 2001-04-27
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of the S2Km-inactivated gene (vdhS2Km) with the deletion-inactivated gene
(vdh0)
was verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR199 aat0 was obtained after conjugating
Pseudomonas sp. HR199 aatS2Km with E. coli S17-1 (pHEaat~). The replacement
of the SZKm-inactivated gene (aatS2Km) with the deletion-inactivated gene
(aat~)
was verified by means of DNA sequencing.
... Example 8
to
Biotransforming eugenol into vanillin using the mutant Pseudomonas sp. HR199
vdh S2Km.
The strain Pseudomonas sp. HR199 vdhS2Km was propagated in 50 ml of HR-MM
containing 6 mM eugenol up to an optical density of approx. OD600nm = 0.6.
After
17 h, it was possible to detect 2.9 mM vanillin, 1.4 mM ferulic acid and 0.4
mM
vanillic acid in the culture supernatant.
Example 9
2o Biotransforming eugenol into ferulic acid using the mutant Pseudomonas sp.
HR199 vdhSZGmaatS2Km.
The strain Pseudomonas sp. I-IR199 vdhSZGmaatS2Km was propagated in 50 ml of
HR-MM containing 6 mM eugenol up to an optical density of approx.OD600nm =
0.6. After 18 h, it was possible to detect 1.9 mM vanillin, 2.4 mM ferulic
acid and
0.6 mM vanillic acid in the culture supernatant.
CA 02348962 2001-04-27
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Example 10
Biotransforming eugenol into coniferyl alcohol using the mutant Pseudomonas
sp. HR199 vdhS2GmaatSZKm.
The strain Pseudomonas sp. HR199 vdhS2GmaatS2,Km was propagated in 50 ml of
HR-MM containing 6 mM eugenol up to an optical density of approx. OD600nm =
0.4. After 15 h, it was possible to detect 1.7 mM coniferyl alcohol, 1.4 mM
vanillin,
1.4 mM ferulic acid and 0.2 mM vanillic acid in the culture supernatant.
to Example 11
Fermentatively producing natural vanillin from eugenol in a 10 1 fermenter
using mutant Pseudomonas sp. HR 199 vdhS2Km.
The production fermenter was inoculated with 100 ml of a 24-hour-old
preliminary
culture which had been propagated at 32°C on a shaking incubator ( 120
rpm) in a
medium which was adjusted to pH 7.0 and which consisted of 12.5 g of
glycerol/1,
10 g of yeast extract/1 and 0.37 g of acetic acid/l. The fermenter contained
9.9 1 of
medium of the following composition: 1.5 g of yeast extract/l, 1.6 g of
KH2P04/1, 0.2
g of NaCI/1, 0.2 g of MgS04/1. The pH was adjusted to pH 7.0 with sodium
2o hydroxide solution. After sterilization, 4 g of eugenol were added to the
medium. The
temperature was 32°C, the aeration was 3 Nl/min and the stirrer speed
was 600 rpm.
The pH was maintained at pH 6.5 with sodium hydroxide solution.
At 4 hours after the inoculation, continuous addition of eugenol was begun
such that
255 g of eugenol had been added to the culture when fermentation ended after
65
hours. 40 g of yeast extract were also fed in during the fermentation. At the
end of the
fermentation, the concentration of eugenol was 0.2 g/1. The content of
vanillin was
2.6 g/l. 3.4 g of ferulic acid/1 were also present.
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The vanillin which is obtained in this way can be isolated by known physical
methods such as chromatography, distillation and/or extraction and used for
preparing natural flavourings.
E~lanatory notes regardin tgy he fi ug res:
FIG. la to lr:
y Gene struktures for isolating organisms and mutants
to
calA*: Part of the inactivated gene for coniferyl alcohol dehydrogenase
calB*: Part of the inactivated gene for coniferyl aldehyde dehydrogenase
fcs*: Part of the inactivated gene for feruloyl-CoA synthetase
ech*: Part of the inactivated gene for enoyl-CoA hydratase-aldolase
vdh*: Part of the inactivated gene for vanillin dehydrogenase
aat*: Part of the inactivated gene for beta-ketothiolase
While the restriction enzyme cleavage sites labelled "*" were used for the
construction, they are no longer functional in the resulting construct.
CA 02348962 2001-04-27
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FIG 2a~ Nucleotideuence of the gene structure
seq calA52Km
FIG 2b' Nucleotideuence of the gene structure:
seg calAS2Gm
FIG. 2c: Nucleotideuence of the
seq calA~ gene structure
FIG 2d' Nucleotideuence of the gene structure
sea calBS2Km
FIG 2e' Nucleotideuence of the gene structure
seq caIBSZGm
FIG 2f~ Nucleotideuence of the
sea calB~ gene structure
FIG 2g' Nucleotidequence of the ene structure
se fcsSZKm g
FIG 2h' Nucleotidequence of the ene structure
se fcsS2Gm g
FIG 2i: Nucleotide sequence of the fcs~ gene structure
to FIG. 2j: Nucleotide sequence of the echSZKm gene structure
FIG. Zk: Nucleotide uence of the echS2Gm gene
seq structure
FIG. 21: Nucleotide uence of the echo glene
sea structure
FIG. 2m: Nucleotide quence of the vdhSZKm
se gene structure
FIG. 2n: Nucleotide uence of the vdhS2Gm gene
seq structure
15 FIG. 20: Nucleotideuence of the vdh0 gene
seq structure
FIG 2y Nucleotide se quence of the aatS2Km
gene structure
FIG 2cL Nucleotide uence of the aatS2Gm gene
seq structure
FIG. 2r: Nucleotide uence of the aat0 gene
seq structure
