EP1828416A1

EP1828416A1 - Nucleic acid-binding chips for the detection of phosphate deficiency conditions in the framework of bioprocess monitoring

Info

Publication number: EP1828416A1
Application number: EP05822552A
Authority: EP
Inventors: Thomas Schweder; Britta JÜRGEN; Stefan Evers; Karl-Heinz Maurer; Le Thi Hoi; Michael Hecker; Birgit Voigt; Jörg FEESCHE
Original assignee: Henkel AG and Co KGaA; Ernst Moritz Arndt Universitaet Greifswald
Current assignee: Henkel AG and Co KGaA; Universitaet Greifswald
Priority date: 2004-12-22
Filing date: 2005-12-15
Publication date: 2007-09-05
Also published as: US20070281312A1; JP2008523833A; WO2006069638A1; DE102004061664A1

Abstract

The invention relates to nucleic acid-binding chips for monitoring bioprocesses, especially for detecting phosphate deficiency conditions. Said chips support probes for at least three of the following 47 genes: yhcR, tatCD, ctaC, gene for a putative acetoin reductase, spollGA, nasE, pstA, spollAA, gene for a hypothetical protein, yhbD, cotE, gene for a conserved hypothetical protein,yurl, spoVID, gene for a putative aromatics-specific dioxygenase, yhbE, gene for a putative benzoate transport protein, pstBB, spoIIIAH, gene for a hypothetical protein, spollQ, spollIAG, yvmA, gene for a putative ribonuclease, dhaS, yrbE, gene for a putative decarboxylase/dehydratase, htpG, yfkH, spollAB, spolllAF, alsD, gdh, yfkN, pstC, yfmQ, pstBA, dhaS homologue, gene for a putative phosphatase, phy, cypX, alsS, phoD, pstS, phoB, yvnA, yvmC, the total number of phosphate metabolism-specific different probes on the nucleic acid-binding chips not exceeding 100. The invention further relates to the use of corresponding gene probes, particularly on such chips, as well as corresponding methods and possible uses.

Description

Nucleic acid-binding chips for the detection of phosphate deficiency states in the context of bioprocess control

The present invention relates to nucleic acid-binding chips for monitoring bioprocesses with a special focus on the detection of phosphate deficiency states and to the use of corresponding gene probes, in particular on such chips, or methods and applications based on such probes and chips.

In the technical use of biological processes, the very fundamental problem is to monitor their course in order to achieve the desired result in order to conserve resources and / or to achieve an optimal result in a given time. By biological processes are meant, for example, the culture of microorganisms on an agar plate or in shaking culture, but in particular their fermentation, or the extraction of raw materials via the fermentation of microorganisms. For this there is a rich state of the art, both in terms of unicellular eukaryotes such as yeasts or streptomycetes, as well as what gram-negative or gram-positive bacteria.

The monitoring of such processes (monitoring) is done on the one hand by observing the changing properties and requirements of the organisms under consideration during the process, which is reflected, for example, in the optical density and viscosity of the medium, in absorbed or emitted gases, in changes in the pH or changing nutrient needs. For this purpose, it is also possible to calculate the measurement of enzymatic activities by means of suitable assays, for example the detection of activities of interest in the culture supernatant.

On the other hand, in recent years, various techniques have been developed to follow the metabolic processes of the subject organisms at the gene expression level. A common method for this is the use of genes for easily detected proteins as indicators of the activity of the promoters for the actually interesting genes (promoter analysis, gene expression analysis). For this purpose, corresponding apparatuses (so-called (bio) sensors) have been developed. Other techniques are concerned with the detection of the proteins of interest or of the mRNA coding for these proteins. These include (1.) the proteome analysis, that is, the consideration of the change in the equipment of the cells in question with proteins, which usually takes place via 2-dimensional gel electrophoresis of the cell lysates, (2.) the analysis of the mRNA formed (transcriptome) via a analogously created "genomic DNA array" and (3.) the chip technology.

The latter is in a relatively early stage of development. While the first two methods are based on quantitative isolation and time-consuming analyzes of the macromolecules in question, chip technology is based on the principle of attaching probes for proteins or nucleic acids to physically readable carriers (chips) that directly depend on the presence of the proteins involved , or address nucleic acids. Compared to the two first mentioned technologies, one expects from such chips a timely analysis of the considered process (/ \ f - // ne-analysis). Another advantage is the need for comparatively small sample quantities.

The principle of chip-based measurements is described, for example, in the article "Real-time Electrochemical Monitoring: Towards Green Analytical Chemistry" by J. Wang (Acc. Chem. Res. ISSN 0001-4842; Rec. Sept. 12, 2001, pages A - F) is schematically presented in Figure 2. Thus, the sample to be analyzed is contacted with a recognition layer (biorecognition layer), which may be, for example, enzyme, antibody, receptor or DNA; Transducers, for example an amperometric or potentiometric electrode, are output via an amplifier (amplification / processing) as an electrical voltage or as an electrical potential.The work in question also addresses optical systems in relation to which the electronically evaluable systems can be miniaturized and other benefits seemed more favorable to the author.

Due to the present invention, the protein-specific chips may be disregarded. mRNA-recognizing chips are usually doped with complementary DNA molecules or DNA analogs. Their preparation and use for very detailed questions such as the differentiation of point mutations is described for example in the application WO 95/11995 A1. Among the DNA chip Analyzes, there are those with a PCR amplification of the target sequence and those without amplification. Furthermore, there are those with optical evaluation of the attributable to the detection signals and those with electrical evaluation.

The optical detection methods sometimes require an amplification mechanism of the signals. For example, fluorophores, acridinium esters or indirect detection via secondary binding processes, for example via biotin, avidin / streptavidin or digoxigenin, are described for this purpose. In the latter case, digoxigenin-specific antibodies are used for optical detection, which are labeled with an enzyme. The enzyme activity is detected either colorimetrically or via luminescence. According to Westin et al. (2000), Nature Biotechnol., 18, pp. 199-204, the hybridization can be coupled to a PCR on the DNA chip so as to perform the entire detection reaction on a chip ("Lab-on-a-Cfr / p "concept).

Further work describes the development of DNA chips that miniaturize the principle of capillary electrophoresis for DNA sequencing or separation (Woolley and Mathies (1994), Proc. Natl. Acad., Sci., 91, pp. 11348-11352; Liu et (2000), Proc Natl Acad., Sci., 97, pp. 5369-5374).

Electrically readable DNA chips have already been introduced in principle in some publications (Hoheisel (1999), DECHEMA Annual Report 1999, pp. 8-11, Hintsche et al. (1997), EXS, 80, pp. 267-283). Wright et al. (2000, Anal. Biochem., 282, pp. 70-79) used a "lon channel sensor" (ICS) for DNA detection as first described by Cornell et al., (1997; Nature, 387, p. 580-583), which is a process whereby the conductivity of molecular ion channels is detected by a binding reaction, essentially the sensor is an impedance element. Cheng et al (1998, Nat. Biotechnol. 16, pp. 541-546), electrical impulses can be used to enhance the hybridization reaction on optical DNA chips, and Fritsche et al (2002, Laborwelt II) proposed an electric chip system that uses metallic nanoparticles, the In this system, a so-called "metallic amplification" during the hybridization reaction triggers a decrease in the electrical resistance at the electrode, which is then measurable as a signal. Another approach is based on an electrical detection principle, in which DNA probes are used, which lead by labeling with a suitable enzyme (for example, alkaline phosphatase) after hybridization to an electrically active substrate, which then by a redox reaction at the Electrode is detectable (Hintsche et al. (1997), EXS, 80, pp. 267-283).

If one has decided on a particular nucleic acid-recognizing chip type with regard to the basic structure and the evaluation system, the more concrete problem arises as to which gene activities should be observed. It should be remembered that in the number of simultaneously analyzable genes with a nucleic acid chip type technically related limits exist. Thus, optically readable chips, what the number of probes that can be applied on the chip, the electrically evaluable currently superior. Their limits are set by the miniaturization of the electronic measuring units.

Thus, the biological problem arises, which selection of gene activities maps the considered process in a suitable way. This also includes the monitoring of product formation, if it is for example a fermentative product production. At the same time, control genes should also be included which indicate when the process is developing in a direction that is not intended. In the course of this monitoring, for reasons of practicability, a not too high number of different genes should be observed.

Of particular technical interest are biotechnological processes with Gram-positive bacteria. Because these are used especially for their ability to secretion for the industrial production of recyclables. Among them are those of the genus Bacillus and among these in turn the species ß. subtilis, B. amyloliquefaciens, B. agaradherens, B. licheniformis, B. lentus and B. globigii are currently the most economically important.

For example, the work presented below deals with the simultaneous observation of the activity of several genes in bacteria (multiparameter detection). Schweder et al., (1999) Biotech Bioeng., 65, pp. 151-159, reports that the change in mRNA levels is different in the article "Monitoring of genes that respond to process-related stress in large-scale bioprocesses." by stress factors of inducible genes, namely clpB, dnaK (induced in heat shock), uspA (glucose deficiency), proU (osmotic stress), pfl and frd (O ₂ deficiency) and ackA (glucose excess) in the course of a fermentation of E. coli and in the subsequent concentration phase described. They were collected via a PCR-based, conventionally performed method. In this case, different expression rates were already determined at different points of the reactor and reactions in a matter of seconds to changed conditions.

A further fermentation of E. coli is described in the work "Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations" by Jürgen et al., (2000) Biotech Bioeng., 70 217-224, which considers the expression of the genes Ion, dnaK, ibpB, MrA, ppiB, groEL, tig, s6, 19 and dps, in part on mRNA, partly at the protein level, partly at both levels. The investigation was carried out using 2D-PAGE or the DNA-array technique In view of the results, it is proposed to follow recombinant bioprocesses such as heterologous protein production via (directly) process-relevant proteins and reporter genes such as ibpB.

The work "Genomic analysis of high-cell-density recombinant Escherichia coli fermentation and cell conditioning for improved recombinant protein yield" by RTGill et al. (1989) deals with a further observation of the course of fermentation in the expression of a recombinant protein by E. coli. 2001, Biotech Bioeng., 72, pp. 85-95.) It is described herein that the stress genes are compared to degP, uvrB, alpA, mltB, recA, ftsH, ibpA, aceA and groEL under the conditions of high cell density By reaction intensity, they clustered among themselves into certain clusters, and this was determined by an RT-PCR and DNA microarray-based and dot-blot added approach, which was run on samples from two time points Fermentation was applied just at the beginning at low cell density and towards the end at high cell density, from which "cell conditioning" approaches were developed to assess the stress response to degrade the cells.

Fundamental differences in the expression patterns of Gram-positive organisms over those of Gram-negative bacteria are described in the work "Proteome and transcriptome based analysis of Bacillus suhtilis cells overproducing an insoluble heterologous protein" by Jürgen et al. (2001), Appl. Microbiol. Biotechnol., 55, Pp. 326-332, in which the expression is inter alia of the genes dnaK, groEL, grpE, cIpP, clpC, clpX, rpsB and rplJ in S. subtilis, as they can be determined by the DNA macroarray technique or by two-dimensional polyacrylic gel electrophoresis. According to this, the genes used for the purine and the pyrimidine synthesis as well as the specific ribosomal proteins are expressed more strongly in gram-positive bacteria used for overexpression than could be expected on the basis of the findings on Gram-negative bacteria. Another difference concerns the proteases Lon and CIp.

Individual of these genes or even nucleic acid-binding chips with individual of these genes are now disclosed in several publications, or at least demonstrated the possibility of their production. For example, the two patent applications DE 10136987 A1 and DE 10108841 A1 each disclose a gene from Corynebacterium glutamicum, namely clpC or citB. Both genes are described as relevant to amino acid metabolism, which is why a commercially interesting use of these genes is to inactivate or at least mitigate them in order to optimize the fermentative production of amino acids by this microorganism. Further applications according to these applications may be to provide probes for the relevant gene products on nucleic acid-binding chips.

On the other hand, more and more genome data from various organisms are published which contain such a wealth of sequence data that a representative selection appears desirable. Thus, the patent application WO 02/055655 A2 discloses more than 1,800 DNA sequences which have been determined by the complete sequencing of the genome of the microorganism Methylococcus capsulatus.

In the meantime, for example, the entire genome of gram-positive Bacillus licheniformis has also been sequenced. It is described in the publication "The Complete Genome Sequence of Bacillus Licheniformis DSM13, to Organism with Great Industrial Potential" (2004) by 8. Veith et al., J. Mol. Microbiol. Biotechnol., Vol 7 (4), p to 211, and is additionally listed under entry AE017333 (bases 1 to 4.222.645) in the GenBank database (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA, http: //www.ncbi. nlm.nih.gov, as of 2.12.2004). In the meantime, using the technology of optically analyzable chips, it is even possible to prepare nucleic acid-binding chips which cover an almost complete genome or the associated transcriptome (genomic DNA chips).

The application WO 2004/027092 A2 provides a representative cross-section with a manageable number of genes in order to identify various physiological states which an observed microorganism can undergo in the course of the cultivation. These included, for example, starvation of various nutrients or stress situations such as heat or cold shock, shear stress, oxidative stress or oxygen limitation. These are the following genes: acoA, ahpC, ahpF, citB, clpC, cIpP, codY, cspA, cspB, des, dnaK, eno, glnR, groEL, groL, gsiB, ibpA, ibpB, katA, katE, IctP , Idh, opuAB, phoA, phoD, pstS, purC, purN, pyrB, pyrP, sigB, tnrA, trxA and ydjF. The associated DNA sequences from B. subtilis, E. coli and / or B. licheniformis also emerge from this application. As a result, it has become possible to produce corresponding nucleic acid-binding chips which, when monitoring a bioprocess based on microorganisms, in particular Gram-positive or Gram-negative bacteria, indicate changes in the metabolic activities characterizing this process.

Nucleic acid-binding chips, which are based on this selection of genes, provide a certain, but rather rough, overview of the respective metabolic situation. As a rule, they are unable to shed light on a single subproblem; however, a single positive signal may result from different situations or even be false-positive, which is why it often makes sense - and especially in such an unclear situation - to analyze a selected metabolic aspect separately. On the other hand, especially in the case of electrically readable nucleic acid-binding chips, which have the advantage of a timely analysis, the number of simultaneously assignable sites is limited, so that additional gene probes can not simply be applied to detect additional, specific metabolic situations.

Certain metabolic situations and even deficiencies are exploited in biotechnology. Thus, the application DE 10012283 A1 discloses a useful application of the inducibility of genes by phosphate deficiency. In that the promoters of the genes pstS, phoD, phoB or glpQ from ß. suhtilis is used to regulate transgenes to be activated for heterologous gene expression by gram-positive host bacteria. For this purpose, the PhoP PhoR regulation system from 6. subtilis must be made available at the same time in order to activate the relevant promoters. According to this application, a phosphate deficiency should be artificially induced in order to achieve via PhoP and PhoR to induce the chosen promoter. It is therefore not assumed that cell-specific regulatory systems are able to induce the artificially introduced promoters of B. subtilis genes pstS, phoD, phoB and glpQ. For example, it is known from other studies (not shown here) that the pho genes in B. licheniformis organizes differently, that is also regulated differently.

However, in the course of a bioprocess, as explained above, it is usually not desirable to expose the cells to a stress situation. In particular, phosphate deficiency is a metabolic situation that can be critical for microorganisms and thus limiting for a corresponding bioprocess.

Thus, there is a particular need to perform in this regard, a chip-based, timely analysis and to be able to intervene punctually and thus more targeted in the ongoing bioprocess due to the result to be obtained quickly. This provides for a loss of yield that would result from a short or late recognized phosphate bottleneck.

The prior art describes further / other genes which are expressed to a greater extent in the case of phosphate deficiency, albeit not equally in all microorganisms relevant for biotechnology. Thus, the publication by Ishige et al. Volume Bacteriol., Vol. 185 (No. 15), pages 4519 to 4529, shows a DNA microarray analysis of the phosphate-deficient genes (the "phosphate stimulant") in Corynebacterium glutamicum, in which stimulus is elicited that orthophosphate is the only source of phosphate to change to a phosphate deficient state, which, in agreement with the data on other microorganisms, clearly induces some genes that are related to phosphate metabolism, whereas the induction of other genes is attributed to mere growth effects It was observed that not all of the known genes of the phosphate metabolism but only individual ones are induced. Conversely, some proteins whose homologues are not expressed to a greater extent in other microorganisms, for example a nucleotidase whose homologue has an unchanged expression level in E. coli, and a ferritin-like protein and a probable extracellular nuclease NucH, whose Expression levels, for example, at ß. licheniformis are not significantly elevated (data not shown).

The publication by Antelmann et al. in J. Bacteriol., Vol. 182 (No. 16), pp. 4478 to 4490 examines the proteins inducible by B. subtilis phosphate deficiency at the proteome and transcriptome level using two-dimensional gel electrophoresis; Micro-array technology is referred to herein merely as a potentially complementary technology. Figure 4 reveals a total of ten proteins which are increasingly produced in this organism on a phosphate-deficient stimulus. The strongest signals are those of GIpQ, PhoD and PstS, followed by PhoB and PeI. In contrast, our own investigations in connection with the present application showed that glpQ and pel are only marginally overexpressed in B. licheniformis in the case of phosphate deficiency. Instead, in B. licheniformis, for example, the gene for the phytase (see examples) gave a surprisingly strong signal, which is not the case in B. subtilis.

Therefore, there are a number of fundamental difficulties in designing RNA RNA-detecting chips to monitor bioprocesses. On the one hand, the question of transferability to other organisms arises with each gene: It must be selected genes that provide clear signals in as many microorganisms relevant for such bioprocesses. On the other hand, there is the question of specificity: the strong signals must also be as clearly as possible attributable to the metabolic situation concerned. Such signals, which respond to several different metabolic situations and / or represent general stress signals, should be excluded as much as possible.

The approach chosen for the present invention is to make as representative as possible a selection of a whole series of genes, wherein not all, according to the invention but several probes in the respective observed organism signals. On the other hand, those genes should be excluded which also give strong or even stronger signals in other metabolic situations than in the phosphate deficiency. For the present invention, therefore, the task was to identify genes that can be brought as clearly as possible in organisms, in particular microorganisms with the stress signal of phosphate deficiency in combination. The aim was to develop probes for these genes in order to be able to use them for the monitoring of corresponding bioprocesses.

This should make it possible to prove nucleic acid-binding chips with gene probes for single or several of these genes and thereby to obtain nucleic acid-binding chips which reliably indicate the signal "phosphate deficiency" in the course of a monitored bioprocess (phosphate deficiency sensors) The problem arose in particular for such nucleic acid-binding chips, whose number of assignable sites is comparatively low due to their design, in particular the electrically evaluable ones, because on the other hand they have the advantages of rapid readability and thus enable a / ct analysis This ensures an early intervention, if necessary, in order to optimize the bioprocess concerned with regard to the phosphate supply.

Such a DNA-binding chip should be usable for several comparable processes and be adapted to specific applications with comparatively minor variations. Preferably, it should be directed to bioprocesses based on Bacillus species, especially B.subtilis, B. amyloliquefaciens, B. lentus, B. globigii, and more particularly B. licheniformis. Bioprocesses focused on fermentations, in particular the technical production of products, especially overexpressed proteins.

Furthermore, such a phosphate-deficient sensor should enable appropriate methods for measuring the physiological state of the cells considered as well as corresponding possibilities of use for monitoring the considered biological processes.

To solve this problem, a large number of genes from the biotechnologically important B. licheniformis bacterium were investigated with regard to their activability by the transition of the relevant culture into a phosphate deficient state (Example 1). It was surprisingly found that by far not all genes on the Phosphate metabolism involved, provide a clear signal in this regard. In addition, it has also been observed, as surprisingly, activation of such genes which have not previously been readily associated with phosphatase metabolism, for example sporulation genes; irrespective of their previously known function, these should now also be considered according to the invention as phosphate metabolism genes. Both documents Example 2 of the present application. Very different degrees of induction were observed. According to the teachings of the present invention, genes should be all the more suitable as indicators the stronger this response is. According to the invention, therefore, such genes are selected as phosphate deficiency indicators, which give a clear, significantly above a certain threshold signal lying. The further the result is, the more they are preferred according to the invention, which explains a corresponding staggering with regard to preferred aspects of the invention. At the same time, those genes have been removed that have also produced strong or even stronger signals in situations other than phosphate deficiency (data not shown).

Table 1 shows all of the 235 genes of Bacillus licheniformis DSM13 determined in Example 1 whose induction was observed under phosphate deficiency, with a factor of at least three being considered significant. Of these, all 47 genes are compiled in Table 2 whose induction by phosphate deficiency at least amounted to at least the factor 10 at any of the measured times and in which parallel examinations, not shown here, could conclude that they were comparatively specific for this signal. These are listed again in Table 3 for the magnitude of their observed maximum induction. Their DNA and amino acid sequences are listed in the Sequence Listing of the present application, wherein the odd-numbered numbers represent DNA and the subsequent even-numbered for the respective deduced amino acid sequences. These sequences are also referred to the respective SEQ ID Nos. In Tables 2 and 3. These are the following genes, in the order of decreasing strength of the phosphate-induced induction (see Tables 2 and 3):

- yvmC (similar to proteins of unknown function; SEQ ID NOs 75, 76);

yvnA (similar to proteins from S. subtilis; SEQ ID NO: 77, 78);

phoB (Alkaline Phosphatase III; SEQ ID Nos. 21, 22);

pstS (phosphate ABC transporter / binding protein; SEQ ID NO: 47, 48); phoD (phosphodiesterase / alkaline phosphatase; SEQ ID NO: 23, 24);

as als (alpha-acetolactate synthase; SEQ ID NO: 29, 30);

cypX (cytochrome P450-like enzyme; SEQ ID NO: 3, 4);

phy (phytase; SEQ ID NOs 33, 34);

Gene for a putative phosphatase (SEQ ID Nos. 61, 62);

Homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; SEQ ID Nos. 17, 18);

pstBA (phosphate ABC transporter PstBA; SEQ ID NO: 87, 88);

yfmQ (unknown function, SEQ ID NOs: 69, 70);

pstC (phosphate ABC transporter / permease; SEQ ID NO: 91, 92);

yfkN (similar to 2 ', 3'-cyclo-nucleotide-2'-phosphodiesterase; SEQ ID NO: 11, 12);

gdh (glucose-1-dehydrogenase; SEQ ID Nos. 31, 32);

as D (alpha-acetolactate decarboxylase; SEQ ID NO: 27, 28);

spolllAF (sporulation factor III AF, SEQ ID NO: 79, 80);

spoll AB (anti-sigma F factor / phase II sporulation protein AB, SEQ ID Nos. 37, 38);

yfkH (similar to proteins; SEQ ID NOs: 67, 68);

MpG (Class III heat shock protein; SEQ ID Nos. 1, 2);

Gene for a putative decarboxylase / dehydratase (SEQ ID NO 57, 58);

yrbE (similar to dehydrogenase; SEQ ID Nos. 9, 10);

dhaS (aldehyde dehydrogenase; SEQ ID NO 19, 20);

Gene for a putative ribonuclease (SEQ ID Nos. 93, 94);

yvmA (similar to a multidrug transporter; SEQ ID NOs: 51, 52);

spollLAG (sporulation factor III AG, SEQ ID NOs 81, 82);

spollQ (sporulation factor IIQ, SEQ ID NO 43, 44);

Gene for a hypothetical protein (SEQ ID NOs 63, 64);

spolllAH (sporulation factor III AH, SEQ ID NOs 83, 84);

pstBB (phosphate ABC transporter / ATP binding protein; SEQ ID NO: 89, 90);

Gene for a putative benzoate transport protein (SEQ ID Nos. 49, 50);

yhbE (similar to proteins from B. suhtilis; SEQ ID NO: 73, 74);

Gene for a putative aromatic-specific dioxygenase (SEQ ID Nos. 55, 56);

spoVID (sporulation factor VI D, SEQ ID Nos. 45, 46);

yur1 (similar to ribonuclease; SEQ ID NO: 15, 16); A conserved hypothetical protein gene (SEQ ID NOs: 59, 60);

cotE (outer spore envelope protein; SEQ ID Nos. 39, 40);

yhbD (similar to proteins from B.subtilis; SEQ ID NOs 71, 72);

Gene for a hypothetical protein (SEQ ID NO 65, 66);

spollAA (anti-sigma F-factor antagonist / phase II sporulation protein AA, SEQ ID NOs 35, 36);

pstA (phosphate ABC transporter / permease; SEQ ID NO: 85, 86);

- nasE (Assimilatory Nitrite Reductase subunit; SEQ ID Nos. 7, 8);

spolIGA (sporulation factor II GA, SEQ ID NO: 41, 42);

Gene for a putative acetoin reductase (SEQ ID Nos. 53, 54);

ctaC (cytochrome CAAS oxidase / subunit II; SEQ ID NO: 5, 6);

tatCD (component of the twin arginine translocation pathway, SEQ ID NOs 25, 26);

yhcR (similar to δ 'nucleotidase; SEQ ID NO 13, 14).

A solution to the problem consists of a nucleic acid-binding chip doped with probes for at least three of the following 47 genes: yhcR, tatCD, ctaC, gene for a presumptive acetoin reductase (homolog to SEQ ID NO. 53), spolIGA, nasE , pstA, spollAA, gene for a hypothetical protein (homologue to SEQ ID NO. 65), yhbD, cotE, conserved hypothetical protein gene (homologue to SEQ ID NO. 59), yurl, spoVID, presumptive aromatic gene specific dioxygenase (homologue to SEQ ID NO: 55), yhbE, gene for a putative benzoate transport protein (homologue to SEQ ID NO 49), pstBB, spolllAH, gene for a hypothetical protein (homolog to SEQ ID NO 63), spollQ, spolllAG, yvmA, gene for a putative ribonuclease (homologue to SEQ ID NO: 93), dhaS, yrbE, gene for a putative decarboxylase / dehydratase (homolog to SEQ ID NO 57), MpG, yfkH, spollAB, spolllAF, alsD, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; homolog to SEQ ID NO. 17), gene for a putative phosphatase (homologue to SEQ ID NO: 61), phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC, where the total number of all phosphate metabolism-specific different probes is not more than 100.

Detailed information on these genes can be found in Examples 1 to 3 and Tables 1 to 3 of the present application. In the Sequence Listing, these genes are disclosed as obtainable from B. licheniformis DSM 13. Most of them are also described from other species (see below). However, some are putative (presumed) genes or genes that are putative Encoding (presumed) putative enzymes. According to the invention, these are defined as far as possible on the basis of database comparisons as having an assumed function and additionally as homologues to the B. licheniformis genes found. For this purpose, two things have to be explained:

- First, it could turn out for one or another by a biochemical analysis that this putative function does not match the real function. Then one does not stick to the putative function. On the contrary, these findings do not call into question the invention insofar as according to the invention only the observation of the increased transcription in connection with the phosphate deficiency arrives, so that the gene activity in question can definitely serve as an indicator of phosphate deficiency, irrespective of the ultimate enzyme activity.

On the other hand, in the absence of a gene, no more suitable definition could be found for them than that about the gene itself. That is why homologues are spoken. So it can be assumed that in other species than ß. licheniformis under phosphate deficiency the homologous genes are activated. If it turns out that in one species considered to one of these genes several homologs exist, which are capable of transcript formation in vivo, the term "homolog to SEQ ID NO. ... "to the nearest similar of these various candidate genes.

According to the invention, at least three of these genes are selected in order to obtain as reliable a statement as possible, that is, to exclude a single false-positive signal due to only one type of probe.

According to the invention, a nucleic acid-binding chip means all objects which are provided with nucleic acid-specific probes and in each case deliver an evaluable signal upon binding of one or more specifically recognized nucleic acids.

From the prior art presented in the introduction, the design of chips doped with nucleic acids as probes is known. In principle, they can all be used for embodiments of the present invention. They are based on the principle of nucleic acid hybridization of the mRNA to be detected (or a molecule derived therefrom) with the probe presented on the chip. Depending on the system for evaluating the triggered by the hybridization signal is between chips with an optical and distinguished with an electrical analysis system. In principle, both systems are applicable according to the invention.

Such chips are used as follows for monitoring (monitoring) of the particular bioprocess under consideration: From the process, a sample with the biological material to be analyzed is taken out at a certain point in time. From this, according to methods known per se, for example by cell disruption and use of a denaturing buffer RNA, in particular mRNA isolated. This is itself labeled or used as the starting molecule for a molecule introduced into the measurement (for example cDNA obtained by reverse transcription) and the resulting molecules are advantageously passed over / through the chip in a buffer. Upon hybridization (sandwich labeling) of a prepared RNA or its derivative with the homologous (congruent with respect to their sequence) provided on the chip probe (target nucleic acid, for example, target DNA or target nucleic acid analogue) results in a corresponding optically or electronically evaluable signal. This is based for example on the labeling of the binding mRNA or a transcript thereof with a staining or fluorescence marker, a hybridization with a second probe or on a secondary detection reaction, for example via an RT-PCR.

Since in each case several molecules are bound to the chip by the same probe in general, the strength of the hybridization signal over a certain - in individual cases optionally to be optimized - range is proportional to the number of specific at the time of sampling in the sample specific mRNA. In this way, the strength of the signal is a direct measure of the activity of the gene in question at the time of sampling.

The time interval between sampling and measurement should be kept as short as possible, for example via a largely automated sampling, their processing and management via / through the sensor.

In principle, all plants, animals and microorganisms in question, in particular those which are used commercially, are considered (monitored) organisms with the aid of a chip according to the invention. Thus, for example, from the application DE 19860313 A1 entitled "Method for detecting and characterizing Active substances against plant pathogens "show that there are metabolic situations in plants, in particular useful plants, which must be observed, as can livestock or laboratory animals, for example, eukaryotic cell cultures are of not little commercial interest, for example in the production of monoclonal antibodies, and in particular The fermentative production of foodstuffs, for example via the alcoholic fermentation operated by yeasts Bacteria are used in particular for the technical production of proteins or low molecular weight valuable substances (biotransformation), for example of vitamins or antibiotics.

According to the invention, probes are to be understood as meaning all molecules which are capable of entering into (respectively binding to) nucleic acids in each case with a largely specific interaction. This interaction is exploited according to the invention in order to obtain a largely unambiguous, evaluable signal within the scope of a corresponding arrangement (chip).

From a chemical point of view, a probe according to the invention is usually a compound which is capable of binding mRNA molecules or nucleic acids derived therefrom via hydrogen bonds, as for example also in the interaction of the two strands of a DNA or of the DNA RNA Interaction takes place. This may be, for example, a DNA which is more stable to hydrolysis than RNA.

In addition, other molecules are known in the art, in particular chemically synthesized, which allow biomimetic interaction, but are more stable than DNA, for example, by replacing the phosphate ester linkages of the backbone with less hydrolysis-sensitive bonds. Such nucleic acid analog probes characterize preferred embodiments of the present application (see below). The specific probes in question would be to synthesize, for example, according to the model of the sequence listing associated with this application. This is in contrast to the aspect that chips according to the invention should advantageously be usable several times, in particular during a single observed process in the course of which continuous monitoring is desirable. In each case, the degree of homology between the probe provided and the mRNA or the nucleic acid derived therefrom, which is to be detected by hybridization, is limiting for the usefulness of a probe. Ultimately, the degree of hybridization of the probe with the mRNA to be detected (see above) decides on its usefulness as a probe and must be experimentally optimized in individual cases and / or taken into account by adjusting the signal evaluation. There must be hybridization under the conditions imposed by the design of the measuring apparatus and other factors, which can be specifically attributed only to the gene of interest, strong enough to give a positive signal, and not too strong on the other hand the detected molecule diffused again after generation of the signal to free the binding site for the next molecule, or to allow a decay of the signal; the latter optionally via a corresponding washing step.

However, it is necessary to estimate the degree of homology between the genes in question before using chips according to the invention for an organism of interest, to anchor such on the chip of the invention more closely related genes on the chip if the affinity of the mRNAs to be detected for the preselected probes is insufficient and Perform calibration measurements to obtain reliable information on which signal strength corresponds to which concentration of specific mRNA.

The identification of the 47 genes essential to the present invention is described in the examples of the present application. Their sequences obtainable from Bacillus licheniformis are given in the Sequence Listing of the present application (SEQ ID Nos. 1 to 94), wherein the sequences with odd-numbered numbers are DNA sequences and the sequences each higher by a number are those derived therefrom Amino acid sequences is. While the DNA sequences can be used directly for the production of probes (see above), the amino acid sequences serve, for example, via sequence database comparisons of the checking of gene function and can also be used to detect nucleic acid-recognizing nucleic acids by, for example, back-translation of the genetic code Generate probes. As shown in Example 1, numerous different gene transcripts, that is to say mRNA molecules, were investigated, in particular those of which participation in the phosphate metabolism was generally known. These mRNA molecules were isolated at different times during the transition from B. licheniformis DSM 13 to a phosphate deficient state. Example 1 also describes how the concentration increase of this mRNA inside the cell of B. licheniformis was determined experimentally. Alternative determination possibilities for this may be established in the prior art; decisive for the understanding of the present invention is the compilation in Table 1 (Example 2). It shows the concentration changes associated with the transition for a total of 235 mRNAs. The following threshold values for the ratio of the RNA amount of the respective gene to the control value were considered significant: According to the invention, the genes whose RNA has a ratio of> 3 (ie at least one tripling) are considered induced; a clear induction is present at a ratio of>10; clearly repressed are genes with an RNA ratio <0.3 (that is, a lowering to less than 30%). In the 235 genes listed in Table 1, at least one tripling was observed at any of the time points in question.

Surprisingly, among these 235 genes, as shown in Table 2, there are only 47 genes with at least 10-fold induction at any of the observed time points under the conditions of the phosphate deficiency described in Example 1. These 47 genes are considered according to the invention as representative indicators of phosphate deficiency. Further information on these genes, for example on their function or deviating start codons can be found in Tables 2 and 3 and in the Sequence Listing; the respective German-language designations for the associated proteins have already been listed above in the order of the statements in Table 3.

All of these genes are individually described in the prior art. They can be found for the various organisms from generally accessible databases. As mentioned above, the sequences given in the Sequence Listing for B.licheniformis DSM 13 have been determined from this microorganism and are virtually identical to those described in the publication "The Complete Genome Sequence of Bacillus Licheniformis DSM13, to Organism with Great Industrial Potential" (2004) by B Veith et al., J. Mol. Microbiol. Biotechnol., Vol. 7 (4), pp. 204 to 211, and in addition to the entry AE017333 (bases 1 to 4.222.645) in the database GenBank (see above). The tribe ß. licheniformis DSM 13 is generally available from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 b, 38124 Braunschweig (http://www.dsmz.de). He holds the accession number ATCC 14580 from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (http://www.atcc.org).

The genes from other organisms corresponding to the abovementioned 47 genes are also to a large extent also stored in generally accessible databases, for example for the well-characterized species B. subtilis and E. coli, which are generally regarded as model organisms of Gram-positive or Gram-negative bacteria. The corresponding sequences can be found, for example, in the databases of Institut Pasteur, 25.28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, which can be accessed via the Internet at http://genolist.pasteur.fr/Colibri/ (for E coli), or http://genolist.pasteur.fr/SubtiList/ (for B. subtilis) are accessible (as of December 2, 2004). Other databases suitable for this purpose are those of the EMBL-European Bioinformatics Institute (EBI) in Cambridge, Great Britain (http://www.ebi.ac.uk), Swiss-Prot (Geneva Bioinformatics (GeneBio) SA, Geneva, Switzerland; http: // www. //www.genebio.com/sprot.html) or GenBank (National Center for Biotechnology Information, NCBI, National Institutes of Health, Bethesda, MD, USA).

These "corresponding genes" are to be understood as meaning those which in each case code for the proteins which catalyze the same chemical reaction in the organism under consideration or participate in the same physiological process as the abovementioned proteins in B. licheniformis DSM 13. Most of them carry other organisms have names and abbreviations similar to those given in B. licheniformis in Tables 1 and 2, because these names stand for the function in question, and in case of doubt they identify themselves by their sequence The function assignment depends above all on the similarity of the amino acid sequences to one another, since the amino acids represent the function carriers of the protein and, due to the degeneracy of the genetic code, can code different nucleotide sequences for the same amino acid sequence. Particularly high degrees of kinship exist between closely related species. Thus, in principle, it can be assumed that homologs can be found in most species for most of the 47 genes mentioned, including in cyanobacteria, in eukaryotic cells such as fungi, or in gram-negative species such as E. coli or Klebsiella. This probability is even higher for Gram-positive bacteria, in particular the genus Bacillus, because B. licheniformis DSM 13, of which the sequences listed in the sequence listing are derived, is such a Gram-positive bacterium. In addition, it can be assumed that in increasingly related organisms, the homologous genes are also increasingly subjected to the same or equivalent regulatory mechanisms; thus these homologues should also indicate the same metabolic situation, in particular a phosphate deficiency. In this respect, B. licheniformis is a happily chosen exemplified organism, because the species S. subtilis, B. amyloliquefaciens, B. lentus, B. globigii, which are likewise particularly important commercially, are also Bacilli and thus Gram-positive. This complies with the relevant aspect of the task.

It should be noted that to work through the invention for a particular species not all of the 47 genes mentioned must be known, but only a few of them (see below) are sufficient to map the phosphate metabolism and in particular to detect the transition to a phosphate deficiency state. Nevertheless, as the number of probes increases, the reliability of the statement about the phosphate supply state increases. If several genes, which in principle appear to be suitable based on the present disclosure, are known, it is advisable to carry out an expression study before producing a corresponding chip, in order to check, similar to the representation in Example 1 or also via a Northern analysis, whether the genes in question actually permit significant statements , The less the species considered is related to B. licheniformis, the more likely it would be shifts in the level of expression caused by phosphate deficiency, so that (possibly other subgroups of these genes than those listed below will prove particularly suitable and thus preferred.

In the production of a nucleic acid-binding chip according to the invention for an organism not mentioned here, the associated homologous genes must therefore be identified for at least some of the genes mentioned for B. licheniformis, for example by a comparison of those known for the organism concerned DNA sequences with the sequences given here. These or parts thereof (see below) can then serve as probes or as template for the synthesis of corresponding probes, which are applied to a nucleic acid-binding chip by methods known per se.

If individual homologous sequences are not stored in databases, it is possible for a person skilled in the art to synthesise respective probes on the basis of the sequences disclosed in the sequence listing for the present application, with the aid of which a gene bank prepared for the desired organism (genomically or preferably on the basis of the cDNA ) to search for the relevant homologue according to generally accepted methods. Alternatively, it is also possible to synthesize oligonucleotides using the DNA sequences given in the sequence listing, which serve as PCR primers, to the relevant genes or probes useful parts thereof from a total genomic DNA preparation or a cDNA preparation of the organism of interest herauszuamplifizieren. These or parts thereof (see below) can be used as probes on nucleic acid-specific chips according to the invention.

An essential feature of the present invention is that the total number of all phosphate metabolism-specific different probes is not more than 100. This feature correlates with the stated task, according to which it should focus on nucleic acid-binding chips whose number of assignable sites is comparatively low due to their design. These are in particular the electrically evaluable chips.

It is therefore increasingly preferred if the total number of all phosphate metabolism-specific different probes is not more than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50.

These may also contain probes for other genes, which are not discussed in the present application, and which serve for the control, for example those which are only expressed if there is sufficient phosphate supply. The disappearance of a signal due to this can also indicate the transition to the phosphate deficiency state. Should such a signal be preserved, this serves to control how reliable the signal of the phosphate deficiency to be determined according to the invention is. Among the other phosphates metabolism-specific probes may be, for example, those that are induced by an excess of phosphate, possibly also others that are apparently not directly related to the Phosphatstoffwechsel, but can be defined as such due to this inducibility. Thus, such a chip also provides an evaluable information useful in the considered process, if the lack of phosphate has been overcome, for example, by taking appropriate countermeasures.

Furthermore, nucleic acid-specific probes are usually only fragments of the complete genes (see below). In individual cases, for example in the case of regulation via splicing or large, polyfunctional polypeptides, it may therefore be useful to detect one and the same gene with two or more different probes. Thus, appropriate embodiments are optionally characterized by more than 47 probes, but which respond to no more than these 47 genes.

Depending on the process to be observed, it is also possible for probes for further genes or gene products to be present on chips according to the invention (see below).

On the other hand, the core of the invention lies precisely in the specificity of the respective chip, with which a special metabolic situation should be detected. Producing a chip with more than 100 probes responsive to different genes, or even a chip that mimics most of the genome of an organism, is not part of the invention described herein for such a specific problem because of the expense involved. Rather, both types of chips can be sensibly used side by side in an observed bioprocess: Thus, the chips with a large number of different gene probes or with a representative cross-section of various possibly relevant situations, as provided by the application WO 2004/027092 A2, can be coarse Provide an overview of the state of the organism in question while a chip according to the invention is consulted for control, if there is cause for concern, the cells in question could enter into a phosphate deficiency state.

In a preferred embodiment, it is a nucleic acid-binding chip according to the invention, which is increasingly preferably doped with the probes specified above in the order given there. By this is meant the genes listed in Table 3 in reverse order. Thus, chips with probes to the gene yhcR (similar to the 5'-nucleotidase; SEQ ID NO 13, 14) are still least preferred among the chips according to the invention for gene selection for the formation of probes because this, among the 47 mentioned, is significant due to phosphate deficiency amplified transcribed genes has been induced the weakest. In contrast, those with a probe for the gene yvmC (similar to proteins of unknown function, SEQ ID NOs: 75, 76) are most preferred for gene selection. Because this showed a nearly 150-fold induction compared to the initial level and thus the strongest of all genes. The signal associated with this should therefore be best suited to indicate the metabolic situation "phosphate deficiency" of all genes investigated.

In a preferred embodiment, it is a nucleic acid-binding chip according to the invention, wherein at least three of the probes are selected from the following 39 genes: gene for a hypothetical protein (homolog to SEQ ID NO. 65), yhbD, cotE, gene for a preserved hypothetical protein (homologue to SEQ ID NO: 59), yur1, spoVID, gene for a putative aromatic-specific dioxygenase (homolog to SEQ ID NO: 55), yhbE, gene for a putative benzoate transport protein (homolog to SEQ ID NO 49), pstBB, spolllAH, gene for a hypothetical protein (homologue to SEQ ID NO: 63), spollQ, spolllAG, yvmA, gene for a putative ribonuclease (homologue to SEQ ID NO: 93), dhaS, yrbE, gene for a putative decarboxylase / dehydratase (homologue to SEQ ID NO: 57), MpG, yfkH, spollAB, spolllAF, as D, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; SEQ ID NO: 17), gene for a putative phosphatase (homolog to SEQ ID NO. 61), phy, cypX, as S, phoD, pstS, phoB, yvnA, yvmC.

For these genes showed in the examples carried out in the examples based on B. licheniformis DSM 13, shown in Examples 1 to 3 gene inductions, which were increased by at least a factor of 13. Accordingly, preferred embodiments are characterized by the first 39 of the genes listed in Table 3.

Further preferred are nucleic acid-binding chips according to the invention, wherein at least three of the probes are selected from the following 14 genes: yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; homologue to SEQ ID NO: 17), gene for putative phosphatase (homolog to SEQ ID NO: 61), phy, cypX, as S, phoD, pstS , phoB, yvnA, yvmC.

For these genes showed in the examples carried out in the examples using B. licheniformis DSM 13, shown in Examples 1 to 3 gene inductions, which were increased by at least a factor of 25.

Further preferred are such nucleic acid-binding chips according to the invention, wherein at least three of the probes are selected from the following 8 genes: phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC.

For these genes showed in the examples carried out in the examples based on B. licheniformis DSM 13, shown in Examples 1 to 3 gene inductions, which were increased by at least a factor of 40.

Further preferred are such nucleic acid-binding chips according to the invention, wherein at least one, more preferably two or three, of the probes are / are selected from the following 3 genes: phoB, yvnA, yvmC.

For these showed in the examples using ß. lichenogenis DSM 13 carried out in Examples 1 to 3 Geninduktionen that were increased by at least a factor of 100, in the cases of yvnA and yvmC by more than 115 and in the latter case by significantly more than a factor of 140.

In preferred embodiments, nucleic acid-binding chips according to the invention are at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26 28, 30, 35, 40, 45 or 47 of the probes mentioned in the present invention.

For the more of these probes respond to a corresponding signal, the more reliable is the associated statement about the instantaneous phosphate supply or sub-supply. Thus, it is also useful to combine the particularly meaningful and thus preferred embodiments characterizing probes with seemingly less meaningful to exclude false-positive signals can. Furthermore, it is advantageous, as indicated in Table 2 to combine such probes with each other, which give different strength signals at different times specified therein. Thus, one can arrive at an estimate of how long before the beginning of the sampling of the phosphate deficiency occurs and whether he - while logging the cultivation conditions - may be due to a specific environmental impact.

With regard to their specific sequences, different probes which, however, respond to the same mRNA, for example fragments which hybridize with different regions of the same mRNA (see below), can be represented several times to increase the readability, but are counted only once within the meaning of this aspect of the invention because they are present in the best case equally responsive to the same signal and in principle would be interchangeable.

In preferred embodiments of nucleic acid-binding chips according to the invention, the total number of all different probes is increasingly preferably not more than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50.

This corresponds to the inventive idea outlined above, according to which a special metabolic aspect is to be monitored with the chips described here, so that a larger number of probes than for detecting this situation need not necessarily be applied to the respective chips. This leaves the situation unaffected that in individual cases it may be useful to use more than one probe to detect the same mRNA and / or apply individual probes that are associated with the production of a valuable material of interest. Overall, the present invention moves in said frame to include chips with only a few binding sites due to technical reasons in the scope.

In preferred embodiments of nucleic acid-binding chips according to the invention, the probes considered to be relevant to the invention are those which respond to the respective, or most homologous, in vivo transcribable genes from the organism selected for the bioprocess, preferably those derived from the relevant, or highly homologous, in vivo transcribable genes are derived just this organism. For this purpose, it has already been stated above that the sequences disclosed in the present application were obtained from B. licheniformis and, owing to the generally known relationships, should be suitable in particular for monitoring related species, in particular those of the genus Bacillus. This applies both to the identified genes, to which specific functions could be assigned on the basis of database comparisons, and which in principle should code for the same function in other species, as well as for those who have been defined only by their sequences. For this purpose, according to the invention, the genes closely related in the organism in question are used to derive corresponding probes. Care must be taken, however, that no probes are generated to pseudogenes, but to those that are actually rewritten under / n-wVo conditions in mRNA, that is, which lead to a measurable in the cytoplasmic nucleic acid signal.

Statistically, however, such a chip should be all the more successful the better the selected probes interact with the nucleic acids to be measured. Thus, especially with decreasing degree of relatedness to B. licheniformis, the need not rely on the sequences given in this hybridization but - if there are sequence differences - to use for the homologous genes from the species in question. As explained, these can be obtained by methods known per se, in particular Genbank screening or PCR with primers oriented on the sequences disclosed here (optionally in the form of so-called mismatch primers with certain statistical sequence variations).

In preferred embodiments of nucleic acid-binding chips according to the invention, the organism selected for the bioprocess is a representative of unicellular eukaryotes, Gram-positive or Gram-negative bacteria.

Because in these groups fall the most commercially used organisms, especially if it is the observed bioprocess is a fermentation. These include, for example, fermentation processes, for example for the production of wine or beer, or the biotechnological production of valuable substances such as proteins or low molecular weight compounds.

Depending on the type of product desired, different organisms are chosen for a biotechnological process. These are not within the meaning of the invention to understand only the production strains but also all upstream of the production process organisms, for example, to clone corresponding genes or to select suitable expression vectors. The need to capture the phosphate limitation basically exists during each sub-process.

In preferred embodiments of inventive nucleic acid-binding chips, the unicellular eukaryotes are protozoa or fungi, including in particular yeast, very particularly Saccharomyces or Schizosaccharomyces.

Because these are used in addition to the production of alcoholic beverages and other foods obtained by fermentation intensively as host cells in particular for the gene products of eukaryotes. The latter is particularly advantageous when these gene products are to undergo specific modifications that can be carried out only by these strains, such as, for example, glycosylations of proteins.

This object also includes chips according to the invention which are directed to the monitoring of the course, in particular of the growth of cell cultures of higher eukaryotes, for example of rodents or of humans. They may also be understood, in a sense, as at least largely unicellular eukaryotes, which have considerable commercial significance, in particular in immunology, for example for the production of monoclonal antibodies.

In preferred embodiments of nucleic acid-binding chips according to the invention, the Gram-positive bacteria are Coryneform bacteria or those of the genera Staphylococcus, Corynebacteria or Bacillus, in particular the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. agaradherens, B. stearothermophilus, B. globigii or B. lentus, and especially B. licheniformis.

Because these are technically particularly important production strains. They are used in particular for the production of low molecular weight chemical compounds, such as vitamins or antibiotics or for the production of proteins, in particular enzymes. Particularly noteworthy here are amylases, cellulases, lipases, oxidoreductases and proteases. The special focus on B. licheniformis is explained by the fact that the sequences given in the sequence listing from this Species were obtained and as described in Examples 2 and 3 could be demonstrably associated with the transition to a phosphate deficiency.

In no less preferred embodiments of nucleic acid-binding chips according to the invention, the Gram-negative bacteria are those of the genera E. coli or Klebsiella, in particular derivatives of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, and more particularly derivatives of the strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

Because these serve both on a laboratory scale, for example, the cloning and expression analysis as well as on a large scale of the production of biological valuable materials.

In preferred embodiments of nucleic acid-binding chips according to the invention, at least one, more preferably more, of the probes mentioned in connection with the invention described herein are derived from the sequences which are listed in the sequence listing under the numbers SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 are listed.

For these genes have been shown in B. licheniformis, as described in Examples 2 and 3, proven to be associated with the transition to a phosphate deficiency. In particular, when B. licheniformis or other, especially related Bacillus species are to be monitored, therefore, these sequences should be used.

Preferred embodiments of nucleic acid-binding chips according to the invention are those which are additionally doped with at least one probe for an additional gene, in particular one which is in a metabolic-related relationship to the gene (s) additionally expressed in terms of the process ), especially for one of them or this one. As noted above, the observed processes serve a technical interest, often associated with other specific genes. This is, for example, in the case that a protein is to be produced to the gene for this protein and in the case that a low molecular weight compound is to be produced, by one or more gene products, which lie in the synthesis route of the compound in question or to regulate this. It may also be affected other genes of the cell, such as metabolic genes that must be increasingly formed in the course of product manufacture, such as a cell's own oxidoreductase, if the product is to be obtained from a starting material or an intermediate via oxidation or reduction.

In addition, for certain biological processes, in particular the formation of commercially relevant compounds by microorganisms usually not the wild-type strains are used, but those that are geared to the process in question. In addition to the transformation with the genes responsible for the actual product production, this includes the addition of selection markers or further metabolic adjustments, as well as auxotrophies. Such strains have a particular requirement profile of the growth conditions and in part have metabolic genes mutated in relation to the wild-type genes. Since chips according to the invention should advantageously be aimed at precisely these strains, in particular the bioprocess under consideration, these strain-specific characteristics should be taken into account and may be reflected in the choice of the probes concerned.

In preferred embodiments of such nucleic acid-binding chips according to the invention, the additionally expressed gene for the process is that for a commercially useful protein, in particular an amylase, cellulase, lipase, oxidoreductase, hemicellulase or protease, or one which is synthesized for a low molecular weight chemical compound or at least partially regulated.

These are then particularly geared to those bioprocesses, especially fermentations, in which the said proteins are produced. These are commercially particularly important enzymes, which are used for example in the food industry or the detergent industry. In the latter case, in particular for the removal of soils which are hydrolyzable by amylases, cellulases, lipases, hemicellulases and / or proteases, for the treatment of concerned materials, in particular by cellulases or to provide a based on an oxidoreductase enzymatic bleaching system.

The latter variant falls within the field of biotransformation, according to which certain metabolic activities of microorganisms, if introduced in addition, are exploited for the synthesis of chemical compounds.

In preferred embodiments of nucleic acid-binding chips according to the invention, one, preferably several, of the probes mentioned in connection with the invention described here is / are provided in the form of the single-stranded strand in the form of the codogenic strand.

This embodiment aims to improve the hybridization between the probe and the sample to be detected. This applies in particular to the case where the content of the relevant mRNA is actually determined from the sample. Since this is single-stranded and in its sequence matches the coding strand of the DNA, an optimal hybridization should be done with the complementary, that is the codogenic strand.

In preferred embodiments of nucleic acid-binding chips according to the invention, one, preferably more, of the probes called relevant to the invention is / are provided in the form of a DNA or a nucleic acid analog, preferably a nucleic acid analog. This embodiment aims to improve the durability and multiple use of the chips of the invention. This need arises, in particular, during a single observed process during which constant monitoring is desirable. The durability of chips according to the invention, in particular with respect to nucleic acid-hydrolyzing enzymes, is already increased by the provision of the probes in the form of a DNA, since this per se is less susceptible to hydrolysis than, for example, an RNA. Even more durable are nucleic acid analogs in which, for example, the phosphate of the sugar phosphate backbone is replaced by a chemically different building block which is not hydrolyzable, for example, by natural nucleases. Such compounds are known in principle in the art and are commercially synthesized for desired, respectively to be given sequences of specialized companies on request. The relevant probes are to be synthesized on the model of the sequences given in the Sequence Listing.

In preferred embodiments of nucleic acid-binding chips according to the invention, one or more of the probes referred to as being relevant to the invention comprises gene regions which are transcribed into mRNA by the organism to be examined, in particular the gene regions which are close to the 5 'end of the mRNA.

This takes into account the fact that in many cases the regulatory DNA segments are also assigned to a specific gene. In fact, however, the chip according to the invention should be used to detect the mRNA actually present in the observed cells, so that for the purpose under consideration only the gene segment that is actually translated into mRNA is of importance. On the other hand, it must be taken into account that introns occur, in particular in the case of eukaryotes, ie the coding region is interrupted by sections which are not translated into mRNA. Probes containing introns are therefore unlikely to respond poorly to the mRNA in question. To realize this aspect, it is advisable not to resort to genomic DNA sequences, but to cDNA sequences, that is, those obtained from the actual mRNA.

Furthermore, hybridization over the entire length of the sequence is often unnecessary to detect mRNA. The specific probes therefore usually only need to comprise a smaller of the transcribed into mRNA gene. It is advantageous for this a selection of a region that is close to the 5 'end of the mRNA, since this is first transcribed into mRNA and thus is the first detectable after activation of the gene. This is contrary to a timely proof.

In preferred embodiments of nucleic acid-binding chips according to the invention, one or more of the probes called relevant to the invention are / are responsive to fragments of the respective nucleic acids, in particular to those which have a low degree of secondary folding in the relevant mRNA, based on the respective total mRNA ,

This is another aspect to optimize hybridization between the probes and the mRNA to be detected. Because mRNA molecules are often present in a secondary structure, which is based on hybridization of individual mRNA regions with their own other areas. For example, it comes to loop or Stem-Ioop structures. However, such regions are generally less likely to hybridize to other nucleic acid molecules, even if they are homologous. Such areas can be calculated quite accurately from computer programs directed thereto (see below). In order to realize this aspect, one should analyze the gene whose activity one wishes to determine for an organism of interest from such a program and resort to sections for obtaining a suitable - usually only a subset (see below) - probe sections which predicts a low level of mRNA secondary structures.

In preferred embodiments of nucleic acid-binding chips according to the invention, one, preferably more, of the probes called relevant to the invention has a length of increasingly preferably less than 200, 150, 125 or 100 nucleotides, preferably from 20 to 60 nucleotides, particularly preferably from 45 to 55 nucleotides on.

Because the probes used for the detection reaction need only to include parts of the mRNA to be detected, if the signal available on them is still specific enough. This specificity, the distinctness of different mRNA sets the lower limit for the length of the respective probes and must be determined experimentally if necessary in preliminary experiments. The identification of suitable probe lengths and regions is known per se to the person skilled in the art and is normally carried out with the aid of specialized software. Examples of such software are the programs array designer of the company. Premier Biosoft International, USA, and Vector NTI ^® Suite, V. 7, available from InforMax, Inc., Bethesda, USA. In addition to the secondary structures already mentioned, these software programs also take into account, for example, given probe lengths and melting temperatures.

In preferred embodiments of nucleic acid-binding chips according to the invention, an electrical signal is triggered by the binding of the mRNA to the probe in question as relevant to the invention.

J. Wang (Acc Chem Res, ISSN 0001-4842, Rec., Sept. 12, 2001, pp. A-F) discusses the advantages of an electrically evaluable system over an optical system. Further reference is made to various embodiments of such sensors developed in the prior art.

Thus, at the present time, the time from sampling to measuring the signal for optically analyzable chips is about 24 hours. Using an electrical system, the time required is currently less than 2 h (see Figure 1). In contrast, the number of simultaneously analyzable samples in electrically analyzable chips is currently in the double digits, but a rapid development suggests that this magnitude can be exceeded soon. Limiting this are the electronic evaluation units for the various signals.

A method for mRNA quantification established in the prior art is, for example, RT-PCT. This is described in the article "Quantification of Bacterial mRNA by One-Step RT-PCR Using the LightCycler System" (2003) by S.Tobisch, T Koburger, B. Jürgen, S. Léja, M. Hecker and T. Schweder in BIOCHEMICA, Volume 3, pages 5 to 8. In contrast, the detection of electrical chips has another advantage, namely the higher reliability of the data these have significantly lower fluctuation ranges compared to the RT-PCR.

The production of corresponding electronically evaluable chips is described, for example, in the patent applications WO 00/62048 A2, WO 00/67026 A1 and WO 02/41992 whose disclosure content is fully incorporated into the present application.

The mode of operation of electrically readable chips of a particularly preferred embodiment can be described as follows: The gene-specific probes are covalently bonded in a manner known per se to magnetic beads which are located in chambers of the chips provided for this purpose. The specific hybridization of the corresponding mRNA to the respective beads takes place in this hybridization chamber, which can be tempered and can be flushed through by the solutions in question. The beads are held in this chamber by a magnet. After hybridization of the RNA samples to the beads-bound DNA probes, a washing step is carried out to remove the unbound RNA so that only specific hybrids are still present in the incubation chamber, bound to the magnetic beads.

After washing, a detection probe is introduced into the incubation chamber labeled with a biotin extravidin-linked alkaline phosphatase. This probe binds to a second free region of the hybridized mRNA. This hybrid is then washed again and incubated with the substrate of the alkaline phosphatase para-aminophenol phosphate (pAPP). The enzymatic reaction in the incubation chamber leads to the release of the redox-active product para-aminophenol (pAP). This is now passed over the Red / Ox electrode on the electrical chip and sent the signal to a potentiostat.

System-specific software (for example MCDDE32) reads the data obtained and the results can be evaluated and displayed on another computer using another program (for example, Origin).

Of course, this process is variable both in terms of the technical design of the chips and the evaluation. For example, the detection reaction can also be carried out by another, but preferably a redox reaction because of the electrical measuring principle.

It is an object of the present invention to have identified phosphate metabolism-specific and therefore process-critical genes and made them available for analysis via correspondingly designed biochips. The advantage of chips Compared to conventional detection methods, in addition to gaining time and accuracy, with the provision of multiple probes on one support simultaneously in the same sample, the activities of several different genes can be detected and a more solid and detailed picture as described herein for a specific problem for example, the time at which a phosphate deficiency occurred.

A separate subject of the invention is thus the simultaneous use of nucleic acid or nucleic acid analog probes for at least three of the following 47 genes: yhcR, tatCD, ctaC, gene for a presumptive acetoin reductase (homolog to SEQ ID NO. 53), spollGA, nasE, pstA, spollAA, a hypothetical protein gene (homologue to SEQ ID NO: 65), yhbD, cotE, a conserved hypothetical protein gene (homologue to SEQ ID NO: 59), yurl, spoVID, a presumptive aromatic gene specific dioxygenase (homologue to SEQ ID NO: 55), yhbE, gene for a putative benzoate transport protein (homologue to SEQ ID NO: 49), pstBB, spolllAH, gene for a hypothetical protein (homolog to SEQ ID NO: 63) , spollQ, spolllAG, yvmA, gene for a putative ribonuclease (homologue to SEQ ID NO: 93), dhaS, yrbE, gene for a putative decarboxylase / dehydratase (homolog to SEQ ID NO 57), MpG, yfkH, spollAB, spolllAF , alsD, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde-De hydrogenase DhaS; Homolog to SEQ ID NO. 17), gene for a putative phosphatase (homolog to SEQ ID NO: 61), phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC, bound to a nucleic acid-binding chip, preferably to the same, for determination of physiological State of a biological process going through an organism.

As explained above, these genes are selected so as to provide an idea of the situation of the phosphate metabolism of the organism under consideration because they are involved in the transition of the Gram-positive bacterium, β, as described in Examples 1 to 3. licheniformis in the phosphate deficient state significantly and comparatively specific induced. A similar statement is to be expected for other organisms that have the homologous genes or proteins with essentially the same metabolism-relevant properties.

As already described in detail, such gene activities can in principle be determined in various ways, for example by Northern hybridization. The analysis using a nucleic acid-binding chip, in particular one described above, however, offers the possibility to determine several gene activities in a very efficient manner at the same time and very promptly. As a result, the metabolic changes of an organism undergoing a biological process can be monitored promptly and, if necessary, intervened by regulatory means.

The statements made above on nucleic acid-binding chips apply correspondingly to the uses of the probes described here.

Accordingly, these are preferably such uses, wherein the total number of all phosphate metabolism-specific different probes is not more than 100.

It is increasingly preferred that the total number of all phosphate metabolism-specific different probes does not exceed 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50, again including positive controls from the other phosphate metabolism.

Accordingly, these are also preferably uses where the probes previously indicated as being in accordance with the invention are increasingly preferably used in the sequence previously indicated as preferred (inversely to the order in Table 3).

According to the statements made above, the following uses of nucleic acid or nucleic acid analog probes just mentioned are increasingly preferred:

Use, wherein at least three of the nucleic acid or nucleic acid analogue probes are specific for genes from the following 39 genes: gene for a hypothetical protein (homologue to SEQ ID NO: 65), yhbD, cotE, conserved gene hypothetical protein (homologue to SEQ ID NO: 59), yur1, spoVID, gene for a probable aromatic-specific dioxygenase (homolog to SEQ ID NO: 55), yhbE, gene for a putative benzoate transport protein (homolog to SEQ ID NO. 49), pstBB, spolllAH, gene for a hypothetical protein (homologue to SEQ ID NO: 63), spollQ, spolllAG, yvmA, gene for a putative ribonuclease (homolog to SEQ ID NO: 93), dhaS, yrbE, gene for a presumptive decarboxylase / dehydratase (homolog to SEQ ID NO. 57), MpG, yfkH, spoll AB, spolllAF, alsD, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; homologue to SEQ ID NO: 17), gene for a putative phosphatase (homolog to SEQ ID NO. 61), phy, cypX, as S, phoD, pstS, phoB, yvnA, yvmC;

preferably including at least three of the following genes: yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS, homologue to SEQ ID NO: 17), gene for a putative phosphatase (homolog to SEQ ID NO 61), phy, cypX, as S, phoD, pstS, phoB, yvnA, yvmC;

- Of these, particularly preferred for at least three of the following 8 genes: phy, cypX, alsS, phoD, pstS, phoB, yvnA, yvmC; and

- Of these, very particularly preferred for at least one of the following 3 genes: phoB, yvnA, yvmC.

In accordance with the above, it is preferable to use according to the invention at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26 28, 30, 35, 40, 45 or 47 of said probes.

In accordance with the above, they are preferably uses according to the invention, wherein the total number of all different probes is increasingly preferably not more than 100, 95, 90, 80, 75, 70, 65, 60, 55, 50, 40, 30, 20 or 10 ,

According to the above, it is furthermore preferable to use according to the invention for determining a change in the phosphate metabolism of the organism passing through the biological process, preferably for the detection of a phosphate deficiency state.

In accordance with the above, it is furthermore preferred to use according to the invention, wherein at least one, more preferably more of the said probes are / is derived from the sequences which are listed in the sequence listing under the numbers SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 are listed. A separate subject of the invention are methods of determining the physiological state of an organism passing through a biological process by using a nucleic acid-binding chip according to the invention.

These procedures are in principle such that during the process and without interrupting it, taken samples of the observed organism and isolated from the mRNA. These or optionally derived therefrom compounds such as cDNA are passed over a nucleic acid-binding chip described above, which, taking into account the above-mentioned process steps - such as sufficient incubation time or washing off unspecifically binding nucleic acids - treated and finally fed to the detection device. Such a method protocol is illustrated in principle by the example of electrically evaluable chips in FIG.

The statements made above on nucleic acid-binding chips apply correspondingly to the methods described herein for the determination of the physiological state of an organism passing through a biological process.

Preferably, it is process according to the invention, wherein a change in the phosphate metabolism of the organism passing through the biological process is determined, preferably a phosphate deficiency state.

Because with respect to this application, the genes described in the examples and listed above have been selected. Their significant induction goes at least at ß. licheniformis associated with the occurrence of a phosphate deficient state, so that they can be detected with the above methods particularly reliable, not only in B. licheniformis but with increasingly better prospects of success even in increasingly related species (see above). In addition, this metabolic situation represents a critical point in the life cycle of many microorganisms. Thus, as explained in the examples, the transition to the stationary growth phase was always associated with the respective onset of phosphate deficiency. Procedures that help to detect this point in time serve to delay this transition and to extend the phase of the production of a valuable material, especially for large-scale fermentations. According to the above, such methods according to the invention are preferred, wherein the organism selected for the bioprocess is a representative of unicellular eukaryotes, Gram-positive or Gram-negative bacteria.

According to the above, such methods according to the invention are preferred among them, wherein the unicellular eukaryotes are protozoa or fungi, including in particular yeast, especially Saccharomyces or Schizosaccharomyces.

According to the above, such methods according to the invention are also preferred, whereby the Gram-positive bacteria are Coryneform bacteria or those of the genera Staphylococcus, Corynebacteria or Bacillus, in particular of the species Staphylococcus carnosus,

Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. agaradherens, B. stearothermophilus, B. globigii or B. lentus, and especially B. licheniformis.

According to the above, such methods according to the invention are no less preferred, the gram-negative bacteria being those of the genera E. coli or Klebsiella, in particular derivatives of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, and whole especially derivatives of strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

In accordance with the above, such methods according to the invention are preferred, those of the named probes being used which are of the SEQ ID NO. 1, 3, 5, 7, 9, 11, 15, 19, 23, 25, 29, 31, 33, 37, 43, 45, 49, 51, 53, 55, 59, 61, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91 or 93 are derived.

Among these, in turn, those methods are preferred in which the probes mentioned here are used for the Gram-positive bacteria listed above, in particular B. licheniformis, since these sequences have been isolated from this very organism and thus can be used most successfully on these species. According to the above, such methods according to the invention are preferred, wherein the determination of the physiological state is carried out at different times of the same process, preferably using a plurality of identically constructed nucleic acid-binding chips, particularly preferably the same nucleic acid-binding chip.

According to the above, such processes according to the invention are furthermore preferred, the process being a fermentation, in particular the fermentative production of a commercially useful product, more preferably the production of a protein or a low-molecular-weight chemical compound.

According to the above, such methods according to the invention are preferred, wherein the low-molecular chemical compound is a natural substance, a food supplement or a pharmaceutically relevant compound.

According to the above, such methods according to the invention are alternatively preferred, wherein the protein is an enzyme, in particular one from the group of α-amylases, proteases, cellulases, lipases, oxidoreductases, peroxidases, laccases, oxidases and hemicellulases.

An independent subject of the invention is also the possible uses of nucleic acid-binding chips according to the invention, as described in detail above, for determining the physiological state of an organism passing through a biological process.

The statements made above on nucleic acid-binding chips apply correspondingly to the uses described herein for determining the physiological state of an organism passing through a biological process.

According to the above, such uses according to the invention are preferred, wherein a change in the phosphate metabolism of the organism passing through the biological process is determined, preferably a phosphate deficient state. According to the above, such uses according to the invention are furthermore preferred, wherein the organism selected for the bioprocess is a representative of unicellular eukaryotes, Gram-positive or Gram-negative bacteria.

According to the above, such uses according to the invention are preferred, the unicellular eukaryotes being protozoa or fungi, in particular yeast, especially Saccharomyces or Schizosaccharomyces.

According to the above, such uses according to the invention are alternatively no less preferred, the Gram-positive bacteria being Coryneform bacteria or those of the genera Staphylococcus, Corynebacteria or Bacillus, in particular the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis B. amyloliquefaciens, B. agaradherens, B. stearothermophilus, B. globigii or B. lentus, and more particularly β. licheniformis.

According to the above, such uses according to the invention are no less preferred, the Gram-negative bacteria being those of the genera E. coli or Klebsiella, in particular derivatives of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, and more particularly Derivatives of strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. CO // XL-1 or Klebsiella planticola (Rf).

In accordance with the above, such uses according to the invention are preferred using those of said probes which are of the SEQ ID NO. 1, 3, 5, 7, 9, 11, 15, 19, 23, 25, 29, 31, 33, 37, 43, 45, 49, 51, 53, 55, 59, 61, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91 or 93 are derived.

Among these, again, for the reason already mentioned, those uses are preferred in which the probes mentioned here are used for the previously mentioned Gram-positive bacteria, in particular B. licheniformis. According to the above, such uses according to the invention are preferred, wherein the determination of the physiological state is carried out at different times of the same process, preferably using a plurality of identically constructed nucleic acid-binding chips, particularly preferably the same nucleic acid-binding chip.

According to the above, such uses according to the invention are furthermore preferred, the process being a fermentation, in particular the fermentative production of a commercially useful product, more preferably the production of a protein or a low-molecular chemical compound.

According to the above, such uses according to the invention are preferred, wherein the low-molecular chemical compound is a natural substance, a food supplement or a pharmaceutically relevant compound.

According to the above, such uses according to the invention are alternatively preferred, wherein the protein is an enzyme, in particular one from the group of α-amylases, proteases, cellulases, lipases, oxidoreductases, peroxidases, laccases, oxidases and hemicellulases.

The present invention is further illustrated by the following examples.

Examples

All molecular biology procedures are followed by standard methods as described, for example, in the Handbook of Fritsch, Sambrook and Maniatis "Molecular Cloning: a Laboratory Manual", CoId Spring Harbor Laboratory Press, New York, 1989, or similar pertinent works ) are used according to the specifications of the respective manufacturer.

example 1

Identification of gene probes by chip analysis

Cultivation of bacteria and sample collection

Cells of the strain Bacillus licheniformis DSM13 (available from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany) were synthesized in phosphate-limited, synthetic Belitsky minimal medium (0.28 mM Final concentration) at 37 ° C with constant shaking at 270 rpm. This medium has the following composition: (1.) Base medium (pH 7.5): 0.015 M (NH ₄ ) ₂ SO ₄ , 0.008 M MgSO ₄ × 7H ₂ O, 0.027 M KCl, 0.007 M Na ₃ citrate × 2H ₂ O, 0.050 M Tris-HCl and 0.009 M glutamic acid; (2) Supplements: 0.2 M KH ₂ PO ₄ , 0.039 M L-Tϊyptophan-HCL, 1 M CaCl ₂ × 2H ₂ O, 0.0005 M FeSO ₄ × 7H ₂ O, 0.025 M MnSO ₄ × 4H ₂ O and 20% (w / v) glucose; and as (3) medium supplementation scheme: 0.14 ml KH ₂ PO ₄ , 0.2 ml CaCl ₂ , 0.2 ml FeSO ₄ , 0.04 ml MnSO ₄ , 1 ml glucose. All values are based on 100 ml of basal medium.

In the course of the growth curve, at an optical density of 500 nm (OD ₅₀ o) of 0.4 to 0.5, the control sample and in the transient growth _phase at an OD _50O of 0.8 to 0.9 another sample ("transition The above-mentioned supplementation scheme for the Belitzky minimal medium is adjusted such that from an OD ₅₀ O of 0.8 to 1, 0 phosphate deficiency occurs and thus the stationary phase is introduced further samples were taken after another 30, 60 and 120 min The samples were immediately removed with the same volume of Killing buffer cooled to 0 ^° C. (20 mM NaN ₃ , 20 mM Tris-HCl, pH 7.5, 5 mM MgCl ₂ ), after immediate removal by centrifugation of the cell pellet The supernatant was discarded for 5 min at 4 ° C and 15,000 rpm and the pellet either frozen at -8O ⁰ C or immediately worked up further. cell disruption

The cell disruption was carried out with the "Hybaid RiboLyser ™ Cell Disruptor" (Thermo Electron Corporation, Dreieich, Germany) .This method is based on the mechanical destruction of the cell wall and the cell membrane with the aid of approximately 0.1 mm small glass beads (Fa. Sartorius BBI, Melsungen, Germany) The cells to be disrupted, which had previously been resuspended in lysis buffer II (3 mM EDTA, 200 mM NaCl), were placed together with the glass beads in a glass (stem) tube acidic phenol was added to prevent RNA degradation by RNases, which was then clamped into the RiboLyser, shaking vigorously with the glass beads colliding with the cells, causing cell disruption.

Total RNA isolation

After the cell disruption, the RNA-containing aqueous phase is separated from the protein and cell fragments, the chromosomal DNA and the glass beads by centrifugation and the RNA is purified therefrom by means of the KingFisher ml_ apparatus (Thermo Electron Corporation, Dreieich, Germany) using the apparatus MagNA Pure LC RNA Isolation Kit I (Roche Diagnostics, Penzberg, Germany). This purification is based on the binding of the RNA to magnetic glass particles in the presence of chaotropic salts, which also cause the inactivation of RNases. The magnetic particles serve as a means of transport of RNA between different reaction vessels, which are filled with binding, washing and Elutionpuffem. The KingFisher mL used for this purpose is a kind of pipetting robot that uses magnets to transport the particles with the bound RNA between the vessels and also to use them for mixing the samples. Finally, the RNA is released from the magnetic particles and is purified.

RNA quality and quantity control

The purification of the RNA is followed by quality and quantity control. The Agilent Bioanalyzer 2100 apparatus (Agilent Technologies, Berlin, Germany), which is used for this purpose, makes it possible to analyze RNA on a lab-on-a-chip scale. Together with Agilent's "RNA 6000 Nano Kit", the total RNA is separated by gel electrophoresis and thus offers the opportunity to study the quality of partial degradation and contamination by using ribosomal RNA (16 S and 23 S rRNA). detected. If these are clear bands, one can assume that the RNA was not degraded in the course of processing and thus is intact and can be introduced into the subsequent explorations. In addition, the exact concentration is determined.

transcriptome

Transcriptome analyzes were performed on whole genomic B. licheniformis DSM13 DNA microarrays which had been prepared in a conventional manner (for example according to WO 95/11995 A1) and can be evaluated by means of an optical system. Almost every B. licheniformis gene was duplicated on these DNA microarrays so that two samples could be analyzed in parallel on the same chip and the values obtained could be averaged.

The principle of the measurement carried out is that the respective mRNA molecules are transcribed from the sample taken in vitro via a reverse transcription into DNA, wherein one of the added deoxyribonucleotides carries a color marker. These labeled molecules are then hybridized with the known, lying on known locations of the chip probes and optically detected the respective strength of the attributable to the relevant sites on the fluorescent marker signal. By using two different fluorescent markers for a control and for a sample actually to be examined, which are simultaneously hybridized and thus compete for binding to the probe presented, different color values are obtained, which are a measure of the concentration of the control Provide concentration of the sample.

For this label, dUTP was selected, which was labeled with the fluorescent dye cyanine 3 or with cyanine 5. Thus, in each case 25 .mu.g total RNA of the control (OD ₅₀₀ 0.4) with the fluorescent dye cyanine 3 (Amersham Biosciences Europe GmbH, Freiburg, Germany) and 25 ug total RNA of the respective stress test (transient phase, 30, 60 and 120 min) with the fluorescent dye cyanine 5 (GE Healthcare, Freiburg, Germany). Competitive hybridization of the two samples on the conventional total genomic B. // c / 7en / 7br / 77 / s DSM13 DNA microarray was for at least 16 hours at 42 ° C. After washing the arrays to remove non-specific binding, the optical readout of the arrays was performed using the ScanArray® Express Laser Scanner (PerkinElmer Life and Analytical Sciences, Rodgau-Jügesheim, Germany). All hybridizations were repeated, with the samples labeled with the respective other dye (dye swap method). The quantitative evaluation of the arrays was carried out using the software ScanArray® Express (available from the company PerkinElmer Life and Analytical Sciences Rodgau-Jügesheim, Germany) according to the manufacturer and under standard parameters.

Using the controls of the Lucidea Score Card (GE Healthcare, Freiburg, Germany), the arrays were normalized and evaluated. With the help of this "score card", which serves to control the efficiency and quality of the hybridization, known control DNA and so-called spikes in the form of oligos were applied to the array according to the manufacturer's instructions and in the hybridization with complementary sequences were added After scanning, the controls should have the same amount of hybridization and dye incorporation, and the controls should be the same amount in both samples and appear yellow after scanning, or have a ratio between both channels of 1. The spikes are for each sample specifically, and are applied at various dilutions, that is they appear red or green after scanning for the particular sample.

For expression of the genes, mean values from the two hybridizations and the respective standard deviations were calculated. For a significant induction or a significant repression, the following threshold values are considered for the ratio of the RNA amount of the respective gene to the control value: The genes whose RNA has a ratio of> 3 (ie at least one tripling) are considered as induced; clearly repressed are genes with an RNA ratio <0.3 (that is, a lowering to less than 30%). These results are listed in Example 2. Example 2

Phosphate-deficient genes

Table 1 below lists all of the 235 genes of Bacillus licheniformis DSM13 determined in Example 1 whose induction (amounting to at least the factor 3) was observed under the conditions of the phosphate deficiency described in Example 1. In the first two columns the respective name of the derived protein or (if available) its abbreviation are given; the "bli number" corresponds to the "locus_tag" of the entry AE017333 (bases 1 to 4.222.645) in the database GenBank (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA; http: // /www.ncbi.nlm.nih.gov, as of December 2, 2004). licheniformis; This is followed by the factors observed at the times indicated above for increasing the concentration of the respectively associated mRNAs.

Table 1: The determined in Example 1 235 genes of Bacillus licheniformis DSM13, whose (at least the factor of 3 amounts) induction was observed under phosphate deficiency (for explanations see text).

Example 3

Specially induced genes under phosphate gel

Table 2 below lists all the genes of Bacillus licheniformis DSM13 determined in Example 1 whose induction under the conditions of the phosphate deficiency described in Example 1 at any of the measured times was at least a factor of 10 and those based on comparative experiments (data not shown) comparatively specific for phosphate deficiency can be classified. This is a total of 47.

The column names are the same as in the previous example. In addition, in the first column, the sequence numbers are given which carry the respective DNA or amino acid sequences in the sequence listing belonging to the present application. Particularities of the respective sequences, which appear as free text in the sequence listing, are supplemented under the category gene name / gene function.

Table 2: The 47 genes of Bacillus licheniformis DSM13 determined in Example 1, whose specifically induced by phosphate deficiency induction at any of the measured times was at least a factor of 10 (for explanations: see text).

In Table 3 below, the same 47 genes are listed again in the order of magnitude of induction observed. In this order, the corresponding terms are given in German in the description section. The staggering of the claims is based on the reverse order.

Table 3: The 47 genes of Bacillus licheniformis DSM13 determined in Example 1 whose specifically induced by phosphate deficiency induction at any of the measured times has been at least the factor 10, in descending order of the measured maximum value (last column).

Example 4

Real-time RT-PCR quantification of selected genes

With the help of real-time RT-PCR, it is possible to determine the absolute molecular numbers of specific transcripts in a sample. The LightCycler (Roche Diagnostics, Penzberg, Germany) was used in conjunction with the "SYBR Green I" kit (Roche Diagnostics). This method is based on the detection of binding of the fluorescent dye to double-stranded DNA. The quantification of specific mRNA molecules were carried out as described in Tobisch et al. (2003; Quantification of Bacterial mRNA by One-Step RT-PCR Using the LightCycler System; BIOCHEMICA, Volume 3. pages 5 to 8), wherein an external standard curve was generated for each mRNA to be examined.

For this dilutions of known concentrations of //> v # ro transcripts of the mRNA determined in Example 1 and listed in Example 2 were measured with the aid of the LightCycler and then the standard curve was created using the device-specific software. For this, specific primers had to be selected in advance for each mRNA to be investigated (with the aid of the software "Array Designer", available from PREMIER Biosoft International, Paolo Alto, USA), which synthesizes intra-vitro transcripts and the PCR Enable amplification in the LightCycler.

After making the external standard curve, the measurement started in the LightCycler. For the measurements, two different dilutions were used for each sample to be analyzed. In the LightCycler run, the specific transcript was then amplified with the respective primers and the incorporation of the dye was measured.

Using the LightCycler software and the script available on the website http://molbiol.ru/ger/scripts/01_07.html (December 2, 2004), it was possible to determine the exact number of molecules for selected transcripts. With the values obtained in this way and their relations to one another, the induction values indicated in Tables 1 and 2 were confirmed for the selected transcripts.

Description of the figures

FIG. 1: Schematic representation of the yAM / ne monitoring of a bioprocess with electrical DNA chips according to the invention

The timely monitoring of the bioprocess takes place advantageously via the following steps:

1. Sampling, for example, from the fermentation of a microorganism;

2. cell digestion via routine methods;

3. RNA isolation via routine methods;

4. hybridization to a chip loaded according to the invention with nucleic acids (for example DNA) or nucleic acid analogs (for example, compounds which are difficult to hydrolyze, analogously constructed);

5. detection of the electrical signals of a correspondingly designed electric chip; Alternatively, the recording of optical signals from an optical DNA chip would be possible;

6. Preferably computer-aided data evaluation.

When using electrical chips results in the current state of development an approximate total analysis time of less than 2 h, with the use of conventional optical DNA chips of about 12 h.

Claims

claims

1. nucleic acid-binding chip doped with probes for at least three of the following 47 genes: yhcR, tatCD, ctaC, gene for a presumptive acetoin reductase (homologue to SEQ ID NO. 53), spollGA, nasE, pstA, spollAA, gene for a hypothetical protein (homologue to SEQ ID NO: 65), yhbD, cotE, conserved hypothetical protein gene (homolog to SEQ ID NO: 59), yur1, spoVID, presumptive aromatic-specific dioxygenase gene (homolog to SEQ ID No. 55), yhbE, gene for a putative benzoate transport protein (homologue to SEQ ID NO: 49), pstBB, spolllAH, gene for a hypothetical protein (homolog to SEQ ID NO: 63), spollQ, spolllAG, yvmA, Gene for a putative ribonuclease (homologue to SEQ ID NO: 93), dhaS, yrbE, gene for a putative decarboxylase / dehydratase (homolog to SEQ ID NO 57), htpG, yfkH, spollAB, spolllAF, as D, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; homologue to SEQ ID NO: 17), gene for putative Pho sphatase (homologue to SEQ ID NO. 61), phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC, where the total number of all phosphate metabolism-specific different probes is not more than 100.

2. Nucleic acid-binding chip according to claim 1, increasingly preferably doped with the probes specified therein in the order given there.

3. Nucleic acid-binding chip according to claim 1 or 2, doped with probes for at least three of the following 39 genes: gene for a hypothetical protein (homolog to SEQ ID NO: 65), yhbD, cotE, gene for a conserved hypothetical potein (homolog to SEQ ID NO: 59), yur1, spoVID, gene for a probable aromatic-specific dioxygenase (homolog to SEQ ID NO: 55), yhbE, gene for a putative benzoate transport protein (homolog to SEQ ID NO: 49), pstBB , spolllAH, gene for a hypothetical protein (homolog to SEQ ID NO: 63), spollQ, spolllAG, yvmA, gene for a putative ribonuclease (homolog to SEQ ID NO: 93), dhaS, yrbE, gene for a putative decarboxylase / dehydratase (Homologue to SEQ ID NO 57), htpG, yfkH, spollAB, spolllAF, as D, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; homologue to SEQ ID NO 17), A putative phosphatase gene (homologue to SEQ ID NO: 61), phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC, preferably f for at least three of the following 14 genes: yfkN, pstC, yfmQ, pstBA, homolog to dhaS (Homolog to aldehyde dehydrogenase DhaS; homologue to SEQ ID NO: 17), gene for a putative phosphatase (homolog to SEQ ID NO: 61), phy, cypX, as S, phoD, pstS, phoB, yvnA, yvmC, most preferred for at least three of the following 8 genes: phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC, most preferably for at least one of the following 3 genes: phoB, yvnA, yvmC.

4. Nucleic acid-binding chip according to one of claims 1 to 3, doped with at least 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 28, 30, 35, 40, 45 or 47 of said probes.

The nucleic acid-binding chip according to any one of claims 1 to 4, wherein the total number of all different probes is increasingly preferably not more than 100, 90, 80, 70, 60 or 50.

6. Nucleic acid-binding chip according to one of claims 1 to 5, wherein said probes are those which are responsive to the relevant, or most homologous, in vivo transcribable genes from the organism selected for the bioprocess, preferably those which are derived from the relevant, or most homologous, in vivo transcribable genes just this organism.

The nucleic acid-binding chip according to claim 6, wherein the organism selected for the bioprocess is a representative of unicellular eukaryotes, Gram-positive or Gram-negative bacteria.

8. A nucleic acid-binding chip according to claim 7, wherein the unicellular eukaryotes are protozoa or fungi, including in particular yeast, especially Saccharomyces or Schizosaccharomyces.

Nucleic acid-binding chip according to claim 7, wherein the Gram-positive bacteria are Coryneform bacteria or those of the genera Staphylococcus, Corynebacteria or Bacillus, in particular of the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. agaradherens, B. stearothermophilus, B. globigii or S. lentus, and more particularly β. licheniformis.

10. Nucleic acid-binding chip according to claim 7, wherein the Gram-negative bacteria are those of the genera E. coli or Klebsiella, in particular derivatives of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, and especially derivatives of the Strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

11. A nucleic acid-binding chip according to any one of claims 1 to 10, wherein at least one, more preferably more of said probes is / are derived from the sequences shown in the sequence listing under the numbers SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 are listed.

The nucleic acid-binding chip according to any one of claims 1 to 11, additionally doped with at least one probe for an additional gene, in particular one which is in a metabolic-related relation to the process-related additionally expressed gene (s) especially for one of these or this self.

13. Nucleic acid-binding chip according to claim 12, wherein the process-related additionally expressed gene is that for a commercially useful protein, in particular an amylase, cellulase, lipase, oxidoreductase, a hemicellulase or protease, or one of which a synthesis route for a low molecular weight chemical compound or at least partially regulated.

14. Nucleic acid-binding chip according to one of claims 1 to 13, wherein one, preferably a plurality of said probes single-stranded, is provided in the form of the codogenic strand / are.

15. Nucleic acid-binding chip according to one of claims 1 to 14, wherein one, preferably more of said probes in the form of a DNA or a nucleic acid analog, preferably a nucleic acid analogue is provided / become.

16. Nucleic acid-binding chip according to one of claims 1 to 15, wherein one, preferably a plurality of said probes gene / includes gene regions / which are rewritten by the organism to be examined in mRNA, in particular the gene regions which are near the 5 'end of lie mRNA.

17. nucleic acid-binding chip according to one of claims 1 to 16, wherein one, preferably more of said probes responsive to fragments of the respective nucleic acids / responsive, in particular to those in the respective mRNA, based on the respective total mRNA a small Have a degree of secondary folding.

18. Nucleic acid-binding chip according to one of claims 1 to 17, wherein one, preferably more of said probes has a length of less than 200 nucleotides, preferably less than 100 nucleotides, more preferably from 20 to 60 nucleotides, most preferably from 45 to 55 nucleotides.

19. Nucleic acid-binding chip according to one of claims 1 to 18, wherein an electrical signal is triggered by the binding of the mRNA to the relevant said probe.

20. Simultaneous Use of Nucleic Acid or Nucleic Acid Analogue Probes for at Least Three of the Following 47 Genes: yhcR, tatCD, ctaC, Gene for a Presumptive Acetoin Reductase (Homolog to SEQ ID NO: 53), spollGA, nasE, pstA, spollAA, a hypothetical protein gene (homologue to SEQ ID NO: 65), yhbD, cotE, a conserved hypothetical protein gene (homologue to SEQ ID NO: 59), yurl, spoVID, a probable aromatic-specific dioxygenase gene ( Homolog to SEQ ID NO: 55), yhbE, gene for a putative benzoate transport protein (homolog to SEQ ID NO: 49), pstBB, spolllAH, gene for a hypothetical protein (homolog to SEQ ID NO. 63), spollQ, spolllAG, yvmA, gene for a putative ribonuclease (homologue to SEQ ID NO: 93), dhaS, yrbE, gene for a putative decarboxylase / dehydratase (homolog to SEQ ID NO 57), MpG, yfkH, spollAB , spolllAF, alsD, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS, homologue to SEQ ID NO: 17), gene for a putative phosphatase (homologue to SEQ ID NO: 61), phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC, bound to, preferably to, a nucleic acid-binding chip for determining the physiological state of a biological process-going organism.

21. Use according to claim 20, wherein the total number of all phosphate metabolism-specific different probes is not more than 100.

22. Use according to claim 20 or 21, wherein the probes specified in claim 20 are increasingly used in the order given there.

23. Use according to any one of claims 20 to 22, which are nucleic acid or nucleic acid analogue probes for at least three of the following 39 genes: gene for a hypothetical protein (homologue to SEQ ID NO: 65), yhbD, cotE , Preserved hypothetical protein gene (homologue to SEQ ID NO: 59), yur1, spoVID, presumptive aromatic-specific dioxygenase gene (homologue to SEQ ID NO: 55), yhbE, presumptive benzoate transport protein gene (homolog to SEQ ID NO: 49), pstBB, spolllAH, gene for a hypothetical protein (homolog to SEQ ID NO: 63), spollQ, spolllAG, yvmA, gene for a putative ribonuclease (homologue to SEQ ID NO: 93), dhaS, yrbE, gene for a putative decarboxylase / dehydratase (homolog to SEQ ID NO 57), WpG ₁ yfkH, spollAB, spolllAF, as D, gdh, yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; Homologue to SEQ ID NO: 17), gene for a putative phosphatase (homolog to SEQ ID NO: 61), phy, cypX , as S, phoD, pstS, phoB, yvnA, yvmC, preferably for at least three of the following 14 genes: yfkN, pstC, yfmQ, pstBA, homolog to dhaS (homolog to aldehyde dehydrogenase DhaS; Homolog to SEQ ID NO. 17), gene for a putative phosphatase (homologue to SEQ ID NO 61), phy, cypX, alsS, phoD, pstS, phoB, yvnA, yvmC, particularly preferred for at least three of the following 8 genes: phy, cypX, asS, phoD, pstS, phoB, yvnA, yvmC, most preferably for at least one of the following 3 genes: phoB, yvnA, yvmC.

24. Use according to one of claims 20 to 23 of at least 4, 5, 6, 8, 10, 12,

14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45 or 47 of said probes.

25. Use according to one of claims 20 to 24, wherein the total number of all different probes increasingly preferably does not exceed 100, 90, 80, 75, 70, 65,

60, 55, 50, 40, 30, 20 or 10 is located.

26. Use according to any one of claims 20 to 25 for determining a change in the phosphate metabolism of the organism passing through the biological process, preferably for the detection of a phosphate deficiency state.

27. Use according to any one of claims 20 to 26, wherein at least one, more preferably more of said probes is / are derived from the sequences which are listed in the sequence listing under the numbers SEQ ID NO. 1, 3, 5, 7, 9, 11, 13,

15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,

61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 are listed.

28. A method of determining the physiological state of a biological process-passing organism by use of a nucleic acid-binding chip according to any one of claims 1 to 19.

29. The method of claim 28, wherein a change in the phosphate metabolism of the organism passing through the biological process is determined, preferably a phosphate deficient state.

30. The method of claim 28 or 29, wherein the organism selected for the bioprocess is a representative of unicellular eukaryotes, Gram-positive or Gram-negative bacteria.

31. The method of claim 30, wherein the unicellular eukaryotes are protozoa or fungi, including in particular yeast, especially Saccharomyces or Schizosaccharomyces.

32. The method according to claim 30, wherein the Gram-positive bacteria are Coryneform bacteria or those of the genera Staphylococcus, Corynebacteria or Bacillus, in particular the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. agaradherens, B. stearothermophilus, B. globigii or ß. lentus, and especially around B. licheniformis.

33. The method of claim 30, wherein the gram-negative bacteria are those of the genera E. coli or Klebsiella, in particular derivatives of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, and more particularly derivatives of the strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E coli XL-1 or Klebsiella planticola (Rf).

34. The method of claim 33 or preferably 32, wherein those of said probes are used which are of the SEQ ID NO. 1, 3, 5, 7, 9, 11, 15, 19, 23, 25, 29, 31, 33, 37, 43, 45, 49, 51, 53, 55, 59, 61, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91 or 93 are derived.

35. The method according to any one of claims 28 to 34, wherein the determination of the physiological state at different times of the same process is performed, preferably using a plurality of identical nucleic acid-binding chips, more preferably the same nucleic acid-binding chip.

36. The method according to any one of claims 28 to 35, wherein the process is a fermentation, in particular the fermentative production of a commercially useful product, more preferably is the preparation of a protein or a low molecular weight chemical compound.

37. The method of claim 36, wherein the low molecular weight chemical compound is a natural product, a food supplement or a pharmaceutically relevant compound.

38. The method of claim 36, wherein the protein is an enzyme, in particular one of the group of α-amylases, proteases, cellulases, lipases, oxidoreductases, peroxidases, laccases, oxidases and hemicellulases.

39. Use of a nucleic acid-binding chip according to any one of claims 1 to 19 for the determination of the physiological state of a biological process going through an organism.

40. Use according to claim 39, wherein a change in the phosphate metabolism of the organism passing through the biological process is determined, preferably a phosphate deficient state.

41. Use according to claim 39 or 40, wherein the organism selected for the bioprocess is a representative of unicellular eukaryotes, Gram-positive or Gram-negative bacteria.

42. Use according to claim 41, wherein the unicellular eukaryotes are protozoa or fungi, including in particular yeast, especially Saccharomyces or Schizosaccharomyces.

43. Use according to claim 41, wherein the Gram-positive bacteria are Coryneform bacteria or those of the genera Staphylococcus, Corynebacteria or Bacillus, in particular of the species Staphylococcus carnosus, Corynehacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. agaradherens, B. stearothermophilus, B. globigii or B. lentus, and more particularly β. licheniformis.

44. Use according to claim 41, wherein the Gram-negative bacteria are those of the genera E. coli or Klebsiella, in particular derivatives of Escherichia coli K12, of Escherichia coli B or Klebsiella planticola, and whole especially derivatives of strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

45. Use according to claim 44 or preferably 43, wherein those of said probes are used which are of the SEQ ID NO. 1, 3, 5, 7, 9, 11, 15, 19, 23, 25, 29, 31, 33, 37, 43, 45, 49, 51, 53, 55, 59, 61, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91 or 93 are derived.

46. Use according to any one of claims 39 to 45, wherein the determination of the physiological state at different times of the same process is carried out, preferably using a plurality of identical nucleic acid-binding chips, more preferably the same nucleic acid-binding chip.

47. Use according to any one of claims 39 to 46, wherein the process is a fermentation, in particular the fermentative production of a commercially useful product, more preferably the production of a protein or a low-molecular chemical compound.

48. Use according to claim 47, wherein the low molecular weight chemical compound is a natural product, a food supplement or a pharmaceutically relevant compound.

49. Use according to claim 47, wherein the protein is an enzyme, in particular one of the group of α-amylases, proteases, cellulases, lipases, oxidoreductases, peroxidases, laccases, oxidases and hemicellulases.