GB2389363A - Promoter and expression vector for Rhodococcus - Google Patents

Abstract

The invention provides a constitutive promoter of Rhodococcus, and vectors comprising this promoter. It is used in an Rhodococcus/E. coli shuttle vector which also comprises a multiple cloning site after the promoter, the genes rep and para which encode proteins involved in the replication and maintenance of the vector in Rhodococcus, a genetic marker selected from an antibiotic resistance gene or a cadmium resistance gene (from the cad operon), and the replication origin of E. coli. Preferably, the vector is used to express the genes of the sox operon (soxA, soxB and soxC) from the constitutive promoter. Bacteria selected from Rhodococcus, Gordona and Nocardia may be transformed with this vector and used for the removal of organic sulphur from fossil fuels.

Description

PROMOTER AND EXPRESSION VECTOR IN RHODOCOCCUS AND ES-

CHERICHIA COLT.

The present invention relates to a new constitutive 10 promoter, a plasmid vector functional in Rhodococcus and Escherichia cold containing said promoter and micro organisms transformed with the plasmid vector for the ex-..DTD: pression of homologous and heterologous proteins.

Bacteria of the Rhodococcus species are of great in 15 tersest in the field of the biodegradation and Liotransfor

matio. of organic compounds (Warhurst and Ffe;on, 1994, Crit. Rev. Biottechnol., 14:29-73i.

Processes are known, for example, which use strains of Rhodocccus for the selective removal of organ c sulfur _ 20 from Fossil fuels, (US 5,358,%70, US 5 132,219, PCT/US9/01868, EP-445896) and for the production of en zymes involved in the production of acrylamide (Kobayashi et al., 1992, Tends Biotechnol., 10:402-408), carboxylic acids, L-amino-acids (WO9804733) and enantiomers of chiral 25 compounds (US 5,672,504).

The main limiting factor in the optimization of bio-

catalysis processes which use these bacteria, however, is the lack of suitable genetic instruments.

This term refers to expression vectors in Rhodococcus 5 which: - are present in the cells in multiple copies; - are kept stable inside the cells in the absence of costly selective agents (for example antibiotics), which considerably influence the economical aspect of an indus lo trial process; and - contain a strong promoter, i.e. capable of allowing an effective expression of a gene, or a strong constitutive promoter which does not require the use of inducers and is not susceptible to repressors.

15 A limit in the removal of organic sulfur from fossil fu els with strains of Rhodococcus which produce the sox enzy matic complex, is in fact due to the presence, before the corresponding genes, of a strongly sulfate inhibited pro moter. 20 In order to overcome this drawback, the genes encoding this enzymatic complex were placed in Rhodococcus vectors under the control of heterologous constitutive promoters such as that of the gene for resistance to chloramphenicol of Rhodococcus fascians (Piddington, C.S. et al., 1995, 25 Appl. and Env. Microbiol., 61,2,:468475) or the gene sacB - 2 -

of B.subtilis (Denis-Larose, C. et al, 1998, Appl. and Env.

Microbiol., 64,11:4363-43G7) and of the gene for resistance to Kanamycin of E.coli (Serbolisca et al., Appl. Microbiol.

Biotechnol. 1999, 52:122-126). The maintenance of the vec 5 tors, however, required the presence of a selective agent in the culture medium and, furthermore, the expression of the sox operon placed under the control of the sac B pro-

moter proved to be extremely low (Lau. P. et al. 1999 ACS Fuel Chem.:3233).

10 It has now been found that the drawbacks of the known art cited above can be overcome by means of the promoter of the present invention.

In accordance with this, an objective of the present invention relates to a new constitutive promoter of 15 Rhodococcus characterized by the sequence SEQ. ID, Nr. 1.

A further objective of the present invention relate<' to an expression vector in Rhodococcus and Escherichia cold which comprises said constitutive promoter.

Another objective of the present invention relates to 20 a micro-organism transformed with said expression vector.

Yet another objective of the present invention relates to a process for the production of homologous and heterolo gous proteins in microorganisms transformed with said ex pression vector.

25 Further objectives of the present invention will ap

pear evident from the following description and examples.

Brief descriptlon_of the figures Figure 1: this indicates the restriction map of the plasmid pSM873 5 igure 2: this provides a photo of a gel relating to the expression of the protein XylE under the control of the promoter 57 or promoter of the soxABC operon. The figure shows the A600 nm values of the cultures at the times at which they were removed.

lo Figure 3: Accumulation of the protein XylE during growth in a medium rich in the presence (+) or absence (no) of Cd++.

The number of generations at the moment of removal of each sample, is indicated. The protein XylE is indicated by the arrow. It Figure 4: provides the restriction map of the plasmid pSM892. Figure 5: indicates a gel of the expression of the sox pro-

teins of SMV128 in minimum medium.

The optical density values of the cultures (expressed as A 20 600 nm) are provided at the moment of removal of each sam ple. (l) negative control; (2) Low Molecular Weights; (3) DS7 1.05 A 600 nm; (4) SMV128 1.20 A 600 nm; (5) DS7 2.35 A600 nm; (6) SMV128 2.17 A 600 nm; (7) DS7 3.03 A 600 nm; (8) 25 SMV128 3.20 A 600 nm; (9) DS7 4.24 A 600 nm; (10) SMVI28

4.49 A 600 nm; (11) DS7 7.12 A 600 nm; (12) SMV128 6.56 A 600 nm.

Figure 6: Expression of the sox proteins in SMV128 in rich medium (LB). The optical density values (A600nm) of the 5 culture are indicated at the moment of removal of each sam-

ple. (1) negative control; (2) Low Molecular Weight Mark-

ers; (3) DS7 1.00 A600nm; (4) SMV128 1.06 A600nm; (5) DS7 2.02 A 600 nm; (6) SMV128 1.86 A600 nm; (7) DS7 4.59 A600 nm; (8) SMV128 3.00 A600 nm; (9) DS7 5.72 A600 nm; (10) 10 SMV128 4.19 A600 nm; (11) DS7 6.65 A600nm; (12) SMV128 4.96 A 600 nm.

Figure 7: Expression of dszD in SMV118, under the control of the promoter 57, and in DS7, under the control of the wild promoter.

15 Detailed description of the invention

Various strategies can be used in the search for new constitutive promoters of the chromosomal DNA of Rhodococ cus. - A strategy was preferably used which is based on the 20 construction of a shuttle vector which can be replicated both in Rhodococcus and in E.coli containing a gene re porter without its own promoter and on the cloning, before said gene, of random fragments of chromosomal DNA of Rhodococcus. 25 In particular, the shuttle vector (pSM73) contains: - 5

(a) rep and par genes which encode proteins implicated in the replication and maintenance of the plasmid in Rhodococ cus; (b) a gene reporter; 5 (c) a multiple cloning site before the gene reporter; (d) at least one gene which encodes a genetic marker se-

lected, for example, from genes of the cad operon, which confer resistance to cadmium or genes which encode resis-

tance to an antibiotic; and 10 (e) the replication origin in E.coli.

Fragments of chromosomal DNA of Rhodococcus having relatively reduced dimensions (-2 kb) are preferably cloned in this vector.

The use of these fragments provides the following ad 15 vantages: - the promoter is in a circumscribed area and there is therefore no difficulty in identifying it after analyzing the sequence; - the lasmid is not magnified with energy saving for 20 the cell; - reduced possibility of integration, by homologous re-

combination, in the chromosome.

A gene reporter refers to a fragment of DNA which en-

codes a product that allows the selection of the clones 25 which express it. Examples of gene reporters which can be

used for this purpose can be selected from those which en code resistance to antibiotics or heavy metals or enzymes such as XylE or the same Sox proteins.

XylE is preferably used as gene reporter.

5 The clones in which xylE is under the control of an active promoter can be easily identified by spraying a so lution of pyrocatechol on the colonies. This compound is transformed by the protein XylE in 2-hydroxymuconic semi aldehyde which gives the colony a yellow colouring.

10 The intensity of the colour depends on the activity level of XylE which is considered as being proportional to the quantity of protein and consequently indirectly to the strength of the promoter.

More accurate measurements of the activity of XylE are 15 effected with the cells in suspension and following, with a spectrophotometer, the transformat on kinetics of the pyro catechol through an increase in the absorbance at 375 mn, which represents a maximum absorption for aldebyde.

With this method, a constitutive promoter of a gene of 20 Rhodococcus was identified, having the sequence SEQ. ID Nr.1. This promoter was further characterized and used for the development of an expression vector optimal for Rhodococcus. 25 The segregational and structural stability of this ex

pression vector in strains of Rhodococcus was determined by operating as described in Example 6. The results demon-

strated that the vector is maintained in over 90 of the cellular population even after subsequent passages (for at 5 least 40 generations) in a culture free of selective agents, thus showing a considerable segregational stabil-

ity. Furthermore, analysis of the plasmids isolated from the transformed strains showed that, even after various 10 generations in liquid culture, this plasmid remains struc-

turally stable in different strains of Rhodococcus.

The expression vector of the present invention can be used for the expression of genes which encode proteins of interest such as, for example, the enzymes involved in the 15 selective removal of organic sulfur from fossil fuels (SoxA, SoxB, SoxC, SoxD), the production of Lamino-acids (amidase, aspartame), the production of enantiomers of chiral compounds (epoxide hydrolase, ketoester-reductase), the production of carboxylic acids (nitrilase), etc. 20 The capacity o- the promoter of directing the consti tutive expression of the gene placed under its control was verified by putting the sox operon, isolated according to what is specified by Serbolisca, L., de Ferra, F., Marga rit, I., Appl. Microbiol. Biotechnol., 1999,52:122-126, af 25 ter said promoter.

The results obtained showed that this promoter allows an effective and constitutive expression of Sox proteins in the presence of inorganic sulfur in the culture medium.

The strain containing the vector pSM886 was deposited 5 at the Centraalbureau Voor Schimmelcultures as E.coli SMC527 where it received the number CBS 110267.

In the following experiments, the following strains were used: the strain of Rhodococcus sp. DS7 10 - the strain of Rhodococcus DS-2, obtained from Rhodococcus sp. DS7, incapable of desulfurating as it is without the plasmid of 130 kb (Margarit, I. et al., 1997, gth Proceedings of the International Conference on Coal Science: 1579-1582).

15 The following experimental examples are illustrative and non-limiting for the invention itself.

EXAMPLE 1

Construction of the plasmid pP600XC A fragment of about 600 bp, containing a constitutive 20 promoter in Rhodococcus DS7, was previously cloned in pUC18 digested with NdeI and HindIII (Margarit et Al. IT-MIOOA-

000332). In this plasmid, the promoter precedes the MCS of pUClr8 so that the HindIII site is the first of the MCS.

1 fig of said plasmid was digested with 5 units (U) of 25 HindIII and BamHI in 50 l of buffer B (Roche) for 2 hours 9

at 37 C.

A fragment was parallelly amplified, containing the gene xylE with the following primers: For: 5' TACCACAAGC TTCCCATACAGG 3' 5 Rev: 5, ACTAGTGGAT CCTCAGGTCA 3' The amplification was carried out with PWO polymerase (Roche) under the conditions suggested by the producer with 30 cycles effected as described below: - 30" at 94 C 10- 30" at S4 C - 45, at 72 C The sample was extracted with phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) and with chloroform/isoamyl alcohol (24:1, v/v). After precipitating the DNA with 2.5 volumes 15 of ethanol in the presence of K acetate 0.3 M, pH 5 and dissolving it in 50 Al of buffer B (Roche), it was digested as described for the plasmid.

The DNA of the two samples was purified from agarose gel (0.8%) according to the method of Tantz D. and Renz M. 20 (Anal. biochem., 132, 14-19, t983) and then dissolved in 30 of H2O.

1 Al of plasmid DNA and 7 Al of fragment containing xylE were ligated in 10 Al of buffer (Roche), in the pres ence of 0.5 U of T4 DNA polymerase, at 14 C for 16 hours.

25 Aliquots (2 Al) of the ligase mixtures were used to -

transform electrocompetent cells of E.col1 XL1-blue, ob tained using an electroporator BIO RAT Gene Pulser II at 2500V, 200 ohm, 25 OF. The cells were resuspended in 2 ml of SOB (Bacto triptone 2%, Yeast Extract 0.%, NaCl 10 mM, 5 KCl 2.5 mM, MgCl2 10 ma, MgSO4 10 mM) and incubated at 37 C for l hour. The recombinant clones were selected on LB me dium in the presence of ampicillin (100 g/ml). A plasmid, called pPr600X, which contains the gene reporter xylE under the control of the promoter Pr600, was isolated from one of 10 the positive clones.

This plasmid was used to clone a fragment containing the gene which encodes resistance to cd++ isolated from pSM843 CBS 102445.

l fig of pPr600X and 2 fig of pSM843 were ingested 15 separately in 50 Al of buffer M (Roche) in the presence of 5 U of Asp7i8 for 2 hours at 37 C. The samples were treated as in the previous passage (purification, ligase, transfor-

mation). The plasmid selected, called pPr600XC, had the genes cadC-cdA in the same orientation as the gene xylE 20 situated in I'.

The plasmid pPr600XC contains the genes for resistance to Cd++ (cadCA), the gene for resistance to ampicillin for selection in E.coli and the replication origin oriC for propagation in E.coli; this plasmid cannot be propagated in 25 Rhodococcus sp.

EXAMPLE 2

Construction of the shuttle plasmid pSM873 The plasmid pPr600XC (1 fig) was digested with 5 units (U) of the restriction enzyme SspI (Roche) in 50 pl of 5 buffer under the conditions suggested by the producer.

2 fig of the plasmid pSM836 (IT-MIOOA-000332) were par-

allelly digested with the restriction enzymes SspI and PvuII (5 U) in 50 Al of buffer.

The enzymatic reactions were then blocked at 65 C for 10 10 minutes and the digestion mixtures were charged on aga rose gel (Seakem LE, BRA) at O. 81.

The linearized DNA of pPr600XC and the fragment SspI PvuII of about kb (containing the genes for the replica tion of the plasmid in Rhodococcus) were elated as de 15 scribed by D. Tantz and M. Renz (Anal. Biochem., 132, 14 19, 1983). The purified fragments were ligated in 50 lin earized of buffer for T4 DNA ligase (Roche) in the presence of 0.3 units of T4 DNA ligate (Roche) and incubated at 14 C for 10 hours.

0 Electrocompetent cells of Rhodoc Ecus DS-2 (100 1), obtained using the electroporator BIO RED Gene Pulser II at 2500V, 600 ohm, 25 ELF, were transformed with 3 Al of ligase mixture. Immediately after the transformation, the cells were resuspended in 2 ml of SOB (Bacto triptone 2%, Yeast 25 Extract O.5%/ NaCl 10 ma, KCl 2.5 me, MgCl2 10 ma, MgSO 10

> ma) and incubated at 30 C for 3 hours.

The recombinant clones were selected on plates of modified LB medium (10 g/1 Bacto triptone (DIFCO), 5 g/1 Bacto Yeast Extract (DIFCO), 5 g/1 NaCl, 15 g/1 agar) con-

5 Gaining CdC12 0.5 ma.

The plasmid DNA was extracted from the recombinant pool with the following method.

The cells were resuspended in 950 q1 of TE (Tris/C1 10 ma, pH8, EDTA 1 ma) containing lysozyme (2 mg/ml) and the 1O cellular suspension was incubated at 37 C for 15 minutes.

50 pi of SDS 10t (in H2O) and 5 pi of proteinase K (stock 20 mg/ml) were then added. The sample was incubated at 37 C for 1 hour.

The suspension was subsequently extracted with phe 15 nol/chloroform/isoamyl alcohol (25:24:1) and the aqueous phase was extracted again with 1 volume of chloro form/isoamyl alcohol (24:).

K acetate 3 M, pH 5 (0.1 M final) and 2.5 volumes of ethanol were added to the aqueous phase to precipitate the 20 DNA which was resuspended in 500 pi of H2O.

An aliquot (1 pi) was used to transform electrocompe tent cells of E.coli XL1-Blue (BRL). The cells were resus pended in SOB and incubated for 1 hour at 37 C before being plated. 25 The transformants were selected on plates of agarized

LB medium containing 100 g/ml of Ampicillin.

The double passage guaranteed that all the recombi-

nants contained the plasmid in the correct form.

The plasmid DNA extracted from some of the clones thus 5 obtained was analyzed by means of restriction analysis.

The results indicated that the gene xylE in the plas-

mid was under the control of the promoter Pr600.

The fragment NcoI-HindIII containing the promoter Pr600 before xylE was subsequently substituted with a syn-

lO thetic MCS (Multiple Cloning Site) sequence 5' CCATGGCCGG CCCGGGCGGC CGCTCTAGAA GATCTATGCA TAAGCTT 3'

containing the sites for the restriction enzymes NcoI, FseI, SrfI, SfiI, XbaI, BglII, Nail and HindIII.

The map of the resulting plasmid, called pSM873, is 15 indicated in figure 1.

EXAM 'LE 3

Construction of the bank in pSM873 - The plasmid pSM873 was used in the search for new pro-

moters, by cloning fragments of genomic DNA of Rhodococcus 20 DS7 before the gene reporter xylE. Fragments containing a promoter should give xylE activity in cells of Rhodococcus.

The genomic DNA of Rhodococcus DS7 was prepared from a culture in 50 ml of modified LB medium in exponential growth according to the method described herein.

25The culture was centrifuged at 6000 RPM for 10 minutes

at 4 C and the cells were resuspended in 5.7 ml of TE (Trig/Cl lO me, pH 8, EDTA I ma) containing lysezyme (2 mg/ml). The cellular suspension was incubated at 37 C for 15 minutes, 300 ill of SDS 10 (in H2O) and 30 1 of pro-

5 teinase K (stock 20 mg/ml) were then added and the mixture was incubated at 37 C for 1 hour. After adding K acetate 3M, pH 5 (0.3M final) , the suspension was incubated in ice for 10 minutes and then centrifuged at 8,000 RPM for 20 minutes at 4 C, to recover the supernatant liquid.

10 The DNA was precipitated with 1 volume of isopropanol and dissolved in 3 ml of TE in the presence of Rnasi A (20 Ug/ml). The sample was incubated at 37 C for 5 minutes and then extracted with 1 volume of phenol/chloroform/isoamyl 15 alcohol (25:24:1). The aqueous phase was extracted again with 1 volume of chloroform/isoamyl alcohol (24:1) and the DNA was precipitated with a volume of isopropanol after adding K acetate (0.3 M final), pH 5. At the end, the DNA was dissolved in 500 Al of H2O.

20 Aliquots (1 fig) of genomic ONA were then digested with combinations of the following enzymes: - NcoI + BspHI + BspLUllI (Roche): to produce the 5' ends of the fragments. The enzymes BspHI and BspLUllI pro duce ends compatible with NcoI; 25 - BglII + BamHI + Ball (Roche): the enzymes BamHI and

BclI produce ends compatible with BglII.

A reaction mixture was prepared, containing 10 fig of ge nomic DNA of DS7 in 100 Al of buffer A (Roche), with 10 U of each of the enzymes NcoI, BspHI, BamHI, BglII. The sam 5 ple was incubated for 2 hors at 37 C. 10 U of BspLUllI and BclI were subsequently added and the incubation was contin-

ued for a further 2 hours at ARC. At the end, the sample was extracted with 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1) and subsequently with 1 volume of chlor 10 form/isoamyl alcohol (24:1).

The DNA was precipitated with 2.5 volumes of ethanol (991), after the addition of 0.1 volumes of K acetate 3M, pH 5, and it was then diluted in 100 1 of H2O.

5 fig of the plasmid pSM873 were digested in 100 Al of 15 buffer H (Roche) for 3 hours at 37 C with 15 U of NcoI and Bgl T: (Roche) The fragment corresponding to the vector cut was puri-

fied as described by D. Tantz and M. Renz (Anal. Biochem., 132, 14-19, 198?). At the end, the plasmid was suspended ir 20 30 pi of H2O 6 Al of digested pSM873 and 12 Al of genomic DNA of DS7 digested as described above, were ligated in final 20 Al of buffer for T4 DNA ligase (Roche), in the presence of 0.5 U of T4 DNA ligase (Roche), at 15 C for 16 hours. The 25 DNA was then precipitated and subsequently diluted in 20 pl

of H2O.

In order to increase the transformation efficiency, it was decided to transform first E.coll by exploiting the ca-

pacity of the shuttle plasmid of replicating itself also in 5 this species. The transformation efficiency of E.coli is always a few orders of magnitude higher than that of Rhodo occus. Furthermore, by first transforming E.coli and subsequently Rhodococcus, the possibility is eliminated of obtaining recombinants by the integration of linear plasmid 10 in Rhodococcus which, in any case, would be Cd+±resistant (selection of the plasmid in Rhodococcus).

Aliquots (3 l) of the ligase mixture for a total of 12 Al were used to transform competent cells of E.coli XL1--

blue (BRL).

5 The transformants were selected on plates of LB con taining Ampicillin (loo legal). The p.asmid DNA was ex-

tracted from a group of 1376 colonies, as described in the previous example.

3 fig of plasmid DNA were used to transform 6 aliquots 20 of electrocompetent cells of Rhodococcus DS-2 as described above. The transformants were selected on plates of LB con-

taining CdC12 O.5 ma.

In order to verify the XylE activity present in the various clones (due to the activation of the gene on the 25 part of a promoter), a 10 mM solution of pyrocatechol in

H2O was sprayed on the plates. Among the positive colonies, which developed a yellow colour, three in particular gave an extremely evident response. One of these (c57) was se-

lected for subsequent tests. The plasmid isolated from this 5 colony was called pSM886.

Said plasmid is capable of replicating itself in E.coli and Rhodococcus and contains a constitutive pro mater, as it is not inhibited by the sulphate present in the LB medium.

10 The strain of E.coli containing the plasmid pSM886 was called SMC527.

EXAMPLE 4

Sequencing of the promoter 57 fragment _ The fragment cloned in pSM873, containing the promoter 15 57, was sequenced using an Applied Biosystems 373 sequencer and Big Dye kit of the same manufacturer. Primers both out side (lnslde the plasmid) and inside the fragment were used for the sequencing. The sequence of the primers is indi cated below: 20 873_FOR: 5'CGAACTTTCG CCAACACC3'

873_REV: 5'GCAACTCGAC GTAGTGTTCC3'

57_2F: 5'CGCTGGGCGT CTTGTATTTC3'

The fragment, which proved to be 1003 base pairs long, con tained a promoter sequence SEQ. Nr. 1, indicated below.

25 EXAMPLE 5

- 18

Comparison with the w.t. promoter in minimum medium Two groups of cells of Rhodococcus, which contain the socABC operon integrated in the chromosome, were respec-

tively transformed with the plasmid pSM886 and with the 5 same plasmid in which the promoter 57 was substituted with the promoter of the socABC operon.

The positive clones were inoculated, separately, in 50 ml of minimum medium (10 g/l KHzPO4 pH 7.4, 2.5 g/1 NH4Cl, 0.2 g/l MgCl2.6H2O, 0.02 g/l CaC12, 0.01 g/l FeC13, 0.005 10 g/l MnCl2.2H2O, 0.003 Gil ZnG12, 0.0009 g/l CuC12.2H2O, aga rose 0.81) containing dibenzothiophene (DOT) as sole sul phur source. The cultures were incubated at 30 C for 3 days. Aliquots of the two cultures were removed at different 15 times, to determine the accumulation of the protein XylE.

Each liquot was brought to a density of 1 U A610 in 1 ml with buffer TE (Tris 10 mM, EDTA 1 me, pH 8).

After centrifugation, the cells were resuspended in 150 pi of buffer TE, in the presence of lysozyme (2 mg/ml) 20 and inclbated for 15 minutes at 37 C to allow the ligestion of the cellular wall.

150 pl of loading buffer 2X (SDS sample buffer 2X.

SDS 4%, Tris 125 mM, pH 8, glycerol 10%, Q-mercapto-ethanol 2%) were then added to the suspensions, which were incu 25 bated at 100 C for 4 minutes.

10 pi of each sample were charged on polyacrylamide gel at 10% and subjected to electrophoresis. The gels were coloured with Blu Comassie to reveal the protein bands (Figure 2).

5 From analysis of the gel, it can be seen that in the clone containing the promoter 57, the accumulation of XylE protein is extremely high in the various growth phases, even though there is a drop in the samples extracted under late exponential growth and in stationary phase. XylE also 10 represents the protein accumulated in the highest quantity in the total extracts in all growth phases. In the case of the sox promoter, the quantity expressed is extremely re duced, EXAMPLE 6

15 Stability of the plasmid pSM _ The objective of the experiment was to verify the sta bility of the plasmid pSM886 in the transformed strains of Rhodococcus DS-2 under non-selective conditions (in the ab sence of cadmium).

20 For this purpose, a pre-in<>culum of the strain c57 was prepared by plating on LB agar medium containing CdCl2 (0.5 ma) which represents the selective condition for maintain ing it in _h coccus. The clones were controlled for the XylE activity by spraying a sterile solution of pyrocate 25 chol 10 me on the plate. Some of the clones which were yel

low-coloured (positive clones) were isolated and inoculated into 20 ml of LB in the presence of CdCl2 (O.1 me).

After incubation at 30 C for a night, a control of the activity of XylE was effected and proved to be positive.

5 An aliquot removed from one of the pre-inoculums was diluted in 40 ml of LB so as to have an optical density of about 0.080 U A600 nm and the resulting mixture was then subdivided into two 20 ml aliquots, CdCl2 (O.1 ma) being added to one of these. The two cultures were incubated at 10 30 C. The experiment was carried out for 5 days.

At the end of each day, new cultures were prepared by diluting each culture in the same type of medium while they were still in exponential growth phase. In this way, a more or less exponential growth was obtained for the whol duress 15 Lion of the experiment. The growth was monitored by fcllow-

ing the absorbance at 600 rim (A600).

After about 4 n generations in the absence of flu++, L:le cells were plated on hB without Cd++ by diluting them seri-

ally 1:10. The colonies were collected from one of the 20 plates and transferred onto LB in the presence of Cd++.

About 90% of the colonies was capable of re-growing in the presence of Cd+ +.

It can be declared that 90t of the cells of a culture maintains the plasmid after over 40 generations, calculated 25 considering the passages subsequent to the first plating.

- 21

EXAMPLE 7

Expression of XylE Aliquots of the culture obtained as described in Exam-

ple 5, equal to 1 U A600 nm in 1 ml, were collected the 5 rind, IIIrd, IVth and Vth day after the beginning of the experiment. The cells were collected by centrifugation and resuspended in 150 pi of TE in the presence of lysozyme.

After incubating the samples at 37 C for 15 minutes, the cells were lysed by the addition of 150 pi of SDS sam 10 ple buffer 2X (SDS 4, Tris/Cl 0. 125 M, pH 6.8, Glycerol 20, p-mercapto-ethanol 2). The samples were incubated for 4 minutes, at 95 C before being charged onto polyacrylamide gel at 10 (figure 3).

This experiment shows that the level of XylE protein 15 accumulated in the cells remains high during the whole e>-

periment both in the presence of Cd'+ (selective condition and in the absence of Cd++ (non-selective condition) for at least 44 generations.

EXAMPLE 8

20 Construction of tle plasmid pSM892 2 fig of the plasmid pSM790 (Margarit et al. IT-MIOOA 000332), containing the soxABC operon, were digested in 50 p1 of buffer B (Roche) with the enzymes HindIII and EcoRI (5 U) for 2 hours at 37 C.

25 After digestion, the sample was charged onto agarose - 22

gel 0.8 and a fragment of about 4550 base pairs, contain ing the soxABC operon without its own promoter, was puri fied as described by Tantz D. and Rena M. (Anal. Biochem., 132, 14-19, 1983).

5 A reduced version of the fragment containing the pro moter 57 was parallelly amplified from the plasmid pSM886, using the following primers: PR_57_3F: 5'CCGTCGTCTC CATGGTCCGC G 3'

PR 57_3: 5'GAGGCTCACG AAGCTTACTC CTTCGC 3'

10 57_3F contains a site for NcoI, whereas 57 3' contains a site forHindIII immediately after with respect to the pre sumed Ribosomal Binding Site of the promoter 57.

For the amplification, 20 ng of pSM886 were diluted in 100 pi of buffer for Taq polymerase (Roche) containing 100 15 pmoles of primers and 2.5 U of Taq (Rocks). The sample was subjected to 30 of the following cycles: 5, ' at g6 , 5'' at 54 , 45" at 72 C in a Thermal Controller (MJ Research, Inc.). The amplification product, of about 550 base pairs, was controlled on an agarose gel at 2.

20 The sample was extracted with a mixture of Phe :ol/Chloroform/Isoamyl alcohol (25:24:1) and subsequently with a mixture of Chloroform/Isoamyl alcohol (24:1).

The DNA was precipitated with 2.5 volumes of Ethanol, after the addition of 0.1 volume of K acetate 3M, pH 5, and 25 subsequently digested in 50 Al of buffer H (Roche) in the - 23

presence of 5 U of NcoI, for hours at 37 C.

2 fig of pSM873 were digested under the same conditions described above. The DNA were then precipitated in ethanol and digested with 5 U of HindIII in buffer B (Roche).

5 After digestion, the samples were charged onto an aga rose gel 0.8t and purified as described by Tantz D. and Renz M. (Anal. Biochem., 132, 14-19, 1983). At the end, the DNA was dissolved in 20 Al of H2O.

2 Al of pSM873 digested and 7 pl of the amplification lo product (PCR) were ligated in 10 Al of buffer for T4 DNA ligate (Roche) containing 0.5 U of T4 DNA ligase (Roche), at 15 C for 16 hours.

The ligase mixture was used to transform electrocompe-

tent cells of E.coli XLl-blue.

15 The cellular suspension was incubated in 2 ml of SOC medium for 1 hour at 37 C. before being plated on LB + Agar in the presence of Ampicillin 100 g/ml.

Analysis of the positive clones, effected as described above, showed the presence of a plasmid with the expected 20 characteristics. A fragment HindIII-EcoRI, coming from the plasmid pSM790, which contains the soxABC operon, was cloned in said plasmid, called pSM890.

In practice, after digestion of the plasmid pSM790, the sample was charged on an agarose gel 0.8% and said 25 fragment was purified as described by Tantz D. and Renz M. - 24

The plasmid pSM890 was parallelly digested with the same pair of enzymes under the same conditions and the fragment of about 8000 bp was purified as described above.

The purified fragments were ligated in 20 pi of buffer 5 for T4 DNA ligase (Roche) in the presence of 0.3 units of T4 DNA ligase (Roche), at 15 C for 16 hours.

The DNA was precipitated in ethanol (2.5 volumes) in the presence of K acetate 0.3 M, pH 5. At the end, the pel let was dissolved in 20 Al of H2O.

10The whole ligase mixture was used to transform elec trocompetent cells of Rhodococcus DS-2 (3 pil for each ali--

quot of 100 pi).

The transformed cells were resuspended in 2 ml of SOB (Bacto triptone 2%, Yeast Extract 0.5%, NaCl 10 mM, KC1 2.5 15me, MgCl2 10 me, MgSO4 10 ma), incubated at 3C C for 3 hours and then washed in minimum medium without IT. The recombinant clones were selected on plates of minimum me dium in the presence of DBT as sole S source.

Only the clones containing soxABC activated by the 20 promoter 57 were capable of growing under the se ection conditions. The plasmid obtained was called pSM892 (Figure 4). The strain of Rhodococcus containing the plasmid was called SMV128.

25 EXAMPLE 9

- 25

- Expression of the sox proteins in SMV128 in minimum medium The strain SMV128 was inoculated in minimum medium in the presence of DBT as sole sulphur source in order to ver-

ify the activation of the sox genes on the part of the pro 5 mater 57 during growth in liquid culture. The strain Rhodococcus DS7 was used as positive control. Under the _ cultural conditions described r the sox proteins are nor-

mally expressed in DS7, but not in the host strain of the plasmid pSM892 (Rhodococcus DS-2).

10 The two strains were previously sown on solid minimum medium in the presence of DBT as sole sulphur source. The plates were incubated at 30 C until colonies were obtained, of at least 1 mm in diameter. Some of the colonies were re suspended in 1 ml of medium without DBT. Serial dilutions 15 1:3 were effected from the suspension in 20 ml of minimum medium in the presence of DBT as sole sulphur source. The cultures were incubated under stirring at 30 C.

During the growth of the cultures, the production of 2-HBP was analyzed to verity the activity of the Sox pro 20 teins. The growth with time was also analyzed by measuring the absorbance variation at 600 nm.

Aliquots of each culture were taken at different times to analyze the protein extracts on polyacrylamide gel to verify the accumulation level of the Sox proteins in the 25 various growth phases. The aliquots were diluted until an - 26

optical density was obtained, equal to 1 U of absorbance at 600 nm in 1 ml of suspension. After being collected by cen trifugation, the cells were suspended in 150 pi of TE in the presence of Lysozyme (2 mg/ml). The samples were incu-

5 bated at 37 C for 15 minutes.

1 volume of SDS sample buffer 2X (SDS 4%, Tris/Cl 0.125 M, pH 6.8, pmercapto-ethanol 2%) was then added to the samples, which were subsequently incubated at 95 C for 4 minutes.

10 7 Al of the samples were charged onto a polyacrylamide gel at 10t (Novex NuPage 10t Bis-Tris lmm x 10 wells)- A sample coming from a strain grown under analogous condi tions containing a similar plasmid but without a gene re porter, was charged as negative control. At the end, the 15 gel was coloured with Blu Comassie.

As can be verified in figure 5, the expression level of the Box proteins is greater when the operon is under the control of the promoter 57 with respect to the wild pro moter. The effect is particularly evident in the case of 20 SoxA whose gene is immediately after the promoter itself.

In SMV128 soxA is the protein which has accumulated in the highest quantity at each moment of growth.

EXAMPLE 10

Expression of the son proteins in SMV128 in rich LB medium _ 25 The strain SMV128 was inoculated in rich LB medium in - 27

order to verify the activation of the sox genes on the part of the promoter 57 during growth under conditions normally inhibiting for the control strain, Rhodococcus DS7.

The strains SMV128 and DS7 were previously sown on 5 solid minimum medium in the presence of DBT as sole sulphur source. The plates were incubated at 30 C until colonies were obtained of at least 1 mm in diameter. Some of the colonies were re-suspended in 1 ml of medium without DBT.

Serial dilutions (1:2) were effected from the suspension in 10 20 ml of rich LB medium.

The cultures were incubated under stirring at 30 C, analyzing the growth with time by measuring the absorbance variation at 600 nm.

Aliquots of each culture were taken at different times 15 to verify the accumulation level of the Sox proteins in the various growth phases, by means of electrophoresis on poly-

acrylamide gel of the protein extracts. The aliquots were diluted until an optical density was obtained, equal to 1 U of absorbance at 600 nm in 1 ml of suspension. After being 20 collected by centrifugation, the cells were re-suspended in 150 Al of TE in the presence of Lysozyme (2 mg/ml). The samples were incubated at 37 C for 15 minutes.

1 volume of SDS sample buffer 2X (SDS 4%, Tris/Cl 0.125 M, pH 6.8, pmercapto-ethanol 2%) was then added to 25 the samples, which were subsequently incubated at 95 C for - 28

4 minutes.

15 Al of these samples were charged onto a polyacryla-

mlde gel at 10% (Novex NuPage 10% Bis-Tris lmm x 10 wells).

A sample coming from a strain, grown in minimum medium, 5 containing a similar plasmid but without a gene reporter, was charged as negative control. At the end, the gel was coloured with Blu Comassie.

From figure 6, it can be deduced that soxA is one of the most highly expressed proteins in SMV128 under the dif-

10 ferent growth phases (from the exponential phase to the late exponential phase). The Sox proteins are not detected in DS7 in complete medium (LB).

EXAMPLE 11

Cloning and expression of dszD in pSM873 15 The gene dezD was isolated from DNA of Rhodococcus JS7, by means of amplification with the olymerase Chain Reaction (PCR) technique, (Leung, D.W., Chen, E., Goeddel, D.V. 1989 Technique: A journal methods in cell and molecu llr biology, 1, Nr. 1: 11-15), using the following pair of 20 o igonucleotides:.

1) 5' GCACACCTCGA GATCTAGCCG GAGAG 3' (FORWARD)

2) 5' GCGGTTAATAA AGCTTCACAG TTGTC3' (REVERSE)

The amplification was carried out in a DNA Thermal Cycler 480 apparatus (Perkin-Elmer Cetus) using 100 Al of a reac-

25 tion mixture containing: 5 ng of plasmid DNA, 60 pmoles of - 29

the two oligonucleotides, 200 AM of dNTPs (dATP, dGTP, dTTP, dCTP) and 1 U of Taq polymerase (Boehringher), in the buffer recommended by the producer. After denaturising for 2 minutes at 94 C, the cyclic program was started, which 5 comprises: 1 minute at 98 C, 1 minute at 60=C and 3 minutes at 72OC for 25 overall cycles, followed by 8 minutes at 72 C (final extension).

The amplification product was cloned inside the sites BglII and HindIII of the vector pSM886 (after the promoter 10 57) and the resulting plasmid was called psM891. The strain of Rhodococcus which contains this plasmid was called SMV118.

The product of the dead gene catalyses the reduction reaction from FMN tc FMNH in the presence of NADH. The FMN 15 reductase activity of the strain SMv118 with respect to the parental strain DS7 'as measured in cellular extracts of the two strains grown in the presence of sulphate or in the presence of DBT as sole sulphur source, and collected in exponential growth phase.

20 20 pi of protein extract (at concentrations within the range of 0.100 to 0.400 fig of protein/pi of extract) were incubated at 30 C in the presence of FMN 0.1 ma and NADH 0.1 me in Na Phosphate buffer pH 7.0 in a final volume of 430 A.

25 The reductase activity (measured as absorbance change -

at 340 am, in the first minute of incubation) proved to be for DS7 0.04 U/mg in cells grown in the presence of sul-

phate and 0.1 U/mg in cells grown in the presence of DBT.

In the recombinant strain SMV118, the reductase activity is 5 2.91 Ujmg for cells grown ill the presence of slphate and 2.07 for cells grown in the presence of DBT.

The expression of deed therefore led to an increase in the activity of this enzyme of 72 and 21 times, respec-

tively, in cells grown in the presence or absence of inor 10 ganic sulphur.

The protein pattern of the extracts of SMV118 and DS7 cells grown in the presence of sulphate is shown in Figure 6, where the arrow indicates the position of the product of the gene dezD.

15 An analysis of these data reveals the over-expression of the gene dezD in cells of Rhodococcus transformed with the vector pSM891.

SEQUENCE LISTING

<110> Enitecnologie S.p.A.

<170> Microsoft Word 7 <210> 1

5 c211> 535 c212> DNA <400> 1

tggEcogagg aetgeeatae ggeatcggeg egateatget 40 ectaggaaLg ggcgeeaate teaeeaaegc ggegttegge 80 10 tcctggatee cegaLgtgee eeaggaeaLg ttgoeeagcg 120 gtcaegEegc ageggcgaee gcgotgLaet gotaggagat 160 ettgatatee geeeegeagt ggegteaggt ggtogaeggt 200 eteggatteg tgggggEtge tgegateggt geeagegeac 240 tggeegeaga gEtgagegge eUtUteggag tgatagetgg 280 15 egtUetgate getetggLat gggoeagtgc tgeageeatt 320 ttgaLggeae gategeaeat egeogcgaaa egtgaagege 360 taegeoagga taeggeagea atagagttet eaaggeaeag 400 gageateega eEttagteet aaatatUtgt gatCeagaga 440 tEetageegt aageaetee egegaeggeg gggoaLgteg 480 20 taLgUtetga aLgEeegaa teaagtagtt eeaeagegaa 520 ggageettta eagtg 535 210> 2

c211> 22 c212> DNA 25 <400> 2

- 32

taccacaagc tEcccataca gg 210> 3

<211> 20

<212> DNA

5 <400> 3

actagtggat cctcaggtca 210> 4

c211> 47 <212> DNA

10 <400> 4

* ccatggaegg cccgggcggc cgctatagsa gatataLgca taagcEt 210> 5

<211> 18

c212> DNA 15 <400> 5

cgaacUtCcg ccaacacc 210> 6

<211> 20

c212> DNA 20 <400> 6

goaactcgac gLagtgttcc 210> 7

<211> 20

<212> DNA

25 <400> 7

- 33

cgaUgggcgt cttgLatUtc 210> 7

c211> 26 <212> DNA

5 <400> 7

gcacacctag agatctagcc ggagag 210> c211> 26 <212> DNA

10 <400>

goggEtaata aagatUcaca gUtgtc 210> 9

c211> 21 c2 2> DNA 15 c400> 9 cagEcgEctc caLggtccgc g 210> 10

c211> 26 c212> DNA 20 <400> 10

gaggctcacg aagattactc cttcgc

B'J3AE5- T!?F..TY _N 'I'B:N-ERNATIONA.:

R_COGN:T:-N O IH.: DE?'SI- OF MICROORGAN SM5

FC'F:.-:- -U.?OSES O ?A. ENT --'JCE.<E

_N'Tr!!lA. ICNA_ FORlA En,Tecnotoge. RECETP,'; THE CkSE. h; ORS NA; rEEcs.: Vla F Maritano 26. 1ss_er purs a.,t to Ru:e: y tne 20097 SAN DONATO MILANESE (Mlano) -: EmblA.TT)N^L uEFC,-ARi ^r,-H-RITV Italle l a-c 2 'i 2dress o- de?cs; cor I IDENT7FIr>.EON OF TH1: bCROOaGANISH _.... ..

Iden';catl:'r: rfere._e given by che j Access1on nu.ber glven by the GEPOSITOR.:NTERNATIONAL DI:PGS:TARY ArJT.O-:TY. I SMC 527 I CBS 110267

II SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATIOM

_ he rr.c-oorgen1sir. 1dentlfled u..der I ahove ws acco.npanled by.

X a s_er.t1flc descrpt1on a pro,Eosec: taxonomlc deslgna_ on (.":a_k W_: a =ross where apclcaDle) . _, ItI. RECEIPT ND ACCEPTANCE _ 1 Tt.=s Inernatlcna' Depos_tary accepts the rlaroorcar isr idenlflec unde- above, wh- ch err-ived by lt Gn 17-05-2002 (date ca-lr,tn-yy of 're ro..^al depcs') 1 i IV RECEIPT OF R'EQUEST FOR CONVERSION

te T_crco_ga..lsr de..t_ led ur._er: abov-e ws _ecleC cy tn_s Ir.terr.at-. 21 Depos:tary tuhory on not applcable (date dd-r-yy Ois tne orlinzi aeposlt) and a rec,;es- to ccnrer: the orl9lual deposit to a depos1t under the 3udapes- Treaty was recel'ed by it cn notappllcable (date d-, y; of rece_pt cf recaest fcr corvers_n) _ V I?.TERNAT ON=u DE20SITARY AUTlIORITY N=.e CentraalbureauvoorSchmrrelcultures 59natires; cf pe_sor.:; nav r, e-de FOAC i -o represen t,.e Inter-.alar.z_ _eFCYitary g_,, of aathorlzec cf f,,clz_'s' Aess Uppsalalaan 8 = c':/ P O Box 35167, = //: / 3508 AD JTRECHT, Mrs F.B Sn,ppe-Cla;D/,lpey' The Netherlands Se (id-rm- yyi, 04-06-20//' / ,/ ibee Rule 6 4l_, applies, such date 15 the ate cn ''hlCn:he stats of:rerr.a1onal cepos -ary au'herlty was acquired Forr E?. 4 (sole page) CBS/5'S -35

E-vEAPE5 - -R-A TV CN -HE IiTER:!AT:ONA,, - _C:GNITION cF -.-;E GE?CS:T CMICROCPGANTSMS FCP T!iE =-JRSS_S CF PA:ENI PRCCEDi-'RE IN. El;A- TN.: !R': :- EniTecnclo51e TB:, S A rV; V!aF Maritano26 lciued -u-suan s u:) c b sne 20097 SAN DGNATO,liLANESE (Milano,, , Nr C.--Rv -.'':E2C Itale ra.e ano' =i4ress er the pary to;A,O ti:e ^ 7'v' 5_ te.e.nt s ss..ec I. DEPOSITOR i II I3ENTIF7CATION OF THE lSICROORGA2TISh! - Na. =e EniTecOologle ' Ac-esslcn n mber gven by the |. IN-ERI1TICNAL GEFCSITV AITHCRI:

! -, CBS 1 10267

ddress Via F Mantano 26 20097 SAN CONATO MILANESE Date,ad-r -yy) cf the deposit or of ^e (Mlan ), t.ar.s e- ' 17-05-2002 T I I. VIAB T LI TY S TATEk:N .. -:ee -r:abllty 5' -he 1 croorgan-,sm 1dentl'led.der II abov-e waS tested or 22-05-2002 2 Cn tnat date dd-mm-yy', the sa:d rr_crsoganls:n was _ _3 X viable . no longe-;able 1 _ Indlcate t:e da-e cf he orlglnal depos t or, H ee a new dep3s t or 2 tansfer nas bee-

Tade, tne m.-st r--ent relevant date:cate of the.,ew aepost cr ate o' the transfer).

2 In the cases refer-e to 1n Rule 0.2(a'!1.' ard l), refer to the.most recent v_ab.llty test Ma-< with a coss te applicable box.

orl 3P/9 'fl-st _age! -36

IV CCh=ITIONS UNDER WHICH -HE VIABILITY HAS BEEN PERFORlt, l v, INTE:RTATIONAL DEPOSITARY AUTHORITY: I Wife CentrOalbureaUvoorSchlmmelcultures Slgnatu.e_; Of Dersor.(s 1.av_ng fine power I co represent the Ir.-ernat onal Depositary j Au the 4 h o - z e d o f f 1 7 7 A.-es; Uppsalalaan8: ',.17 /I/' P O Box 85167 Mrs F B Ipers/T 3508 AD UTRECHT /,'

The Netheriands:' ' 94 /,/ -_11 n!f -re nfonat:or. has reen reuested and:= the rest:lts of the tes,r/ere nesat_ve "c rr BEiC seconc an lee a?e -37

Claims (9)

1. A constitutive promoter of Rhodococcus characterized by the sequence SEQ. ID. Nr.1.

2. An expression vector which comprises: 5 (a) the genes rep and pare which encode proteins im plicated in the replication and maintenance of the vector in Rhodococcus; (b) a constitutive promoter of Rhodococcus having the sequence SEQ. rD. Nr. 1; 10 (c) a multiple cloning site after the promoter; (d) at least one gene which encodes a genetic marker selected from genes of the cad Oberon, which confer resistance to cadmium, or genes which encode resis tance to an antibiotic; and (e) the replication origin in E.coli; deposited with the number COBS 110267.

3. The vector according to claim 2, which comprises after the constitutive promoter one or more genes which en code a protein of interest.

20

4 The expression vector according to claim 3, wherein the genes encode a protein selected from enzymes in volved in the selective removal of organic sulphur from fossil fuels or enzymes involved in the produc tion of L-amino-acids, enantiomers of chiral compounds 25 and carboxylic acids.

-38

5. A micro-organism transformed with the expression vec-

tor according to claims 2 to 4, wine-rein said micro-

organism is selected from Rhodococcus and Escherichia coli. 5

6. A process for the production of homologous or het-

erologous proteins of interest which comprises culti-

vating, under suitable conditions, a micro-organism transformed with the expression vector according to claims 2 to 4.

10

7. The process according to claim 6, wherein the protein is selected from enzymes involved in the selective re-

moval of organic sulphur from fossil fuels or enzymes involved in the production of L-amio-acids, enanti-

omers of ehiral compounds and carboxyLic acids.

15

8. The process according to claim 7, wherein the proteins are Sox enzymes.

9. A process for the removal of organic sulphur from fos sil fuels characterized in that it uses a micro organism selected from Rhodococcus, Gordona and Nocar 20 die transformed with the expression vector CBS 110267 containing the son operon after the constitutive pro moter. -39