WO2002053772A1 - Novel biosensor system - Google Patents

Abstract

The present invention relates to the field of qualitative and/or quantitative detection of analytes. In particular, the present invention provides a biosensor system for the evaluation of the presence and quantity of chemical substances desired to be analyzed as well as processes using said biosensor system. The biosensor system according to the present invention uses metabolic/catabolic pathways of living cells which have been transformed to dsplay specific characteristics that can be used to identify the presence and quantity of a broad range of analytes.

Description

Novel biosensor system

The present invention relates to the field of qualitative and/or quantitative analyses and provides a new biosensor system for the detection of analytes which is based on a reporter gene that can also be used as a reporter system in molecularbiological approaches and pharmacological screenings. In particular, the present invention provides a biosensor system for the evaluation of the presence and quantity of chemical substances desired to be analyzed as well as processes using said biosensor system. The biosensor system according to the present invention uses metabolic/catabolic pathways of living cells which have been transformed to display specific characteristics that can be used to identify the presence and quantity of a broad range of analytes.

Numerous test systems are available which enable detection of certain analytes. However, these test sytems are mostly cost and labour intensive and only a small number of them are based on living cells or organisms.

Thus, there is a need to provide novel biosensor systems as well as suitable processes using the same which enable quick, reliable and low cost analyses.

The present invention is primarily based on the use of j?-hydroxyphenyl-pyruvic acid dioxygenase (HPD) cDNA as a reporter gene. cDNAs coding for

acid dioxygenase from different sources were cloned under the control of inducible promoters in appropriate vectors, which, upon transformation, ensure inducible expression of the reporter gene in selected model organisms such as E. coli and Saccharomyces cerevisiae. The enzymatic activity of the translation product HPD catalyzes the metabolization of p- hydroxyphenyl-pyruvic acid and the formation of homogentisic acid which is secreted from the transformed host cell into the extracellular space. Spontaneous oxidation of homogentisic acid by e.g. oxygen and/or NaOH forms ochronotic acid, a brown coloured pigment.

In accordance with the principles of the present invention, hpd-deήved expression vectors can be used as reporter and biosensor systems. Depending on the sensitivity of the promoter to the inducing agent (analyte), the emergence and quantity of the ochronotic pigment are time-dependent and saturable by an excess of substrate. The experiments conducted have shown, that the ochronotic pigment can most easily be measured quantitatively over a spectral range from 350 to 600 nm. To demonstrate, that hpd cDNA can be used as a versatile reporter, eg. for the detection of environmental pollution by heavy metals, in procaryotes and eucaryotes, it was cloned under the control of zinc and copper specific promoters in E.coli and Saccharomyces cerevisiae. The enzymatic activity, measured by the formation of ochronotic pigment, correlated with the transcriptional activity of the promoters. Due to the inherent features of the selected promoters, the detection limit of zinc was at 1 to 2,5 μM, and of copper at 5 μM. Since the analytical detection limit of zinc by ICP-AES is at 200 pM (4), it could be proven that cDNAs encoding j>-hydroxyphenyl- pyruvic acid dioxygenase can be used as reporters in low-cost, reliable, quantitative and easy-to-handle biosensor systems. As a proof-of-concept, another most familiar reporter system (i.e. β-galactosidase) was assayed under the same conditions. The results achieved were comparable but indicated, that this well-established system is more expensive and labour intensive, requires more experimental equipment and cannot be applied to high- thoughput analyses.

According to a first aspect, the present invention provides a novel biosensor system comprising a cell or organism harbouring a DNA molecule enabling said cell or organism to express 7-hydroxyphenyl-pyruvic acid dioxygenase under the control of a promotor which is inducible by the presence of substances or conditions to be analyzed. Preferably, the substances to be analyzed are selected from the group consisting of air, water and soil contaminants, toxins and toxic compounds, whereas said conditions are selected from the group consisting of salt, osmotic and oxidative stress.

As has been exemplified hereinbelow for the procaryotic promoter zntA and the eucaryotic promoter Cupl-2, a vast quantity of specific procaryotic and eucaryotic promoters can be applied in the biosensor system according to the principles of the present invention in dependence of the specific analytes or conditions to be detected and/or quantified. Such promotors can easily be identified by the skilled artisan and are preferably selected from the following also indicating the respective analytes to be detected with the aid of the selected promotor: Procaryotic promoters:

For the detection of the heavy metals zinc (Zn) and cadmium (Cd), zntA-ptomoter from E.coli and caάA-operon from Staphylococcus aureus, respectively, can suitably be selected. For mercury (Hg) and lead (Pb), the mer-4-promoter from E.coli will be appropriate. For the detection of nickel (Ni), the ^'A operon from E.coli can be selected, whereas for chrome (Cr), selection of chr from Alcaligenes eutrophus is preferred. Likewise, the αra-operon from Staphylococcus aureus can be selected for the detection of arsenic, whereas other metals (Fe, Cu, Al, Ni) can be detected by using the^t^'C-promoter from E.coli. The metals manganese (Mn) and cobalt (Co) are preferably detected by using the cor A -promoter from E.coli.

Examples for organic compounds which are detectable by carrying out the present invention comprise phenols (the /mp-operon from Pseudomonas sp.), benzene (the xylR and xylS- promoters from Pseudomonas), paraquat (the^ -9-promoter from E.coli, controlled by the SoxRS-regulon from E.coli).

Eucaryotic promoters:

For the detection of the heavy metals zinc (Zn), cadmium (Cd), and copper (Cu), the zrt- promoter, members of the zip-family from yeast (Zn), the ycfl -promoter from yeast (Cd), and the cupl -promoters from yeast (Cu), respectively, can suitably be selected. For other metals, such as iron (Fe), use of the gefl-mdftrl -promoters from yeast are preferred. For the evaluation of stress conditions, the promoters hall (salt), grel (osmotic), and aad4 (oxidative) from yeast are preferably selected.

It is clear, that the promoters exemplified above can be used in the native or modified form and can be substituted by selection of homologues from other species or organisms without departing from the principles underlying the present invention. Likewise, the present invention can be carried out by using novel promoter sequences which will be identified in the future to be inducible by certain chemical compounds, compositions or conditions to be detected.

Additionally, this soluble reporter system can be integrated into molecularbiological techniques for the detection and analysis of cw-acting elements (promoters, enhancers), signal transduction pathways in eucaryotes, and DNA-protein interactions. In pharmaco- logical approaches it can be applied in affinity screenings for membrane receptors and ligands.

It follows from the above that the reporter system as provided by the present invention can be applied to a broad range of technical fields which are all based on hpd expression analyses and which can briefly be examplified in a non-limiting manner as follows:

Induction studies to identify the features of a procaryotic/eucarvotic promotor According to a preferred embodiment of the present invention, a method is provided to study and monitor the activity of a promoter by use of a vector which comprises hpd cloned 3'- downstream of a multiple cloning site (MCS), followed by a transcriptional terminator. As it is well-known for eucaryotic vector systems, the turnover rate of HPD protein can be influenced by cloning a PEST-domain (Pro-Glu-Ser-Thr; (19)) into the coding sequence of hpd. After cloning a target promoter, whose features are to be identified, into the MCS of an appropriate vector backbone, a functional expression cassette for hpd is generated. Transient transformation of host cells with such a vector and subsequent selection (a selection system, like a constitutively expressed resistance cassette, is also a feature of the vector system) leads to the generation of transformants. The activity of the promoter can then be monitored in the dependence of agents and/or environmental factors (such as, e.g., light, salt, water, temperature). Due to the PEST-domain, which is preferably used in studies of eucaryotic promoters, the response to the exogenous agent and/or transcription factor can be diminished, i.e. HPD protein will undergo degradation within a few hours.

Due to the versatility of the inventive concept, studies can be performed in all living organisms, although eucaryotic cell or tissue cultures, whole plants or parts thereof, yeast and bacteria are preferred.

Screening system for identification of cis-acting elements (i.e. promoters, enhancer sequences) and trans-acting factors (i.e. transcription factors) According to another embodiment of the present invention, insertion of a known or putative promoter element into the multiple cloning site of an appropriate vector will promote the transcription translation (expression) of hpd gene products after transformation of suitable host cells. The expression can subsequently be analyzed by extracellularly detecting the quantity of ochronotic pigment accumulated in the culture medium.

By insertion of different lengths or parts of the overall promoter sequence and quantification of the ochronotic pigment synthesis (s.a.), e.g. the minimally required promoter region can be characterized. Furthermore, mutagenization of the promoter sequence allows the identification of functionally relevant sequence stretches. With the knowledge of the promoter sequence, a putative enhancer sequence 5'-upstream of the promoter can be identified, cloned and analyzed. The dependence of the promoter activity from the presence of a transcriptional activator, e.g. transcription factor, can be quantified by using the same vector for the transformation of two different cell cultures, one of them lacking or being deficient in the transcriptional activator. A comparable system using a different reporter

(secretory alkaline phosphatase) is already commercially available (SEAP reporter system 3;

Clontech, Heidelberg, Germany). In contrast to this well-established system, the screening system according to the invention allows direct analysis without the necessity of additionally performing and evaluating an enzymatic assay.

The general advantages of the system provided herein over state-of-the-art techniques are:

• The quantification of ochronotic pigment is in proportion to cellular RNA levels of hpd and can easily be performed in the cell culture medium. A measurement of promoter activity is thus achieved without lysing transformed cells.

• Kinetics of gene expression can easily and repeatedly be studied by sampling the same cultures.

• After assaying for hpd activity, transformed cells can be further studied using other methods, e.g. Northern blots, RNAse protection assays or Western Blots.

• Sample collection can be automated by using cultures grown in e.g. 96-well plates. Therefore, the system provided herein enables high-throughput screenings.

Analysis of signal transduction pathways Activation analysis of cis-acting enhancer elements in vivo

Vectors designed for this purpose and based on other reporter genes are already commercially available (e.g. Mercury profiling systems, Clontech, Heidelberg, Germany). According to another embodiment of the present invention, appropriate vectors comprise expression cassettes which may be e.g. composed from 5' to 3' of a specific cw-acting enhancer element, the TATA-like region from the thymidine kinase promoter or only its TATA-Box, and the hpd-reporter with transcriptional terminator. There are cw-acting elements described, which can be utilized according to the invention for the detection of transcription factors such as API (PKC-pathway), GRE (steroidhormone pathway), NFkB (NFkB-pathway) and SRE (MAPK-pathway). After transformation of host cells with such vectors and exposure of the host under suitable conditions, the easily detectable expression of the hpd-reporter directly indicates activation of a particular signaling pathway or pathways.

Analysis of DNA-protein interactions by a One-Hybrid Assay According to still another embodiment of the present invention, there is provided a screening method for isolating potential interaction partners of specific DNA-sequences, such as e.g. transcriptional activators, from a One-Hybrid library by preferably using yeast cells transformed with a 2 reporter-system comprising both the his3 -reporter and the hpd-reporter. Positive interactions are indicated by growth of transformed yeast cells on histidin-deficient agar plates. After this selection, false positive clones can be eliminated by growing them in normal yeast medium. The medium of positive clones turn ochre, due to the expression of HPD, whereas the medium of false positive clones does not change colour. Thus, the present invention provides a superior method, because the time consuming control-assay by another reporter (e.g. β-galactosidase-filter assay) can be omitted and positive colonies can directly be subjected to other analytical procedures (e.g. plasmid-rescue).

Receptor based ligand-affinitv-screening According to still another embodiment of the present invention, there is provided a method to screen for agonistic/antagonistic ligands having a specific affinity for a given receptor or extracellular domain or part thereof. Likewise, there is provided a method to perform studies on extracellular receptor domains desired to be characterized. The principle of these methods is based on a known signal transduction cascade, which is triggered by binding of a ligand to an extracellular domain or part of a transmembrane receptor. According to the invention, this binding can be measured by the accumulation of ochronotic pigment in the medium. For this purpose, the cascade will generally comprise a membrane receptor, being a chimeric protein composed of the cytoplasmatic and transmembrane domains of an already known receptor, and the extracellular domain or part of a receptor to be analyzed. An expression cassette for this chimeric receptor is part of a vector used to transform suitable host cells which are either transgene with respect to a hpd expression cassette according to the invention or which are co-transformed with such a genetic construct enabling expression profiling on the same or different vector. After ligand binding to this transmembrane receptor, a transcription factor will be released which activates the transcription of the hpd expression cassette. The translated gene product then catalyzes the synthesis of homogentisic acid, which is released into the medium where it spontaneously oxydizes to ochronotic pigment. The accumulation of ochronotic pigment is quantitatively dependent on the affinity of the ligand to the receptor. By this method, using hpd as a reporter, known receptors can be screened for their affinity to different ligands. It is also possible to screen mutagenized forms of a receptor for their ability to bind a distinct ligand.

An example for a suitable plasmamembrane receptor family in vertebrates, which can be selected as a component of the chimeric receptor, is the epidermal growth factor receptor (EGFR)-family. It consists of four members ErbBl, ErbB2, ErbB3 and ErbB4 (for review see 2). The replacement of the extracellular domain by e.g. the extracellular domain of the insulin-receptor leads to activation of the signaltransduction cascade after insulin-binding (38). In the cytoplasmic region three conserved sequence domains can be recognized: a region for feedback attenuation by protein kinase C (PKC), the tyrosine kinase domain and a C-terminal domain, which contains motifs for internalization and degradation of the receptor. EGF-receptor is activated by ligand-binding to the monomeric form and dimerizes before internalization. The chimeric receptor will consist of two forms, where the extracellular domain will be changed and the tyrosine kinase domain completely removed.

For signal transduction the split ubiquitin system may be used as a sensor of protein interactions in vivo (17; US Patent US 5,503,977). Formerly described for the interaction of soluble proteins in vivo, it has been demonstrated, that this system can be used to analyze the interactions of integral membrane proteins via a short signal transduction cascade (35). In our case, the system will be used for interactions between two different monomeric forms of EGF-receptor. Ubiquitin is a conserved protein of 76 amino acids, which is usually attached to the N terminus of proteins as a signal for their degradation (39).The ubiquitin moiety is recognized by ubiquitin-specific proteases, resulting in the cleavage of the attached protein. The cleavage can be visualized with a transcription factor attached to the C-terminus of ubiquitin, which after release leads to the activation of reporter gene expression (35). The ubiquitin-fusion can be divided and expressed in two parts, a N-terminal part (Nub/, amino acids 1-34, with / being isoleucin at position 13) and a C-terminal part of ubiquitin (Cub, amino acids 35-76 of ubiquitin), fused to a transcription factor. Nub/ and Cwb-reporter assemble in the cell and foπn split-ubiquitin. The split-ubiquitin is recognized by ubiquitin- specific proteases, resulting in the cleavage of the transcription factor attached to Cub. The protein interaction can be provided by two forms of monomeric EGF-receptor after ligand- binding, one fused to Nub and the other fused to Cwb-transcription factor.

The released transcription factor might be proteinA-LexA-VP16 (PLV), as already described in Stagljar et al. (35). It can activate the hpd reporter gene and by this allows the analysis of ligand-receptor binding via extracellular soluble ochronotic pigment.

Indicator of vitality during e.g. fermentation

The quality of the end product of many biotechnological processes is dependent on the vitality of the organism, of whom it has been synthesized. The expression of hpd under the control of a specific promoter in a transgenic organism can be an indicator for this. For example, changes in pH, temperature, osmolarity or vitality can be observed in a change of colour in the medium, being an indicator of stress conditions for the organism. Accordingly, the present invention provides a simple means to monitor the vitality of an organism during cultivation.

Brief description of the Figures Figure 1: Biochemical pathway for the degradation of L-tyrosine to ochronotic pigment. Figure 2: Formation of ochronotic pigment in over night-cultures of E. coli.

E.coli strain JM109 was transformed with pQE60 or pHPDQE, respectively. Cultures were induced with 500 μM IPTG and grown for 24 hours in LB/ampicillin (100 μg ml). Due to the presence of HPD in the culture of pHPDQE, homogentisic acid was formed which was oxidized on air to ochronotic pigment. This colour change was not observed in the control culture of pQE60. Figure 3: Identification of homogentisic acid in bacterial lysates of pHPDQE - cultures. Reversed phase Cig - HPLC was performed as indicated in the example section. (A) The chromatogram of lysates of E.coli transformed with pHPDQE (2) showed a prominent peak after 7,8 min. of separation. The retention time and the spectrum (B) of the component was identical with that of a separated HGA-standard (1), whereas separated lysates of E.coli transformed with pQE60 (3) lacked this component. Figure 4: Spectrum of ochronotic pigment.

The differential spectrum of a cleared lysate of cells transformed with pHPDQE was measured against a control lysate of transformed pQE60 cells by a wavelength scan between 750 to 350 nm in a spectrophotometer. Figure 5 : Detection of HPD (49 kDa) in induced lysates of JM109 (pHPDQE).

25 μg protein of J /O -lysates containing the indicated plasmids were induced for 24 hours (27°C; shaking) by the indicated IPTG-concentrations and separated under denaturing/reducing conditions by 10 % (w/v) SDS-

PAGE/coomassie-staining. JM109 was transformed/induced with: 1: pQE60/500 μM IPTG; 2:pHPDQE/0 μM IPTG; 3: pHPDQE/10 μM IPTG 4:pHPDQE/100 μM IPTG; 5: pHPDQE/500 μM IPTG; 6: pHPDQE/1 mM IPTG; 7: pHPDQE/10 mM IPTG. M: broad range protein marker (No. P7702S, New England Biolabs,

Schwalbach, Germany). Figure 6: Arabinose-induction of HPD expression in E.coli transformed with pBADHPD. 25 μg protein of lysates from Top 10 transformed with pBADHPD or the anti- sense construct pBADDPH and induced with different concentrations of arabinose were analyzed by (A) 10 % SDS-PAGE/coo assie or (B) immuno blot with affinity-purified mouse anti-HPD-antibodies. Whereas in (A) the induction of HPD was not evident, immuno blot analysis (B) revealed a steep increase of HPD expression dependent on the arabinose-concentration. 1: negative control ρBADDPH/2 x 10^"2 % arabinose; 2: pBADHPD/ 0 % arabinose; 3: pBADHPD/2 x 10^"5 % arabinose; 4: pBADHPD/ 2 x 10^"4 % arabinose; 5: pBADHPD/2 x 10^"3 % arabinose; 6: pBADHPD/2 x 10^-2 % arabinose; 7: pBADHPD/2 x 10^"1 % arabinose; M: broad range protein marker. Figure 7: Enzymatic activity measured as formation of ochronotic pigment in induced Top 10 transformed with pBADHPD.

The absoφtion of cleared cell lysates transformed with pBADHPD and induced with different concentrations of arabinose was measured against a Top 10 control (untransformed; induced with 2 x 10^'2 % arabinose). (A) semilogarhythmic plot of absoφtion versus log arabinose-concentration. The absoφtion was measured at defined wavelengths:

— •— optical density at 350 nm ^• - 0 - - optical density at 400 nm ^■ • optical density at 450 nm — 0 - - optical density at 500 nm • optical density at 550 nm — O ^• - optical density at 600 nm

(B) picture of induced cleared lysates. Figure 8: Time dependence of enzymatic activity.

A culture of Top 10 transformed with pBADHPD was induced with 2 x 10^"2 % arabinose and the formation of ochronotic pigment in the cleared lysate was spectroscopically measured against induced (2 x 10^"2 % arabinose) Top

10 control lysates within time (A). The absorption was measured in two independent experiments at defined wave-lengths as indicated. (B) Picture of the time-course of the cleared lysates after induction. Figure 9: Zn²⁺-induction of HPD expression in E.coli transformed with pzntHPD. 25 μg protein of lysates from Top 10 transformed with pzntHPD and induced with different concentrations of ZnCl₂ were analyzed by (A) 10 % (v/v) SDS- PAGE/coomassie or (B) immuno blot with affinity-purified mouse anti-HPD- antibodies. Whereas in (A) the induction of HPD was not evident, immuno blot analysis (B) revealed a steady increase of HPD expression dependent on the Zn (IΙ)-concentration. 1: untransformed Topi 0/50 μM Zn (II); 2: pzntHPD/0 μM Zn (II); 3: pzntHPD/1 μM Zn (II); 4: pzntHPD/2,5 μM Zn (II); 5: pzntHPD/5 μM Zn (II); 6: pzntHPD/10 μM Zn (II); 7: pzntHPD/25 μM Zn (II); 8: pzntHPD/50 μM Zn (II); 9: pzntHPD/100 μM Zn (H); M: prestained protein marker (No. 7708S, New England Biolabs; Schwalbach, Germany). Figure 10: Time dependence of enzymatic activity. A culture of Top 10 transformed with pzntHPD was cultivated in Zn (II)- minimal medium and induced with 20 μM ZnCl₂. The formation of ochronotic pigment in the cleared lysate was measured spectroscopically at 400 nm against an induced (20 μM Zn(II)) Top 10 control lysate within time. Figure 11 : Substrate dependence of enzymatic activity. A culture of Top 10 transformed with pzntHPD was cultivated in Zn (II)- minimal medium and induced with 20 μM ZnCl₂. Different concentrations of L-tyrosin were added and the formation of ochronotic pigment in the cleared lysate was measured spectroscopically at 400 nm against an induced (20 μM Zn(II)) Top 10 control lysate after 24 hours. (A) linear plot of absoφtion versus L-tyrosin-concentration. (B) visible colour change in the clear lysates in 24 well-plates. Abbreviations: M9, M9-medium without cells; C: E.coli ToplO without external addition of ZnCl₂ and L-tyrosine; Z: E.coli Top 10 without external addition of L-tyrosine. Figure 12: Inducibility of enzymatic activity is dependent on log (Zn(II)). Top 10 transformed with pzntHPD were cultivated in Zn (IΙ)-minimal medium, including 500 μM L-tyrosine and induced with different concentrations of ZnCl₂. (A) semilogarhythmic plot of absorption versus logC (Zn(II)) measured spectroscopically at 400 nm against an induced (20 μM Zn(II)) Top 10 control lysate after 24 hours. (B) visible colour change in the clear lysates in 24 well-plates. The formation of ochronotic pigment in the cleared lysate was dependent on the concentration of the inducer and revealed a linear kinetic within the range 1 μM to 10 μM Zn (II). Abbreviations: M9, M9-medium without cells; BL, induced (20 μM Zn(II)), non-transformed BL21DE3(RIL); -T, non-induced, transformed TOP10 in M9-medium without L-tyrosine. Figure 13: The induction of a bacterial hpd by Zn(II) is similar to that of hpd from Arabidopsis.

BL21DE3(RIL) transformed with pzntSYN were cultivated in Zn (II)-minimal medium, including 500 μM L-tyrosine and induced with different concentrations of ZnCl₂. Absorbance was measured spectroscopically at 400 nm against an induced (20 μM Zn(II)) Top 10 control lysate after 24 hours. Like in Zn(II)-induced E.coli transformed with pzntHPD, a linear increase of absorption can be observed within the range of 1 to 10 μM Zn (II) in lysates. Figure 14: Inducibility of CUPl-2 promoter and enzymatic activity of HPD dependent on logC (Cu(II)) in transformed cells with an eucaryotic background (yeast).

Saccharomyces cerevisiae strain INVScl transformed with pYCA were induced for 48 hours with different concentrations of CuSO . The absorbance of cell lysates were measured spectroscopically at 400 nm against an induced (500 μM Cu(II)) yeast control and plotted versus logC (Cu(II)). The formation of ochronotic pigment in the cleared lysate was dependent on the concentration of the inducer and revealed a linear kinetic within the range of 5 μM to 500 μM Cu (II). Figure 15: Comparison of ochronotic pigment formation in yeast supernatants and lysates. Cultures of Saccharomyces cerevisiae strain InVScl were induced for 48 hours with different concentrations of Cu(II) as described. 1 ml-aliquots of cultures were harvested and supernatants harvested after a 20 000 x g centrifugation for 5 min. at room temperature. The "development of colour" was performed by the addition of NaOH to a final concentration of 0.1 N. Lysates were gained by the addition of NaOH, a 10 min. incubation at room temperature and a separation from cell debris by a 20 000 x g centrifugation for 5 min. at room temperature. No significant differences in absoφtion at 400 nm between lysates and supernatants could be measured, as depicted in a bar plot (absorbance versus Cu(II) concentration). Figure 16: Spectrum of ochronotic pigment in lysates of yeast.

The absorbance of yeast lysates transformed with pYCA were measured from 750 nm to 300 nm against an untransformed control after a period of 48 hours induction with 500 μM Cu(II). The spectrum reveals no maximum in absorbance and looks similar to the spectrum of ochronotic pigment in bacteria transformed with a hpd expression cassette (Figure 4). Figure 17 : Induction of β-galactosidase in a reference system.

Top 10 bacterial cells were transfomed with pzntGal, a vector with a β- galactosidase expression cassette in the same genetical background as pzntHPD and induced with different concentrations of Zn(II) for 24 hours. After performance of the β-galactosidase assay, lysates were measured at 420 nm and at 550 nm as a reference. The cell concentration in harvested culture suspensions was constant independent of the concentration of the inducer as indicated by the absorbance at 600 nm.

Δ absorbance at 420 nm vs Zn(II) O absorbance at 550 nm vs Zn(II) • absorbance at 600 nm vs Zn(II)

Figure 18 : Standardization of ochronotic pigment formation.

Over night-cultures of untransformed Top 10 were incubated (24 hours at 37°C with shaking) with different concentrations of standardized homogentisic acid. Afterwards lysates were gained by the addition of NaOH, a 10 min. incubation at room temperature and a separation from cell debris by a 20 000 x g centrifugation for 5 min. at room temperature. As indicated by a plot of absorbance (400 nm) versus concentration of added homogentisic acid the formation of ochronotic pigment can be standardized.

The formation of ochronotic pigment has been linked to the metabolism of aromatic amino acids. In the oxidative degradation of L-tyrosine (Figure 1), it is first converted to p- hydroxyphenyl-pyruvate by transamination with alpha-ketoglutarate, in a reaction catalyzed by aromatic transaminase (20). ;?-hydroxyphenyl-pyruvic acid dioxygenase (HPD) then catalyzes its oxidation to homogentisic acid (HGA) in a complex reaction, involving hydroxylation of the phenyl ring, decarboxylation, oxidation and migration of the side chain (14). The enzyme appears to be ubiquitary in living organisms and has been purified from vertebrates (22, 28, 40), procaryotes (21) and plants (9). cDNAs and encoding genes have been identified from numerous sources, such as mammals, fungi, bacteria and plants (9, 6, 29, 26). They show between 25 % to 95 % identity at the amino acid level (26). With respect to the employed HPDs from Arabidopsis thaliana and Synechocystis sp. PCC6803, there is a 39,7 % identity on cDNA-level and a 32,3 % /40,2 % identity/similarity on the protein-level (GAP-program; GSG software package),

HGA, the product of the reaction, is further catabolized by the next enzyme in the degradation pathway, homogentisic acid 1,2-dioxygenase (Figure 1). This enzyme has also been purified to homogeneity from different sources (32, 37) and the respective genes have been identified (33, 11, 23, 7).

In alkaptonuria, the first described human heriditary metabolic disorder (10), the enzyme homogentisic acid 1,2-dioxygenase is deficient (41). This leads to accumulation of HGA in the urine of alkaptonuric patients, which turns dark when made alkaline and exposed to oxygen. The direct oxidation product of HGA is benzoquinone acetic acid (Figure 1) which is quickly polymerized to an ochre pigment, called ochronotic acid. From this pigment, neither the chemical nature nor the mechanism of its biosynthesis is known. Since it was even extracted and identified from a 3500 years old ochronotic egyptian mummy by spectral comparison of experimentally oxidized homogentisic acid with this ochronotic pigment, it is well characterized in terms of spectral properties, solubility, and stability (36, 5). The spontaneous oxidation of homogentisic acid to ochronotic pigment and its measurement was used to build up the quantifiable biosensor system according to the present invention.

The present invention is explained in more detail in the following examples which should be understood to exemplify but not to limit the broad inventive concept.

DNA manipulations and bacterial strains Standard molecular biological techniques were carried out according to the manufacturers' protocols or according to 21. All cloning was carried out in the E.coli strain Top 10 unless noted otherwise and plasmid construction confirmed by DNA sequencing.

Plasmid construction and expression

Cloning of pHPDQE and purification of HPD

Hpd from Arabidopsis (26, 1) was amplified from an Arabidopsis thaliana Matchmaker cDNA library (Clontech, Heidelberg, Germany) by proof-reading PCR using the primers hpdfor (5'-TGAAATCCATGGGCCACCAAAACGCCGCCGTT-3'; SEQ ID No.l) and hpdback (5'- TCTTCTTGTGGATCCCACTAACTGTTTGGC-3*; SEQ ID No.2). The 1355 bp-PCR-fragment was digested with Ncol and BamΗl and inserted into pQE60 (Qiagen, Hilden, Germany) which had been digested with the same enzymes. The resulting plasmid pHPDQE was transformed into E.coli strain JMl 09. For overexpression of HPD, log-phase JMl 09 (OD₆₀O = 0,7) cells were induced by adding 500 μM IPTG, grown for 5 hours at 27°C and then harvested. Because of the 3'-terminal fusion between the hpd-coding sequence and the Hw₆-codons of the vector, ΗPD could be purified by Talon metal affinity chromatography according to the manufacturer's protocol (Clontech, Heidelberg, Germany). Cloning of pBADHPD The coding sequence of hpd was amplified from pHPDQE by Taq-PCR using the primers hpd5 (5'-ACCATGGGCCACCAAAACGCCGCC-3*; SEQ ID No.3) and hpd3 (5'- TCCCACTAACTGTTTGGCTTCAAG-3'; SEQ ID No.4), obtaining a 1337bp-PCR- fragment. This was inserted into pBADTopo TA (Invitrogen, Groningen, Netherlands) according to the manufacturer's protocol. E.coli strain Top 10 was transformed with the resulting plasmid pBADHPD and cultures were used for the induction experiments.

Cloning of pzntHPD Using the primers znt5 (5'-TCCGTGCGGATATCGCGATTGCTGCGG-3': SEQ ID No.5) and znt3 (5'-CAGGAGTCGCCATGGCATCCTCCGGTT-3*; SEQ ID No.6) the zntA- promoter was amplified from the E.coli chromosome by proof-reading-PCR. The 163 bp fragment was digested with Ncol and EcoRY and cloned into pBADHPD which had been digested with the same enzymes. Thus, the αrα-promoter, the enterokinase-domain and most of the αraC-regulator were deleted. In the resulting vector pzntHPD, the distance and sequence between zHtA-promoter and hpd-coding sequence were the same as between zntA- promoter and z«tA in the native context of the E.coli chromosome. E.coli strain Top 10 was transformed with the resulting plasmid pzntHPD and respective cultures were used for induction experiments.

Cloning ofpSYN Hpd from Synechocystis sp. PCC6803 (open reading frame slr0090) was amplified from 25 ng genomic DΝA by proof-reading-PCR with the Advantage cDΝA PCR Kit (Clontech, Heidelberg, Germany) using the primer pair HPDsyn3 (5*-GGATCCATGGAATTCGA- CTATCTTCATTTA-3*; SEQ ID ΝoJ) and HPDsyn4 (5"-TGGCACTTCTAACTGTTTT- TCTAA-3'; SEQ ID Νo.8). After denaturation for 1 min at 94°C, 30 cycles of denaturation/annealing polymerisation (20s at 94°C/50s at 55°C/150s at 68°C) were performed, followed by an extension at 68°C for 5 min. 3'-A-overhangs necessary for TA- cloning were generated by incubating the gel-purified PCR-product with 0,5 units of Taq- polymerase and an excess of dATP in the appropriate buffer for 10 min at 72°C. The resulting 1023 bp PCR-fragment of hpd was cloned into pBADTopo (Invitrogen, Groningen, Netherlands) according to the manufacturer's protocol, resulting in vector pSYN. Cloning ofpzntSYN Using the primers znt5 (5'-TCCGTGCGGATATCGCGATTGCTGCGG-3'; SEQ ID No.5) and zn^βmod (5'-GTCGGGATCCCATCCTCCGGTTAAGTTTTTTCT-3'; SEQ ID No.9) the zflt-4-promoter was amplified from pzntHPD by proof-reading-PCR as above (denatiiration at 94°C for 2 min; 30 Cycles: 30s at 94°C/45s at 60°C/60s at 68°C; extension at 68°C for 5 min). The 138 bp fragment was digested with BamHl and EcoRV and cloned into pSYN which had been cut with the same enzymes, h the resulting vector pzntSYN the distance and sequence between z«t^-promoter and bpd-coding sequence were the same as between z«t_4-promoter and zntA in the native context of the E.coli genome. E.coli strain B121(DE3)R1L was transformed with pzntSYN and respective cultures were used for induction experiments.

Cloning of pYCUP The CUPl-2 promoter of Saccharomyces cerevisiae, located on chromosome VII coordinates 214718 to 214533, was amplified by proof-reading-PCR from yeast genomic DNA (preparation see 21) with the Advantage cDNA PCR Kit (Clontech, Heidelberg, Germany) according to the manufacturer's protocol. PCR was performed by denaturation for 2 min at 94°C, 30 cycles (30s at 94°C/45s at 60°C/60s at68°C) and an extension of 5 min at 68°C, using primers CUP5 (5'-TTAGGAGCTCGATCCCATTACCG- ACATTTGGGCG-3'; SEQ ID No.10) and CUP3 (5'-TATCGGATCCTACAGTTTG- TTTTTCTTAATATCTATTTCG-3'; SEQ ID No.11). The achieved 430 bp fragment was digested with Sacl and BamHl and ligated into the equally digested vector p426ADH (23), resulting in plasmid pYCUP.

Cloning ofpYCA

For cloning of hpd from Arabidopsis thaliana into pYCUP under the control of the CUPl-2 promoter, hpd was amplified by proof-reading-PCR as above from pBADHPD, using the primers YHPD5 (5'-TATCGGATCCATGGGCCACCAAAACGCCGCC-3'; SEQ ID No.12) and YHPD3 (5'-TTAGAAGCTTTCATCCGACTAACTGTTTGGCTTC-3'; SEQ ID No.13). After denaturation for 2 min at 94°C, 30 cycles ( 30s at 94°C/45s at 60°C/60s at 68°C) and an extension of 5 min. at 68°C were performed. Hpd of 1300 bp was digested with BamHl and Hindlll and ligated into pYCUP which had been cut with the same enzymes. The resulting vector pYCA was transformed into chemocompetent Saccharomyces cerevisiae strain JNNScl (Invitrogen, Groningen, Netherlands) and plated on selective plates without uracile. Incubation of transformed yeast clones of pYCA and induction with Cu (II) was performed in the same selective liquid medium.

Cloning ofpzntGal β-galactosidase (EC: 3.2.1.23; b0344 in E.coli K12 genomic database)-DNA was amplified from genomic DNA of E.coli K12 CGSC strain 6821 by proof-reading PCR (1 min at 94°C; 35 cycles: 15s at 94°C/40s at 50°C/5 min at 68°C; extension for 6 min at 68°C), using the primers Gal5 (5'-ACAGCCATGGCCATGATTACGGATTCACTGGCC-3'; SEQ ID No.14) and Gal3 (5'-TCCCCCGGGCACGTGTTATTTTTGACACCAGACCAACTGGTA-3'; SEQ ID No.15). 3'-dA-overhangs were generated by incubating the purified PCR-product with Taq-polymerase and an excess of dATP in the appropriate buffer for 10 min (72°C). To facilitate digestion with Pmll and Ncol the fragment was cloned into pCR4.1Topo (Invitrogen, Groningen, Netherlands). Nector pzntHPD was digested with Eel 13611 and Ncol and ligated with the respective, repurified fragment of β-galactosidase. Transformants of pzntGal in ToplO were used for induction assays with Zn (II).

Competent Yeast Cells Saccharomyces cerevisiae cells strain INVScl (Invitrogen, Groningen, Netherlands) were grown over night in YPD-medium at 30°C. For inoculating the starter culture the next day 1,5 ml over night-culture were used in 100 ml of YPD-medium. Cells were grown at 30°C until the OD₆oo was 0,6. They were harvested by a 5 min. centifugation at 1600 x g and resuspended in 50 ml solution A (15 g ethylene glycole/5 ml 1 M bicine pH 8.35/91,1 g sorbitol, diluted in water and filtrated in a final volume of 500 ml). The suspension was pelleted again as above, resuspended in 2 ml solution A before adding 110 ml dimethyl- sulfoxide and aliquotated in 200 μl portions. Until transformation they were stored at -80°C.

Yeast transformation Saccharomyces cerevisiae strain INVScl (Invitrogen, Groningen, Netherlands) was transformed by adding 1 μg DNA to 200 μl of frozen competent cells, followed by a 5 min. incubation at 37°C, thereby carefully mixing every minute of incubation. Afterwards, 1,5 ml of solution B (60 g poly ethylene glycole 1000/30 ml 1M bicine pH 8,35 diluted in H₂0_dest. and filtrated in a final volume of 150 ml) was added and the mixture was incubated for one hour at 37°C. Then cells were harvested by a 3 min. centifugation at 900 g and the resulting pellet carefully resuspended in 1,5 ml solution C (1, 32 g 150 mM NaCl/1,5 ml 1 M bicine pH 8,35 diluted in H₂0d_est- and filtrated in a final volume of 150 ml). The cells were again harvested as above, the pellet then resuspended in 200 ml solution C and plated on selective plates. Usually the first cultures developed after 3 days at 30°C.

Protein-analytical methods Affinity-purification ofantisera Purified recombinant HPD was used for immunization in mice. The antiserum was affinify- purified by binding to the electro-transferred overexpressed antigen onto nitrocellulose according to the method described by Smith and Fisher (34). For immunoblot analysis this antibody preparation was used in a 1:5 (v/v) dilution, corresponding to a 1:200 (v/v) dilution with respect to the serum.

SDS-PAGE and Western Blot

Aliquots of 1 ml bacterial culture were centrifuged for 5 min at 21.000 x g, the cell pellet resuspended in 100 μl of 1 mM DTE/10 mM MgCl₂/100 mM Tris-HCl, pH 7,4 by sonication (Branson Sonifier, USA) and quantified according to the method of Lowry, modified by Schacterle and Pollack (31). The protein yield was separated by SDS-PAGE (18) and visualized with Coomassie Brillant Blue R or by silver staining (15).

For immunoblot analyses the proteins were electrotransferred "semidry" onto nitrocellulose membranes using a Multiphor II device (Amersham-Pharmacia, Freiburg, Germany). Blots were controlled by staining reversibly with 0.02 % (w/v) Ponceau S in 3 % (v/v) trichloro- acetic acid (TCA). Analysis was canied out using the ECL-detection system (Amersham- Pharmacia, Germany), according to the manufacturer's instructions. An anti-mouse IgG peroxidase conjugate (A-4416, Sigma, Germany) was used as secondary antibody in a 6000- fold dilution.

Induction of promoter activity and measurements

Induction ofprocaryotes in LB-broth (30) Over night cultures of transformed E.coli strain Top 10 were inoculated in LB-broth, containing 100 μg/ml ampicillin. 1 ml of these cultures was inoculated in 100 ml LB/ampicillin and grown at 37°C for 2 hours with shaking (180 rpm). Cultures were cooled for 5 min. on ice. When a time course was performed, defined amounts of inducer were added. Final concentrations in the cultures were 500 μM IPTG, 2 x 10^"2 % (w/v) arabinose or 5 μM ZnCl₂, depending on the vector system. Non-transformed Top 10 control cultures in LB-broth were treated likewise.

In promoter induction assays, defined concentrations of inducer were added to the transformed cultures after aliquoting in 10 ml to sterile 50 ml-tubes (Greiner, Frickenhausen, Germany). The Top 10 control cultures were induced by 1 mM IPTG, 2 x 10^"2 % (w/v) arabinose or 50 μM ZnCl₂.

Cultures were then grown at 22°C with shaking (180 φm) for 24 hours (promoter induction assays) or for a different time interval. Afterwards, 20 μl 5 N NaOH were added to 1 ml of culture to give a final concentration of 0,1 N NaOH, vortexed and incubated at room temperature for 10 min. Supernatants were cleared by a short centrifugation (5 min., 21.000 x g) at room temperature and wavelength scanned in a 2-beam Uvikon 941plus spectrophotometer (Kontron Instruments, Milan, Italy) against the induced ToplO control.

Induction ofprocaryotes in Zn (II) -minimal medium The Zn (IΙ)-minimal medium was a sterile filtrated M9 minimal medium (30), containing 2

% (w/v) glucose/ 1 mM MgSO₄x7H₂O/ 15 mM thiamine pyrophosphate and a dropout supplement (30 μg/ml L-isoleucin, 150 μg/ml L-valin, 20 μg/ml L-adenine hemisulfate, 20 μg/ml L-arginine HC1, 20 μg/ml L-histidin monohydrate, 100 μg/ml L-leucin, 30 μg/ml L- lysine HC1, 20 μg/ml L-methionine, 50 μg/ml L-phenylalanine, 200 μg/ml L-threonine, 20 μg/ml L-tryptophane, 30 μg/ml L-tyrosine, 20 μg/ml L-uracil). E. coli strains Top 10 and

BL21(DE3)RIL transformed with Zn (IΙ)-inducible vectors (see above), were grown in this medium/ 100 μg/ml ampicillin at 37°C and shaking (180 rpm) to an OD (600 nm) = 0,5 prior to induction. In time course measurements the culture was induced with 20 μM ZnCl₂, in the induction course with different concentrations of Zn (II). For the assays concerning substrate dependence, different amounts of L-tyrosine were added at the time point of induction together with 20 μM Zn (II). After induction cultures were grown in test tubes or 24 well plates (Greiner, Frickenhausen, Germany) at 37°C with shaking (180 rpm) for 24 hours, unless time courses were made. Harvesting of the cultures, "development of colour" by NaOH and spectrophotometrical measurements were performed like described before.

β-Galactosidase-assay ofprocaryotes after Zn (II) induction For assays using the pzntGal-vector construct, the detection of β-Galactosidase activity was performed as recommended in most laboratory manuals (30): 1 ml of the previously induced transformed Top 10 culture was pelleted by a short centrifugation (5 min., 21.000 x g) and resuspended in 800 μl Z-Buffer. The cell mix was diluted 1/100 (v/v) with Z-Buffer, then 40 μl of 0,1% (w/v) SDS and 60 μl of chloroforme were added on ice and the mixture was vortexed for 15 sec. After a 15 min. equilibration at 30°C 160 μl of 4mg/ml o-nitrophenyl-β- D-galactopyranoside was added and the mixture vortexed again for 10 sec. Timing started with incubation at 30°C. The reaction was stopped by adding 400 μl of 1 M NA CO₃ and supernatants cleared from cell debris by centrifugation (10 min., 21.000 x g). Supernatants were analyzed at 420 nm in a 2-beam Uvikon 941plus spectrophotometer (Kontron Instruments, Milan, Italy) against the induced ToplO control.

Induction of transformed Saccharomyces cerevisiae cells in Cu (II) -minimal medium The Cu (II)-minimal medium was a sterile filtrated YNB (Yeast Nitrogen Base)-medium, containing 2% (w/v) glucose, supplemented with 20 mg/ml L-histidine-HCl, 30 mg/ml L- leucine and 20 mg/ml L-tryptophan (1,5 % agar was added for plates) to ensure selection for His^", Leu^", Tφ^" and Ura⁺ (selective marker on p426ADH).

Induction of yeast with Cu (II) Yeast liquid cultures were grown for 2 days at 30°C post inoculation and induced with different amounts of Cu (II) by adding diluted solutions of CuSO . Substrate dependency was measured by adding different amounts of L-tyrosine together with 100 mM Cu(II).

After induction, cultures were grown for 48 hours at 30°C. Harvesting of the cultures, "development of colour" by NaOH and spectrophotometrical measurements were performed as described above. Identification of HGA bv HPLC The identification of HGA in supernatants of bacterial cultures was performed according to Denoya et al. (5). log-cultures of E.coli, transformed either with pHPDQE or pQE60, were induced at OD(600 nm) = 0,5 for 4 hours with 500 μM IPTG. Aliquots of 1 ml were harvested, mixed with 100 μl glacial acetic acid and centrifuged 5 min at 21.000 x g. The recovered supernatants were diluted threefold with 10 mM acetic acid and filtered with a Millipore HV filter (0,2 μm pore size). 20 μl of the probes were applied to a HPLC system consisting of a Nucleosil Cι₈ column (5 μm; 200 mm; Macherey -Nagel, Dϋren, Germany) and a linear gradient system using 10 mM acetic acid/methanol (85:15, by vol.) at a flow rate of 0,8 ml/min. UV/VIS spectra were monitored by a photodiode array detector (Waters 986, Eschborn, Germany) and chromatograms analyzed at 290 nm using the Millennium software package PDA software (Waters, Eschborn, Gennany). Products were identified by chromatographic comparison to a 500 μM HGA-Standard (Sigma-Aldrich, Germany), diluted in LB-brotiVlOmM acetic acid. The spectrum of HGA had a maximal absoφtion at 290 nm.

Functional analysis of HGA and spectrum of ochronotic pigment The already identified hpd-cDNA from Arabidopsis (26, 1) was cloned into the procaryotic expression vectors pQE60 and transfered into E.coli. Overexpressed enzyme was functionally analyzed. As already reported for this gens in a different expression vector system (26), cultures of E.coli transformed with pHPDQE developed a dark-brown colour over night after induction, whereas cultures transformed with the empty vector pQE60 and treated as well, did not (Figure 2). This pigment could be separated from the bacteria by a 5 minute- centrifugation at 21.000 x g and remained soluble in the supernatant. Addition of alkali to the cell-free supernatant enhanced the oxidation of HGA to ochronotic pigment. When E.coli cells transformed with pHPDQE were grown on LB-plates supplemented with antibiotics and L-tyrosine (60 μg ml), the ochronotic pigment was detectable in the agar, whereas the bacteria did not change colour.

These two observations indicate that HGA is secreted to the medium and then will be oxidized. To prove that the formation of the pigment was due to the enzymatic activity of HPD in the transformed cells, cleared lysates from cultures transformed with pHPDQE and pQE60 respectively were analyzed by HPLC (Figure 3). These cultures were only induced for a short time period (4 hours) and acidified before centrifugation to allow the detection of HGA and to inhibit its oxidation to ochronotic pigment as described (36, 24). The separation of the pHPDQE lysate (Figure 3 A; curve 2) revealed a prominent peak at 7.8 min., that co- migrated with the HGA standard (Figure 3A; curve 1) and had the identical spectrum and absoφtion maximum of 290 nm (Figure 3B, curves 1 and 2). As the separation of pQE60 control lysate only revealed a minor peak at 7,6 min. (Figure 3, curve 3) with a different spectrum (absorbance maximum at 257 nm), these results indicate an enzymatically active HPD in E.coli transformed with pHPDQE.

To analyze the dark brown pigment, cultures of E. coli transformed with pHPDQE and pQE60 were harvested after 24 hours of induction with 500 μM IPTG and equilibrated to a final concentration of 0,1 N NaOH to allow the quantitative oxidation of HGA at high pH. The differential spectrum of the lysates (Figure 4) revealed an increase of absoφtion within the range of 600 to 350 nm in pHPDQE lysates, which is a characteristic for the formation of ochronotic pigment (36). The spectrum describes a hyperbolic curve without any absolute spectral maximum.

Although the foe-promoter in pHPDQE is an inducible promoter, it is not tightly regulated.

The basic expression levels of HPD in transfonned cells were already quite high without induction (Figure 5). Thus, overexpressed protein could be detected already in non-induced cells (Figure 5; lane 2) compared to the control (Figure 5; lane 1). When inducing with 100 μM IPTG, the concentration of the protein in lysates of cultures transformed with pHPDQE increased to a maximum, were half of the protein in the lysate was HPD (Figure 5; lane 4).

Even if the foe-promoter was repressed by adding 2 % (w/v) glucose to the medium, translational activity in the culture was still present. Because of the enormous expression of

HPD in transformed cells, the pQE60-vector system was inadequate for quantification.

Time linearity and inducibility To improve the measurements in terms of time-linearity and inducibility a different promoter for regulated expression of HPD was chosen. For this puφose hpd-cDNA from pHPDQE was cloned under the control of the rα-promoter in pBADTopoTA. This expression system is described as being tightly regulated by the araC gene product encoded on the same vector (3, 13). The resulting vector pBADHPD was transferred into E.coli cells and cultures induced by different concentrations of arabinose. When separating the respective lysates by 10 % SDS-gelelectrbphoresis HPD was not detectable after coomassie-staining of the gel (Figure 6A). However in a Western Blot, using affinity-purified mouse-anti-HPD-antibodies, the induction of the αra-promoter could be demonstrated (Figure 6B). According to HPD expression, this promoter is tightly regulated, showing only marginal expression in the non- induced sample (Figure 6B; lane 2) and a steep increase from 2 x 10^"5 % to 2 x 10^"1 % (w/v) arabinose (Figure 6B; lanes 3 to 7).

These results indicate a much lower protein expression of HPD in the used vector system in comparison to the foe-vector system. Nevertheless the system was sensitive enough, that the protein expression was also reflected in the enzymatic activity of the induced probes (Figure 7), which could also be detected by a visible colour change (Figure 7B). Inducing in a range of 2 x 10^"4 % to 2 x 10^"2 % arabinose gives linearity of absoφtion in a wavelength range from 600 to 350 nm in a semilogarhythmic plot compared to the non-induced control (Figure 7 A). This demonstrates clearly that the formation of ochronotic pigment can be used as a quantitative indicator of promoter activity.

Another feature of enzymatic activity is its time-dependency. When a culture of E.coli transformed with pBADHPD was induced with 2 x 10^"2 % arabinose the concentration of ochronotic pigment increased within time (Figure 8). The increase was linear between 17 to 30 hours after induction and then reached saturation (Figure 8 A). This could also be noticed by eye without the utilization of a spectrophotometer (Figure 8B). For cultivation of the bacteria only the standard LB-broth is required. The addition of L- tyrosine to the medium leads to a prolongation of time linearity and to an increase in absorption. Since already an absoφtion of > 1 can be measured in induced cultures, supplementation with L-tyrosine is not necessary. The sole requirement of cultivation medium for promoter analysis makes it to an unexpensive detection system.

Example for a biosensor system for environmental pollution The sensor system based on hpd-cDNA and an αrα-promoter might appear quite artificial.

Therefore, as a proof-of-concept, a biosensor system was established which can directly be applied for measurements of environmental heavy-metal pollution. The transcriptional control protein MerR in E.coli is a metalloregulatory switch, activating transcription of a mercury resistance operon in the presence of mercuric ions and repressing transcription in their absence (4). It is a direct Hg(II) sensor that catalyzes transcriptional activation of a Hg(II) efflux gene. Since working with Hg(II) is harmful, we chose a Zn(II)-responsive MerR homologue in E.coli, ZntR. ZntR is a trans activator of zntA transcription and binds to the z«tA-promoter. The induction of this promoter by Zn(II) via ZntR is well characterized on a transcriptional level by run-off transcription assays (27).

The zrctA-promoter was amplified from E.coli K12 - DNA by proof-reading-PCR and cloned into pBADHPD, thereby deleting the tfrø-promoter, the enterokinase-domain and most of the røC-regulator. Cloning of the zntR-gene in the resulting vector pzntHPD was unnecessary, since it is already an endogeneous feature of E.coli genome. Nevertheless, it was tried to generate a vector consisting of a z/.tR-gene under the constitutive control of the nptll- promoter. Only expression cassettes with zntR in the wrong orientation were obtained indicating, that a constitutive overexpression of the regulator might be harmful in E.coli.

Vector pzntHPD was transformed into E. coli, cultures grown in LB-broth and induced with different concentrations of Zn (II) ranging from 0 to 300 μM. When 25 μg protein of the cell lysates were separated by a 10 % SDS-gelelectrophoresis and stained with coomassie the expression of HPD was undetectable compared to an induced negative control (Figure 9A). However Western Blot -analysis revealed a clear Zn(II)-dependent induction of HPD (Figure 9B). Unlike the steep increase under αrø-promoter-control, a steady increase of HPD protein was detectable from 1/ 2,5/5/10 to 25 μM Zn (II), reaching a plateau of expression (Figure 9B; lanes 3 to 9).

The formation of ochronotic pigment in these cultures was analyzed 24 hours after induction. A high basal level of absorption could already be observed in non-induced, transformed E.coli, when compared to an induced untransformed control (data not shown). Since in lysates of non-induced cultures HPD expression could already be seen on Western Blot (Figure 9B; lane 2), it was concluded that the high background was a consequence of the Zn (IΙ)-content in LB-broth.

Therefore the same assay was repeated in an optimized Zn (IΙ)-minimal medium. E.coli was growing normal in this novel medium, showing a time course comparable to time kinetics with pBADHPD when induced with 20 μM Zn (II) (Figure 10): Increase in ochronotic pigment was linear in the time interval between 8 and 24 hours.

Since the culture medium in use was well defined, different amounts of the substrate L- tyrosine were added to cultures induced with 20 μM Zn (II) and harvested in time linearity. A steady, linear increase of ochronotic pigment formation could be observed up to a final concentration of 500 μM L-tyrosine, reaching saturating concentrations afterwards (Figure 11).

When cultures transformed with pzntHPD were induced with different concentrations of Zn (II), treated as described above and measured against induced non-transformed controls, a linear increase in absoφtion between 1 μM to 10 μM Zn (II) was observable (Figure 12). It can be concluded that the expression of HPD on the protein level, analyzed by Western Blot (Figure 9), was directly reflected by the enzymatic activity (Figure 12). The transcriptional activity of the zntA -promoter, demonstrated by Outten et al. (27), is linearily induced in the range of 4 to 10 μM Zn (II). This result can be directly correlated with the translational activity and enzymatic activity after Zn (ID-induction. Summarizing the above, these results define a biosensor system for Zn (II) heavy metal pollution.

Example for the specificity of pigment formation

The formation of ochronotic pigment is not only a feature of plant HPDs (eg. from Arabidopsis; see above) or eucaryotic HPDs. It could be shown that bacterial HPDs, cloned under the same genetical background, reveal the same kinetics of time, substrate dependency and induction. For this puφose, hpd from Synechocystis sp. PCC6803 was cloned under the control of the Zn (ID-promoter (see above). When pzntSYN was expressed in BL21(DE3)RIL, the increase in ochronotic pigment formation was linear in the time interval between 8 and 24 hours, it increased linearily to a final concentration of 500 μM L-tyrosine and revealed a linear increase in absoφtion between 1 μM to 10 μM Zn (II) (Figure 13).

Example for the versatility of the biosensor system and its soluble character

To demonstrate that the transformation of the biosensor system is versatile and can also be utilized in eucaryotes (i.e. cells, cell cultures, protoplasts, animals and plants) further experiments were conducted employing Saccharomyces cerevisiae as a model organism. In yeast, a different subset of metallothionein genes is transcriptionally activated when the extracellular copper concentration exceeds 1 μM (12). As CUP1 is the dominant locus that confers the ability of yeast cells to propagate in medium containing copper salts, the CUPl-2 promoter was chosen for the demonstration of the biosensor according to the present invention. In induction experiments with Cu (II), a linear increase in ochronotic pigment formation was observable between 5 μM and 200 μM, reaching saturation at 500 μM (Figure 14). These data were consistent with published mRNA-analysis experiments concerning the same promoter (16). Ochronotic pigment was soluble and detectable in the medium in a quantifiable manner, i.e. as in E.coli the end product of the enzymatic reaction was secreted into the medium (Figure 15). Spectra of pigment mprocaryotes and eucaryotes are identical (Figures 4 and 16). The increase in ochronotic pigment formation in transformed yeast cell culture was independent of the exogenous addition of tyrosin, indicating that there is already a huge amount of substrate precursor available from the medium or that tyrosin cannot be incoφorated by yeast.

Example for the sensitiveness of a comparable biosensor system under control of the same promoter

The hpd reporter system according to the invention is as sensitive as other reporter systems already commercially available. As a reference, β-galactosidase from E.coli, the most common reporter system, was chosen. This reporter was cloned under the same genetical background as the hpd reporter and analyzed under the same conditions. Measurements of β- galactosidase activity were performed 2 and 24 hours after induction. Kinetics of this reporter system were equivalent to the hpd reporter showing a linear increase between 1 to 10 μM (Figure 17). In both systems the amount of accumulated end product is solely a feature of transcriptional promoter activity and can be calibrated by exogeneous standards (Figure 18). This also demonstrates that the spontaneous oxidation process leading to ochronotic pigment formation is comparable to the enzymatic reaction of β-galactosidase.

In contrast to the hpd reporter system provided in accordance with the present invention, the β-galactosidase technique requires cell lysis prior to perform the enzymatic assay. This enzymatic assay, which is not necessary in the case of hpd reporter, is time-consuming, needs personal trained in laboratory practice and far more equipment than the hpd system provided herein. Additionally, this β-galactosidase assay cannot be performed as a high troughput assay like the hpd biosensor. Furthermore, the use of organic solvents that might be harmful to the technicians represents an additional drawback inherent in comparable assay techniques.

It should have become apparent from the above, that the fundamental principle underlying the different aspects of the present invention is the direct coupling of an enzymatic reaction with a fast non-enzymatic reaction, leading to the quantitative formation of a coloured pigment which can easily be measured. Normally, these kind of couplings are not wanted by the experimentator and react quite artificially. However, it could be shown according to the present invention that the formation of ochronotic pigr ent surprisingly behaves like an enzymatic reaction, when specific conditions are applied.

The suφrising and therefore unpredictable features of the system provided hereinbefore are:

• The non-enzymatic reaction is faster than the enzymatic reaction. Pigment formation therefore behaves in a quantifiable manner. Its formation can be standardized in equivalents to the enzymatic reaction.

• The enzymatic activity of the gene product (i.e. hydroxyphenyl pyruvate dioxygenase) is very high, so that a minimal induction of the expression cassette in procaryotes as in eucaryotes already leads to discrete amounts of homogentisic acid. Thus, promoter- analyses and/or biosensor systems can be provided.

• Homogentisic acid is soluble. Unexpectedly, it is secreted into the extracellular space in procaryotes as in eucaryotes and is not retained in cells. For analysis, the fast non- enzymatic reaction to the soluble and quantifiable ochronotic pigment has to be performed. This happens spontaneously in the medium and can be increased to a maximum by the addition of basic agents, such as addition of NaOH to a final concentration of 0,1 N.

• The colour change can be measured over a wide spectral range and is not restricted to a specific wavelength, so that there are several possibilities of quantification dependent on the absoφtion of the inducer. • Time for quantitative measurements is in the range of 24 hours post induction; variabilities of cell cultures will diminish within this time, as E.coli will already arrest to the fog-phase. • Most suφrisingly, the system needs only a minimal medium where the transformed organisms can survive. The substrate of the enzymatic reaction or its biochemical precursors don't need to be components of the medium. They also don't need to be added exogenously before, within or after induction or for the development" of the system. Synthesis of the substrate is an endogenous feature of the amino acid-metabolism in all living organisms. Suφrisingly, the amount of fonned substrate in the assayed transformed organisms is high enough to allow quantification after induction.

The above disclosure is given to exemplify the principles of the present invention and shall not be construed to limit the scope of protection. It is clear for a person skilled in the art that suitable cDNAs encoding Hpd are available from a broad range of organisms and can be used according to the invention. Likewise, the selection of an appropriate test cell or organism is only restricted by its applicability in the context of the present invention. Thus, appropriate test cells or organisms can easily be selected from eucaryotes and procaryotes, with bacteria, fungi and small higher organisms which can easily be cultured being preferred. Although the present invention is disclosed in connection with certain promotors it is to be understood, that any promotor can be used according to the invention which is inducible by a substance desired to be analyzed.

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Claims

Claims:

1. Biosensor system comprising a cell or organism harbouring a DNA molecule enabling said cell or organism to express -hydroxyphenyl-pyruvic acid dioxygenase under the control of a promotor which is inducible by the presence of substances or conditions to be analyzed.

2. Biosensor system according to claim 1, characterized in that the substances to be analyzed are selected from the group consisting of air, water and soil contaminants, toxins and toxic compounds.

3. Biosensor system according to claim 2, characterized in that the substances are selected from the group consisting of zinc, cadmium, mercury, lead, nickel, chrome, arsenic, iron, copper, aluminum, manganese, cobalt, phenols, benzene and paraquat.

4. Biosensor system according to claim 2, characterized in that the conditions are selected from the group consisting of salt, osmotic and oxidative stress conditions.

5. Biosensor system according to claim 1, characterized in that the promoter is selected from the group consisting of zntA, cadA, merA, nic, chr, ars,fliC, cor A, dmpR, xylR, xylS,pqiAB, SoxRS, zrt, zip,ycfl, cupl-1, cupl-2, gefl trl, hall, grel and aad4.

6. Biosensor system according to claim 1, characterized in that the cell or organism is procaryotic or eucaryotic.

7. Biosensor system according to claim 6, characterized in that the procaryotic cell or organism is E. coli.

8. Biosensor system according to claim 6, characterized in that the eucaryotic cell or organism is selected from yeast.

9. Biosensor system according to claim 8, characterized in that the eucaryotic cell or organism is Saccharomyces cerevisiae.

10. An isolated DNA molecule comprising a nucleotide sequence providing an expression cassette capable of directing the expression of p-hydroxyphenyl-pyruvic acid dioxygenase, wherein said expression cassette comprises from 5' to 3':

(a) an inducible promoter capable of expressing a downstream coding sequence; (b) a DNA sequence coding for the expression ofp-hydroxyphenyl-pyruvic acid dioxygenase; and (c) a 3' termination sequence, characterized in that the inducible promoter (a) is selected from the group consisting of zntA, cadA, merA, nic, chr, ars,βiC, cor A, dmpR, xylR, xylS, pqiAB, SoxRS, zrt, zip, ycfl, cupl-1, cupl-2, gefl trl, hall, grel, and aad4.

11. A plasmid or vector system comprising a DNA molecule according to claim 10.

12. A transgenic cell or organism containing a DNA molecule according to claim 10.

13. Process for detecting the presence and quantity of a substance to be analyzed, comprising the steps of incubating the biosensor system according to any of claims 1 to 9 with the substance and detecting the presence and quantity of said substance photometrically.

14. Process according to claim 13, wherein the substances to be analyzed are selected from the group consisting of air, water and soil contaminants, toxins and toxic compounds.

15. Process according to claim 14, wherein the substances are selected from the group consisting of zinc, cadmium, mercury, lead, nickel, chrome, arsenic, iron, copper, aluminum, manganese, cobalt, phenols, benzene and paraquat.

16. Process for studying and/or monitoring the activity of a promoter or functional part thereof by the steps of using of a vector which comprises a DNA sequence coding for the expression of p-hydroxyphenyl-pyruvic acid dioxygenase under the control of said promoter or functional part thereof and a transcriptional terminator, wherein the

DNA sequence is, for the transformation of a host cell or organism, cultivating the transformants and monitoring the activity of said promoter or functional part thereof by measuring the content of ochronotic pigment accumulated in the culture medium.