Hydroxylation of methyl groups in aromatic heterocycles by microorganisms

A process using microorganisms which contain genes, which form an active xylene monooxygenase, which form no effective, chromosomally or plasmid-coded alcohol hydrogenase, and which are, thus, capable of hydroxylating methyl groups on aromatic 5- or 6-atom heterocycles to the corresponding hydroxymethyl derivatives, for the production of hydroxymethylated 5- or 6-atom heterocycles.

BACKGROUND OF THE INVENTION 
1. Field Of The Invention 
The invention relates to a new microbiological process for hydroxylating 
methyl groups in aromatic 5- or 6-atom heterocycles as well as to new 
hybrid plasmids and new production strains especially suited for the 
process. 
2. Prior Art 
A microbiological process for the terminal hydroxylation of aliphatic side 
chains by genetically changed microorganisms is known from European 
Published Patent Application No. 0277674. This reaction is catalyzed by 
the alkane hydroxylase, coded by genes alkBA from the OCT-plasmid of 
Pseudomonas oleovorans. These microorganisms were changed genetically so 
that they are no longer capable of further oxidizing the resulting 
hydroxyl groups to the acid. But the natural expression and regulation 
(alkR) of these genes were maintained. These microorganisms have no 
activity for the oxidation of methyl groups in heterocycles, but catalyze 
only the hydroxylation of alkanes and alkylated compounds with alkyl 
radicals with 6 to 12 carbon atoms. 
Further, it is known from Harayama et al., J. Bacteriol. 171, (1989), pages 
5048 to 5055, that microorganisms of the species Pseudomonas putida with 
plasmid pWWO can oxidize the methyl group on toluene in three steps to 
benzoic acid. By the action of xylene monooxygenase (xylMA), benzyl 
alcohol first results, which in two further steps is then catalyzed by an 
alcohol dehydrogenase (xylB) and converted by an aldehyde dehydrogenase 
(xylC) to the acid. Both the xyl genes, which code for the enzymes of the 
xylene catabolism, and the genes which are responsible for the regulation 
of the xyl genes on plasmid pWWO, are in this strain. Thus, the 
properties, the identification, the cloning, the selection and the 
restriction map of the genes xylMABCN responsible for the oxidation of the 
methyl group are known from it. The function of gene xylN is still 
unknown. But no microbiological process is known which can hydroxylate 
methyl groups in aromatic 5- or 6-atom heterocycles. Moreover, those 
specifically hydroxymethylated heterocycles are chemically difficult to 
obtain. 
BROAD DESCRIPTION OF THE INVENTION 
The main object of the invention is to provide a microbiological process 
for specific hydroxylation of methyl groups in aromatic 5- or 6-atom 
heterocycles to the correspondingly pure hydroxymethylated derivatives, 
and the products must not be further catabolized. Other objects and 
advantages of the invention are set out herein or are obvious herefrom to 
one skilled in the art. 
The objects and advantages of the invention are achieved by the process, 
hybrid plasmids and production strains of the invention. 
The invention involves a microbiological process of hydroxylating the 
methyl group or groups in a 5- and 6-atom aromatic heterocycle. The 
invention process is performed with microorganisms, which: 
(a) contain the genes of a Pseudomonas TOL plasmid, which form an active 
xylene monooxygenase, and 
(b) form no effective chromosomally or plasmid coded alcohol dehydrogenase, 
and 
thus, are capable of hydroxylating methyl groups of aromatic 5- or 6-atom 
heterocycles to the corresponding hydroxymethyl derivative, and the 
heterocycle is used as substrate for the reaction and exhibits no 
substituents on the carbon atom adjacent to the methyl group to be 
hydroxylated and the hydroxymethyl derivative is not further catabolized. 
The hydroxymethylated heterocycles produced by the invention process are, 
for example, important intermediate products for the production of 
pharmaceutical agents and agricultural chemicals.

DETAILED DESCRIPTION OF THE INVENTION 
The microorganisms used in the invention process suitably contain the genes 
for forming a xylene monooxygenase of Pseudomonas TOL plasmid pWWO of the 
FIGURE, which are characterized by the following restriction map, and have 
already been described in J. Bacteriol., 171, (1989), pages 5048 to 5055: 
Source of the xylene monooxygenase genes 
As a source for the xylene monooxygenase genes, Pseudomonas putida can be 
used with the TOL plasmid pWWO, which, e.g., can be obtained under ATCC 
33015 in the American Type Culture Collection. 
The genetic data, which is the code for the xylene monooxygenase, can then 
thus be obtained by (a) the TOL plasmid DNA being isolated from this 
microorganism, which is used as a source for the DNA, then (b) this TOL 
plasmid DNA being digested to isolate the gene for the xylene 
monooxygenase and the specific gene sequence, then (c) being introduced in 
an expression vector, and as a result, (d) a hybrid plasmid resulting. 
This hybrid plasmid can then be introduced in a microorganism (e) (host 
strain), suitable for the process, by transformation (f). This transformed 
host strain then forms production strain (g) [after selection (h)] for 
fermentation process (i) according to the invention. 
(a) Isolation of the TOL plasmid DNA 
The TOL plasmid DNA can be obtained according to methods usual and known to 
one skilled in the art, such as, according to the method of Hansen and 
Olsen [J. Bacteriol., 135, (1978), pages 227 to 238] or Humphreys et al. 
[Biochim. Biophys. Acta, 383, (1975), pages 457 to 463]. The method of 
Humphreys et al. [Biochim. Biophys. Acta, 383, (1975), pages 457-463] is 
suitably used for the isolation of large amounts of TOL plasmid DNA, by 
Pseudomonas putida (ATCC 33015) being completely lysed and then the TOL 
plasmid being isolated by density gradient centrifuging. 
(b) Cleavage with restriction enzymes and isolation of DNA by agarose gel 
electrophoresis. 
After isolation of the TOL plasmid DNA, the TOL plasmid DNA is suitably 
cleaved with restriction enzymes SalI and HindIII, and then the DNA 
section, which is the code for the xylene monooxygenase, can be isolated 
by agarose gel electrophoresis, according to Current Protocols in 
Molecular Biology, John Wiley and Sons, New York, 1987), section 2.6, 
"Isolation and Purification of Large DNA-Restriction Fragments from 
Agarose Gels." 
This DNA section is characterized, as already described before, by the 
following restriction map of the FIGURE and contains no genes which are 
coded for an effective alcohol dehydrogenase: 
(c) Ligation of the DNA section in expression vectors 
The thus-obtained gene section can be ligated to a hybrid plasmid by the 
usual and known molecular biological techniques with a previously equally 
cut expression vector DNA. Expression vectors usually contain a suitable, 
mostly adjustable promoter. One or more singular cutting sites for 
restriction enzymes advantageously lie behind this promoter in the 
transcription direction. Then, the desired gene section, in whose 
expression there is interest, is usually inserted in these cutting sites. 
Listed in Table 1 are suitable expression vectors. For the process 
according to the invention, expression vectors with a broad host range, 
such as, pME285, pKT240, pMMB67EH or pMMB67EH*, are suitably used. 
These expression vectors with restriction enzymes SalI and HindIII are 
suitably cut, and the resulting restriction ends with the isolated TOL 
plasmid DNA are then ligated by, for example, T4 DNA ligase. Also, 
optionally, other methods for ligation can be used, such as, those which 
are described in Current Protocols in Molecular Biology, John Wiley and 
Sons, New York, (1989), section 3.16,"Subcloning Of DNA Fragments". 
(d) Hybrid plasmids 
Hybrid plasmids pL03, pL04 and pL05, suitably thus resulting, are also a 
component of the invention, exhibit a broad host range and can 
consequently be used in host strains with high substrate and feedstock 
tolerance. These hybrid plasmids are suitably decoupled from the natural 
regulation system. Consequently, hybrid plasmid pL04 (consisting of 
expression vector pMMB67EH and the TOL plasmid gene) is characterized by 
the above-described restriction map with promoter P.sub.tac controlled by 
repressor gene lacIo. The expression of the TOL plasmid genes can 
consequently be induced with isopropyl thiogalactoside (IPTG). 
The expression of the TOL plasmid genes in hybrid plasmid pL05 [consisting 
of expression vector pMMB67EH* and the TOL plasmid gene characterized by 
the restriction map set out above and noted as previously being described 
in J. Bacteriol., 171, (1989), pages 5048 to 5055], with promoter 
P.sub.tac, is permanently (constitutively) induced because of the missing 
repressor gene lacIg. 
Repressor gene lacIg in pMMB67EH* is suitably mutated for this purpose by 
introducing a kanamycin resistance. It is also possible to use a hybrid 
plasmid with a narrow host range. Suitably pGSH2836 with promoter lambda 
P.sub.L is used as a hybrid plasmid with a narrower host range, and the 
expression of the TOL plasmid genes is permanently (constitutively) 
induced. If, for example, Escherichia coli (E. coli) K12* is used as a 
host for pGSH2836, repressor gene cI857, integrated chromosomally there, 
has to be deactivated by temperature to achieve an expression of promoter 
lambda P.sub.L. 
Hybrid plasmid pGSH2836 is deposited in E. coli K12* under deposit number 
DSM 6154 in the German Collection for Microorganisms and Cell Cultures 
GmbH, Mascheroderweg lb, D-3300 Braunschweig. Hybrid plasmids pL04 and 
pL05 are deposited in E. coli K12* (pL04) or in Pseudomonas putida (pL05), 
as described in the following sections. 
(e) Host strains 
Because of the broad host range, hybrid plasmids (pL03, pL04, pL05) thus 
resulting can be introduced in a multiplicity of host strains. Host 
strains with high substrate and feedstock tolerance are suitably used, 
such as, those of genus Pseudomonas, Acinetobacter, Rhizobium, 
Aorobacterium or Escherichia. 
(f) Transformation 
The introduction of the hybrid plasmids in the above-described host strains 
can take place according to the usual and known methods, preferably 
according to the method of Lederberg and Cohen [J. Bacteriol., 119, 
(1974), pages 1072 to 1074]. 
(g) Production strains 
As production strains, all of those listed in Table 2 are suitably used. 
The microorganisms transformed with hybrid plasmids pL03, pL04 and pL05 
(Table 2) are new and, thus, also a component of the invention. 
Microorganism E. coli K12* is preferably used, transformed with hybrid 
plasmid pL04, deposited on Aug. 29, 1990, under deposit number DSM 6153, 
or microorganism Pseudomonas putida JD7 is used, transformed with hybrid 
plasmid pL05, deposited on Aug. 29, 1990, under deposit number DSM 6152, 
as well as their descendants and mutants. The two deposits took place at 
the German Collection of Microorganisms and Cell Cultures GnbH, 
Mascheroderweg lb, D-3300 Braunschweig. 
Also, these microorganisms can be used as production strains which contain 
a natural TOL plasmid and in which then, the gene, which is the code for 
an effective alcohol dehydrogenase, is removed or deactivated. The 
deactivation or removal can take place by standard mutation, e.g., with 
acridine orange by a transposon insertion or by the method below of "gene 
replacement" with homologous recombination [A. Zimmermann et al., 
Molecular Microbiology, 5, (1991), pages 1483 to 1490]. 
In these production strains, the expression of the TOL plasmid genes takes 
place, for example, by induction with compounds such as toluene, xylene or 
cymene. 
The removal of the alcohol dehydrogenase gene suitably takes place so that 
a previously produced auxiliary hybrid plasmid, in which the alcohol 
dehydrogenase gene is already removed, is taken up by homologous 
recombination in the natural TOL plasmid ("gene replacement" with 
homologous recombination). Then, the alcohol dehydrogenase is suitably 
removed in the natural TOL plasmid by this taking up. 
(h) Selection of the transformed microorganisms (production strains) 
The transformants can usually be selected on a minimum medium glucose agar 
with corresponding inhibition concentration of suitable antibiotics. The 
antibiotic-resistant markers used are listed in Table 1. 
(i) Fermentation process 
According to the invention, the production strains obtained according to 
the above-described processes, as well as their descendants and mutants, 
are used for the process according to the invention for hydroxylating 
methyl groups in aromatic 5- or 6- atom heterocycles. 
As substrates for the reaction, methylated aromatic 5- or 6-atom 
heterocycles can be used which contain one or more heteroatoms from the 
series oxygen, nitrogen and sulfur. Suitable 5-atom heterocycles are, for 
example, methylated thiophene, methylated furan, methylated pyrrole, 
methylated thiazole, methylated pyrazole and methylated imidazole 
derivatives, all of which have no substituents on the carbon atom adjacent 
to the methyl group to be hydroxylated. Preferably, 3,5-dimethylpyrazole, 
4-methylthiazole and 2,5-dimethyl-thiophene are used as the 5-atom 
heterocycles. 
Suitable 6-atom heterocycles are, for example, methylated pyridine, 
methylated pyrimidine, methylated pyrazine and methylated pyridazine 
derivatives, which have no substituents on the adjacent carbon atom to the 
methyl group to be hydroxylated. Preferably, 2-chloro-5-methylpyridine, 
2,5-dimethylpyrazine and 2,6-dimethylpyrimidine are used as the 6-atom 
heterocycles. 
Before the addition of the substrate, the cells are cultured up to an 
optical density at 650 nm (OD.sub.650) of 1 to 200 in the culture medium, 
preferably up to an optical density of 5 to 100. 
The reaction can take place either under the single or continuous addition 
of the substrate, so that the substrate concentration in the culture 
medium does not exceed 20 percent (w/v) or (v/v) for liquid substrates. 
Preferably, the addition of the substrate takes place so that the 
substrate concentration in the culture medium does not exceed 5 percent 
(w/v) or (v/v). 
The reaction is usually performed with resting cells in a pH range of 4 to 
11, preferably 6 to 10. The reaction is usually performed at a temperature 
of 15.degree. to 50.degree. C., preferably at a temperature of 25.degree. 
to 45.degree. C. 
After the reaction, the corresponding hydroxymethyl derivative can be 
isolated in the known manner. 
EXAMPLE 1 
Cloning of Genes xylMA 
1.1. Plasmid preparation 
[Humphreys et al., Biochim. Biophys. Acta, 383 (1975), pages 457 to 483]. 
The cells of 1 1 of a fully grown bacteria culture, Pseudomonas putida pWWO 
(ATCC 33015), were centrifuged out. After resuspension of the cells in 10 
ml of 25 percent saccharose in 0.05 mol of tris buffer, pH 8.0, .5 ml of 
lysozyme solution (20 mg/ml in 0.25 mol of tris buffer, pH 8.0) was added. 
Then, the mixture was incubated for 5 minutes on ice; 10 ml of 0.25 mol of 
Na.sub.2 EDTA (pH 8) was added and it was further incubated for 5 minutes 
on ice. Then, 15 ml of Brij.RTM. polyoxyethylene lauryl ether/DCL solution 
(1 percent of Brij.RTM. 58, 0.4 percent of sodium deoxycholate in 0.01 mol 
of tris buffer, 0.001 mol of Na.sub.2 EDTA; pH 8.0) was added. Good, 
uniform, thorough mixing followed, then there was incubation on ice for 30 
minutes until complete cell lysis. 
After centrifuging for 45 minutes at 4.degree. C. at 16,000 rpm, the 
supernatant was decanted in an autoclaved measuring cylinder. 3 percent 
(w/v) of NaCl and of 10 percent PEG (polyethylene glycol) was added. By 
careful turning of the cylinder, which was closed with parafilm, a 
solution was produced. It was incubated for 2 hours at 4.degree. C. and 
then centrifuged for 2 minutes at 5,000 rpm. Then, the supernatant was 
decanted and the precipitate was dissolved in 5 ml of TES- buffer (0.05 
mol of TRIS, 0.005 mol of Na.sub.2 EDTA, 0.05 mol of NaCl, pH 8.0); this 
was followed by conversion in autoclaved 15 ml Corex test tubes with 8.0 g 
of calcium chloride. After adding 0.6 ml of ethidium bromide solution (10 
mg/ml), it was incubated for 30 minutes on ice. 
After centrifuging for 30 minutes at 4.degree. C. at 12,000 rpm, it was 
carefully decanted to remove the precipitated PEG from the solution. After 
ultracentrifuging of the solution in closed test tubes with a 50TI-rotor 
at 40,000 rpm for 30 hours at 18.degree. C., the plasmid band was isolated 
from the CsCl.sub.2 gradients with a cannula in front of the UV 
transilluminator. 
The ethidium bromide was removed from the plasmid preparation by shaking 
out with n-butanol. Next, isopropanol precipitation of the plasmid DNA and 
drying of the precipitate in a Speed VaC.RTM. concentrator was followed by 
resuspension of the plasmid preparation in 0.01 mol of tris buffer (0.001 
mol of Na.sub.2 EDTA, pH 8.0). 
1.2 Isolation of DNA fragments xylMA from agarose gels 
The plasmid DNA cut with SalI and HindIII (4 units each per microgram of 
plasmid DNA) was subjected to a preparative agarose gel electrophoresis 
(0.6 percent (w/v) agarose in TBE buffer [0.09 mol of tris-borate, 2.5 
mmol of Na.sub.2 EDTA, pH 8.3, ethidium bromide (100 micrograms/100 ml)]. 
A DEAE cellulose membrane cut into small strips was prepared in water and 
inserted in slots in the agarose gel directly in front of the desired DNA 
fragment band. DNA was allowed to accumulate in the voltage field on the 
membrane. Optionally, higher DNA bands were retained with additional 
membranes. The accumulated DNA was washed off from the membrane with 500 
microliters of elution buffer (20 mmol of TRIS, pH 7.5, 1 mmol of Na.sub.2 
EDTA, 1.5 mol of NaCl) for 1 hour at 65.degree. C. 
The membrane was removed and washed off. Ethidium bromide was extracted 
with H.sub.2 O-saturated n-butanol from the DNA solution. The DNA was 
precipitated with isopropanol. The precipitate was dried in a Speed 
VaC.RTM. concentrator, followed by resuspension of the fragment 
preparation in 0.01 mol of tris buffer, 0.001 mol of Na.sub.2 EDTA, pH 
8.0. 
1.3 Ligation of DNA fragments xylMA with expression vectors 
[Current Protocols in Molecular Biology, John Wiley and Sons, New York, 
(1989), section 3.16, Subcloning Of DNA Fragments] 
(a) Preparation of hybrid plasmid poL04 
Preparation of the expression vector DNA 
Before the ligation, the pMMB67EH vector DNA (2 micrograms) was cut with 10 
units each of SalI and HindIII in the corresponding ligation buffer [20 
mmol of tris buffer, 10 mmol of DTT (dithioerythritol), 10 mmol of 
MgCl.sub.2 and 0.6 mmol of ATP; pH 7.2]. This cut DNA was then 
dephosphorylated with 4.8 units of alkaline phosphatase. The DNA was 
precipitated and washed repeatedly with isopropanol. 
Ligation of the xylMA-DNA with the expression vector-DNA 
For the ligation, the respective DNA samples (in various quantitative 
ratios in excess of the insert-DNA) were added together, subjected to an 
isopropanol precipitation, and the dried precipitates were taken up in 40 
to 100 microliters of ligation buffer (20 mmol of tris buffer, 10 mmol of 
DTT, 10 mmol of MgCl.sub.2 and 0.6 mmol of ATP, pH 7.2). The ligation took 
place after adding 0.2 units of T4-DNA ligase per microgram of DNA 
overnight with incubation at 12.degree. to 16.degree. C. Then, the 
ligation mixture was used directly for transformation. 
(b) Preparation of hybrid plasmid pL05 
Analogously to Example 1.3 (a), expression vector pMMB67EH* was prepared 
and according to Example 1.3 (a), the xylMA genes were then ligated in 
this vector. 
1.4 Transformation of competent cells with hybrid plasmid DNA (pL04) 
a) Concentration of hybrid plasma DNA (pL04) 
The cells of a 25 ml culture of E. coli S17-1 were harvested as an 
auxiliary strain at an OD.sub.546 =2.0 and were made competent according 
to the method of Lederberg and Cohen [J. Bacteriol., 119, (1974), 1072 to 
1074]. After washing these cells in 10 ml of 0.1 mol of MgCl.sub.2, the 
cells were incubated for 30 minutes in 10 ml of 0.1 CaCl.sub.2 on ice. 
These cells in 1 ml of 0.1 mol of CaCl.sub.2 were centrifuged and 
resuspended. 0.2 ml each of the cell suspension was mixed with 0.5 
microgram of ligated hybrid plasmid DNA for transformation. The suspension 
was incubated for more than 30 minutes on ice, that is, 2-minute thermal 
shock at 42.degree. C. 
Then, the respective cell suspensions were filled up to 5 ml with preheated 
nutrient yeast broth (Oxoid, Wesel, FRG), and incubated for 1 hour without 
shaking and another hour with shaking for the expression of the genes at 
optimum growth temperature of the recipient cells (E. coli 517-1). 
Aliquots of the transformed cultures were placed on corresponding 
selective media (nutrient agar, 100 micrograms of ampicillin per ml). 
(b) Transformation of pL04 in the production strain 
Hybrid plasmid pL04 was isolated from the E. coli S17-1-strain with pL04 
corresponding to Examples 1.1 and 1.2. Then, E. Coli K12* was transformed 
according to the method in Example 1.4 (a) with hybrid plasmid pL04. The 
selection took place in accordance with the selective medium (nutrient 
agar, 100 micrograms of ampicillin per ml). 
1.5 Transformation of competent cells with hybrid plasmid pL05 
Corresponding to Example 1.4, hybrid plasmid pL05 was transformed into 
Pseudomonas putida JD7. The selection took place with selective medium 
(nutrient agar, 50 micrograms of kanamycin per ml). 
EXAMPLES 2 TO 8 
Hybrid plasmid pL03 was produced corresponding to Example 1.3. 
Hybrid plasmids pGSH2836, pL03, pL04 and pL05 in host strains E. coli K12*, 
Pseudomonas aeruginosa 25, Pseudomonas putida JD7 and Pseudomonas 
putida were transformed corresponding to Examples 1.4 and 1.5. 
The reaction rates of these production strains are compiled in Table 2. 
EXAMPLE 9 
Construction of the xylB mutants 
9.1 Construction of plasmid pL010 
Genes xylMABCN were isolated from plasmid pGSH2816 [Harayama et al., J. 
Bacteriol., 171, (1989)] by EcoRI and HpaI restriction and then ligated in 
the equally cut vector pBR322 [Bolivar et al., Gene, 2, (1977), p. 95 ff]. 
9.1.1 Cleavage with restriction enzymes and isolation of the DNA by agarose 
gel electrophoresis 
The pGSH2816-DNA cut with EcoRI and HpaI (5 units each per microgram of 
DNA) was separated by preparative agarose gel electrophoresis (0.7 percent 
agarose in 0.09 mol of tris-borate, and 2.5 mmol of Na-EDTA; pH 8.3), and 
the DNA fragments of the required size were isolated (corresponding to 
Example 1.2). 
9.1.2 Ligation of the DNA fragment with xylMABCN in pBR322 
[Current Protocols in Molecular Biology, John Wiley and Sons, New York, 
(1988), Section 3.16, Subcloning Of DNA Fragments] 
(a) Preparation of the vector-DNA 
The pBR322-DNA (2 micrograms) with 10 units each of EcoRI and ScaI in the 
restriction buffer (50 mmol of TRIS, 10 mmol of MgCl.sub.2, and 100 mmol 
of preparative agarose gel electrophoresis) was separated before the 
ligation. The desired 3850 bp band was isolated as described in Example 
9.1.1. 
(b) Ligation 
For the ligation, the respective DNA samples (in various quantitative 
ratios in excess of the insert-DNA) were added together, mixed with 
ligation buffer (20 mmol of TRIS, 10 mol of DTT, 10 mmol of MgCl.sub.2 and 
0.6 mmol of ATP, pH 7.2) and incubated overnight at 12.degree. to 
16.degree. C. after adding 1 unit of T4 DNA ligase. Then, the ligation 
mixture was used directly for transformation. 
(c) Transformation of E. coli C600 
[According to Example 1.4.] 
The selection took place on nutrient agar with tetracycline (25 
micrograms/microliter). According to restriction control, sizable amounts 
of pLOIO-DNA were purified by CsCl gradient. 
9.2 Design of plasmid pL011 
9.2.1 Cleavage of pL101 DNA with restriction enzymes 
5 micrograms of pL010 DNA was cut with 22 units of HindIII in the 
restriction buffer (10 mmol of TRIS, 10 mmol of MgCl.sub.2, 50 mmol of 
NaCl and 1 mmol of DTT, pH 7.5) and subjected to a preparative agarose gel 
electrophoresis. Two fragments with sizes of 3.8 kb and 2.9 kb were 
isolated as described in Example 9.1.1 and taken up in 45 and 30 
microliters of water, respectively. They contained vector pBR322 and the 
range of xyl genes except for xylB. 
9.2.2 Ligation 
Both fragments isolated in Example 9.2.1 were used for ligation, as 
described in Example 9.1.2b. For this purpose, 45 microliters of fragment 
3.8 kb, 30 microliters of fragment 2.8 kb, 10 microliters of ligation 
buffer, 10 microliters of 10 mmol of ATP and 1 microliter of T4 DNA ligase 
were mixed and incubated overnight at 12.degree. to 16.degree. C. Then, 
the DNA was precipitated with ethanol and taken up in 10 microliters of 
water. 
9.2.3 Transformation of E. coli HB101 
The DNA obtained according to Example 9.2.2 was used directly for 
transformation of E. coli HB101 (corresponding to Example 9.1.2c). 
Transformed cells were selected on nutrient agar with tetracycline (25 
micrograms/microliter). 
9.3 Conversion of pL011 in pRK2013 containing E. coli HB101 
pRK2013 containing E. coli HB101 was selected as a host for pL011. A 
mobilization in other gram-negative bacteria, such as, Pseudomonas putida 
JD7 with pWWO is possible by the functions coded on pRK2013. Isolated 
pL011-DNA was transformed into pRK2013 containing E. coli HB101, as 
described in Example 9.1.2a. The selection took place on nutrient agar 
with tetracycline (25 micrograms/microliter) and kanamycin (25 
micrograms/microliter). 
9.4 Conjugation of pRK2013 pL011 containing E. coli HB101 with pWWO 
containing Pseudomonas putida JD7 
2 ml was centrifuged off from overnight cultures of both conjugation 
partners, washed several times in 0.9 percent NaCl (saline), taken up in 
100 microliters of saline and mixed on nutrient agar plates. The plates 
were incubated for conjugation for 6 hours at 30.degree. C. The resulting 
bacteria lawn was resuspended in 1 ml of saline and plated out in suitable 
dilutions of nutrient agar with tetracycline (50 micrograms/microliter). 
With the resulting transconjugants, some pL011 should have been taken up 
in the TOL plasmid because of homologous recombination. 
9.5 Marker exchange between pL011 and pWWO 
To remove xylB by homologous recombination from TOL plasmid pWWO in 
Pseudomonas putida JD7, approximately over 100 generations of the 
above-obtained E. coli transconjugants were cultured without selection 
pressure by tetracycline. In this case, the exclusion (removal) of vector 
pBR322 and intact xylB gene was desired. 
To increase the number of tetracycline-sensitive xylB mutants, a selection 
from integrated vector pBR322 was then performed: 
Cells were taken up in 25 ml of complex medium nutrient yeast broth (Oxoid, 
Wesel, FRG) of tetracycline (50 micrograms/microliter) and incubated up to 
an OD.sub.650 of about of 3.0 at 30.degree. C. Then, 500 
micrograms/microliter of cycloserine C and 100 micrograms/microliter of 
piperacillin were added. After incubation for several hours at 30.degree. 
C., an almost complete lysis of the cells took place. Surviving cells were 
centrifuged off, washed several times in saline and plated out in suitable 
dilutions on nutrient agar. Up to 85 percent of the resulting colonies 
were sensitive to tetracycline. 
9.6 Test of the colony for the presence of an xylB deletion 
9.6.1 Detection of the deletion with Southern-blot hybridization 
pWWO'-DNA of the resulting clones was isolated according to the method of 
Kado and Liu [J. Bacteriol., 145, (1981), pages 1365 to 1373]. For this 
purpose, 1 ml of overnight culture was centrifuged of and resuspended in 
40 mmol of tris-acetate buffer, 2 mmol of EDTA, pH 7.9. The cells were 
lysed by adding 200 microliters of 3 percent SDS, pH 12.6, incubated for 1 
hour at 65.degree. C. and then extracted several times with phenol 
chloroform (1:1). 
The aqueous DNA solution was freed from phenol by repeated washing with 
diethyl ether and mixed with 1/10 volumes of 3 mol of sodium acetate, pH 
4.8. Then, the DNA was precipitated with ethanol and taken up after drying 
in 100 microliters of water. 
About 40 microliters of this DNA sample was cut with 100 units of EcoRI and 
5 units of HpaI in a digestive buffer (50 mmol of TRIS, 10 mmol of 
MgCl.sub.2, 100 mmol of NaCl and 1 mmol of DTT, pH 7.5) and subjected to 
an agarose gel electrophoresis. The DNA transferred to nitrocellulose 
membranes was hybridized from 500 ng of a pL011 sample labeled .sup.32 
P-ATP. The 1.4 kb xylB deletion was directly recognizable after 
autoradiography. 
EXAMPLE 10 
Production of the hydroxymethylated heterocycles 
E. coli K12* with pL04 (DSM no. 6153) was cultured overnight at 30.degree. 
C. in nutrient yeast broth (Oxoid, Wesel, FRG) by adding the corresponding 
antibiotic agent listed in Table 1 corresponding to the method in Example 
1.4 for stabilizing the plasmids. Then, an aliquot was transferred in a 
fresh medium and incubated for another 2 hours at 30.degree. C., before 
the xylene monooxygenase genes corresponding to the expression system 
(Table 1) were induced. This took place by adding 1 mmol of IPTG for 
induction of the expression by the tac-promoter. The induction phase was 
between 2 and 4 hours in each case. The bacterial suspension was 
centrifuged and the cellular pellet was then resuspended in fresh medium 
without adding antibiotics so that an OD.sub.650 of 10 occurred. This 
suspension was then mixed with 0.1 percent (v/v for liquid substrates, w/v 
for solid substrates) of the heterocycles to be oxidized and further 
incubated at 30.C. After specific periods, the bacterial suspension was 
examined for product formation. 
EXAMPLE 11 
Pseudomonas putida JD7 with pL05 was cultured according to Example 10. 
Because of deficient repressor gene lacIg, an induction with IPTG was able 
to be dispensed with. The strain was used corresponding to Example 10 for 
reaction of heterocycles. 
EXAMPLE 12 
According to Example 10, E. coli K12* with pGSH2836 (DSM no. 6154) was used 
for the reaction. The induction took place by deactivation of repressor 
gene cI857 by temperature effect for 2 hours at 42.degree. C. 
EXAMPLES 13 AND 14 
According to Example 10, the production strains produced in Examples 2 to 8 
were used for the reaction. 
EXAMPLES 15 TO 20 
The results of the conversion rates of the various heterocycles with the 
production strain of Example 12 are compiled in Table 3. 
TABLE 1 
______________________________________ 
Expression 
vectors Hybrid- 
(without) 
Described plasmids Characterized 
Size 
xylMA) in with xylMA by in kb 
______________________________________ 
pLV85 J. Bacteriol., 
pGSH2836 ampicillin- 
5.25 
169, (1987), resistant 
pp. 4457-4462 promoter 
lambda PL 
pME285 Gene, 36, pLO2 kanamycin- 
12.95 
(1985), sensitive 
pp. 27-36 mercuric 
salt re- 
sistant mob.sup.+ 
pKT240 Gene, 26, pLO3 kanamycin- 
15.25 
(1983), sensitive 
pp. 273-282 ampicillin- 
resistant mob.sup.+ 
pMMB67EH Gene, 48, pLO4 ampicillin- 
11.15 
(1986) resistant 
pp. 119-131 promoter 
p.sub.tac lacIq.sup.+ 
pMMB67EH* 
-- pLO5 ampicillin- 
13.5 
resistant 
promoter 
p.sub.tac lacIq.sup.- 
kanamycin- 
resistant 
______________________________________ 
TABLE 2 
______________________________________ 
Production strains for Hydroxylation of Methyl Groups 
Containing Yield in % in the case 
hybrid plas- of reaction of 2- 
mid or DSM chloro-5-methyl-pyri- 
mutated deposit 
midine as substrate in 
Production plamid num- a concentration of 
Ex. strains (PWWO') ber 0.1% (v/v) 
______________________________________ 
2 E. coli K12* 
pGSH2836 6154 80 
3 E. coli K12* 
pLO4 6153 80 
4 E. coli K12* 
pLO3 -- 80 
5 E. coli K12* 
pLO5 -- 80 
6 Pseudomonas 
pLO5 -- 10 
aeruginosa 
PAO25 
7 Pseudomonas 
pLO5 6152 5 
putida JD7 
8 Pseudomonas 
pLO5 -- 5 
putida 
______________________________________ 
TABLE 3 
__________________________________________________________________________ 
Microbiological oxidation of methylated aromatic heterocycles 
with microorganism strain: E. coli K12* containing expression 
vector pGSH2836 
Concentration 
of the substr. 
Reaction 
in the culture 
time in 
End Yield 
Ex. 
Substrate 
medium hours 
Product in % 
__________________________________________________________________________ 
15 2-chloro-5- 
0.1% (v/v) 
16 2-chloro-5- 
80 
methyl- hydroxymethyl- 
pyridine pyridine 
16 2.5-dimethyl- 
0.1% (v/v) 
16 2-hydroxymethyl- 
50 
pyrazine 5-methyl- 
pyrazine 
17 2,6-dimethyl- 
0.1% (w/v) 
16 2-hydroxymethyl- 
10 
pyrimidine 4-methylpyrimidine 
18 3,5-dimethyl- 
0.1% (w/v) 
16 3-hydroxymethyl- 
10 
pyrazone 6-methylpyrazole 
19 4-methylthia- 
0.1% (v/v) 
16 4-hydroxymethyl- 
10 
zole thiazole 
20 2,5-dimethyl- 
0.1% (v/v) 
16 2-hydroxymethyl- 
10 
thiophene 5-methylthiophene 
__________________________________________________________________________