WO2006122655A1 - Nucleic acid-binding chips for detecting glucose deficiency conditions within the scope of bioprocess control - Google Patents

Abstract

The invention relates to nucleic acid-binding chips for monitoring bioprocesses with special orientation toward the detection of glucose deficiency conditions. These chips support probes for at least six of the following 85 genes: yxe/, dppE, fruK, ybdN, yvdE, rbsK, hxIA, yvdH, ywfL, ispA, yvyD, licA, rocD, ycgO, yqiQ, sigX, iolC, licH, iolE, rsiX, mmgE, iolD, yvql, glpF, ysbA, yvqH, bpr, acdA, yvoA, aprE, yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, mmgD, tdh, mmgC, malP, mmgA, yusJ, malA, glpD, etfB, etfA, ywjF, yvdG, yusL, yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, acoB or homologs to SEQ ID NO. 157, 39, 159, 103, 151, 125, 123, 63, 9, 49, 93, 35, 13, 105, 17, 15, 95, 99, 101, 107 or 11 with a maximum of 100 glucose metabolism-specific different probes. The invention also relates to the use of corresponding gene probes, particularly to chips of the aforementioned type, and to corresponding methods and possible uses.

Description

 "Nucleic acid-binding chips for the detection of glucose deficiency states in the context of

bioprocesses "

The present invention relates to nucleic acid-binding chips for monitoring bioprocesses with a special focus on the detection of glucose 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, there is the very fundamental problem of monitoring their progress in order to achieve the desired result, to save resources and / or to achieve an optimal result in a given time Microorganisms on an agar plate or in Schuttelkultur to understand, but especially their fermentation, or the extraction of raw materials via the fermentation of microorganisms There is a rich state of the art, both what single-cell eukaryotes such as yeasts or Streptomyces-, 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 This can also be used to measure enzymatic activities by means of suitable assays, for example the detection of activities of interest in the cultural supernatant

On the other hand, in recent years, various techniques have been developed to track the metabolic processes of the organisms concerned at the level of gene expression. 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 genes of interest ( Promoter analysis, gene expression analysis) For this purpose, appropriate apparatuses (so-called (Biko) 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-dimentional gel electrophoresis of the cell lysates (2) the analysis of the generated mRNA (transknotoma) via an analogously constructed "genomic DNA array" and (3) the chip technology The latter is in a relatively early stage of development While the two former methods are ultimately based on quantitative isolation and time-consuming analyzes of the macromolecules concerned, chip technology is based on the principle of attaching probes for proteins or for nucleic acids on physically readable carriers (chips). The directly on the presence of the relevant proteins, or nucleic acids respond Compared to the two first-mentioned technologies, one promises of such chips a timely analysis of the considered process (At Iine analysis) Another advantage is the need for relatively small amounts of sample

The principle of chip-based measurements is described, for example, in the article "Real-time electrochemical monitoπng towards green analytical chemistry" by J Wang (Acc Chem Res, ISSN 0001-4842, Rec Sept 12, 2001, pages A-F) schematically in FIG Accordingly, the sample to be analyzed is brought into contact with a recognition layer (biorecognition layer), which may be, for example, an enzyme, antibody, receptor or DNA, the signal received thereby being transmitted via a transducer, for example an amperometre or potentiometnsche electrode, via an amplifier (amplification / processing) output as electrical voltage or as an electrical potential in the work in question and optical systems are addressed to which the electronically evaluable systems in terms of Miniatuπsierbarkeit and other advantages the author seemed more favorable

Due to the present invention, the protein-specific chips can be disregarded mRNA-recognizing chips are doped with complementary DNA molecules or DNA analogs Their production and use for very detailed issues such as the differentiation of point mutations, for example, in the application WO 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 signals attributable to the recognition and those with electrical evaluation

For this purpose, for example, fluorophores, Acπdiniumester or indirect detection of secondary binding processes, for example on biotin, avidin / streptavidin or digoxigenm described In the latter case, digoxigenin-specific antibodies are used for optical detection, the The enzyme activity is detected either by colorimetry or by 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 be able to perform the entire detection reaction on a chip ("lab-on-a-chip 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., 9, pp. 11348-11352; Liu et al., (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. I6, 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. In this system, a so-called "metallic amplification" during the hybridization reaction triggers a drop 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, there is the biological problem of which selection of gene activities mimic the process under consideration. This also includes the monitoring of product formation, if it is for example a fermentative product production. At the same time Also included are control genes that indicate when the process is developing in a direction that is not intended. In the course of this monitoring, for practical reasons, a not too high number of different genes should be observed

Of particular technical interest are biotechnological processes with gram-positive bacteria. These are used especially for their ability to secretion for the industrial production of valuable substances. These include those of the genus Bacillus and of these, in turn, the species B subtilis, B amyloliquefaciens, B agaradherens, B licheniformis, B lentus and B globigii currently the most economically significant

The simultaneous observation of the activity of several genes in bacteria (multiparameter detection), for example, deals with the work presented below. In the article "Monitoring of genes that respond to process-related stress in large-scale bioprocesses" by Schweder et al (1999), Biotech Bioeng, 65, pp. 151-159, discloses the change in mRNA levels of various stress-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 described in the subsequent concentration phase. They were detected by a PCR-based method carried out in a conventional manner. Different expression rates were already observed at different sites of the E. coli Reactor and in a matter of seconds reactions to changing conditions

Another 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, p. 217- 224, describes the expression of the genes Ion, dnaK, ibpB, htrA, ppiB, groEL, tig, s6, 19 and dps, partly on mRNA, partly on the protein level, partly on both levels. 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 RT GiII et al. (2001) is further concerned with a further observation of the course of fermentation on expression of a recombinant protein by E. coli. Biotech Bioeng, 72, pp. 85-95). It is described that the stress genes degP, uvrB, alpA, mltB, recA, ftsH, ibpA, aceA and groEL are more intensely expressed under the conditions mentioned at high cell density compared to low cell density Strength of the reaction, they grouped themselves among certain clusters RT-PCR and DNA microarray-based and complemented by dot-blot analysis approach applied to samples from two time points of fermentation, just at the beginning at low cell density and towards end at high cell density. This has become "cell conditioning" approaches designed to minimize the stress response of the cells

Fundamental differences in the expression patterns of Gram-positive organisms over those of Gram-negative bacteria are the work "Proteome and transcnptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein" by Jürgen et al (2001), Appl Microbiol Biotechnol, 55, p 326-332 The expression of the genes dnaK, groEL, grpE, cIpP, clpC, clpX, rpsB and rplJ is described in 8 subtilis, as they can be determined by the DNA-macro-array technique or by two-dimensional polyacrylic gel electrophoresis Gram-positive bacteria used for overexpression, the genes for the purine and Pyπmidin synthesis and the particular πbosomaler proteins more expnmiert than 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 it is at least shown the possibility of their production Thus, for example, the two patent applications DE 10136987 A1 and DE 10108841 A1 each reveal a gene from Corynebacterium glutamicum, namely clpC or citB Both genes are described as relevant for the amino acid metabolism, which is why a commercially interesting use of these genes should then consist of inactivating or at least attenuating them in order to optimize the fermentative production of amino acids by this microorganism. Further application possibilities can be found in these applications then submit probes for the relevant gene products on nuclear acid-damaging chips

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

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, on Organism with Great Industrial Potential" (2004) by B Veith et al in J Mol Microbiol Biotechnol, Vol 7 (4), pages 204 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, Booth 2 12 2004)

Using the technology of optically evaluable chips, it is now even possible to produce nucleic acid-binding chips that cover an almost complete genome or the associated transcptome (genomic DNA chips).

With the application WO 2004/027092 A2, a representative cross-section with a manageable number of genes is provided to identify various physiological conditions which an observed microorganism can undergo in the course of cultivation. These include, for example, starvation conditions for various nutrients or stress situations such as for example 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, of, dnaK, eno, glnR, groEL, groL, gsiB, ibpA, ibpB, katA, catE, IctP, Idh, opuAB, phoA, phoD, pstS, purC, purN, pyrB, pyrP, sigB, tnrA, trxA and ydjF. From this application also the corresponding DNA sequences from B subtihs As a result, it has become possible to also produce corresponding nucleic acid-binding chips which are used in the monitoring of a microorganism In particular, Gram-positive or Gram-negative bacteria-based bioprocesses indicate changes in the metabolic activities characterizing this process

Nucleic acid-binding chips, which are based on this selection of genes, provide a certain overall but rather rough overview of the respective metabolic situation. Thus, among the genes designated WO 2004/027092 A2, only one (acoA), which is also associated with In addition, a single positive signal may result from different situations or even be false-positive, which is why it often-and in particular In such an unclear situation - it makes sense to analyze a selected metabolic aspect separately. On the other hand, the number of simultaneously assignable sites is limited especially for electrically readable nucleic acid-containing chips, which have the advantage of a timely analysis, so that additional, special metabolic situations can be detected not easy extra Liche gene probes can be applied

Nos. DE 102004061664 7 and DE 102005042572 0 disclose nucleic acid-binding chips for monitoring bioprocesses with a special focus on the detection of phosphate deficiency or nitrogen deficiency states. They each carry a limited number of probes in order to display these specific stress situations A metabolic situation that may be critical for microorganisms and thus limiting for a corresponding bioprocess is that of glucose deficiency. Because glucose is the most important carbon (C), that is energy source in most nutrient media, or is obtained by digesting the more complex carbohydrates contained in the media by the microorganisms in question before it is metabolized. Glucose deficiency therefore represents a lack of carbon and energy supply and can quickly lead to the death of cells or at least prevent the achievement of higher cell densities.

For example, the publication by Bernhardt et al. Deals with this metabolic situation. "Bacillus subtilis During Feast and Famine: Visualization of the Overall Regulation of Protein Synthesis During Glucose Starvation by Proteome Analysis"; Genome Research (2003), 13 (2), 224-237. It shows that in glucose deficiency 150 proteins be formed de novo.

The publication "Genome-wide mRNA profiling in glucose-starved Bacillus subtilis cells", Mol. Gen. Genomics (2005), 274, 1-12, by Koburger et al., Relates to a complex study at mRNA level where clusters of each In total, it is described that approximately 900 genes are strongly up-regulated in the case of glucose deficiency.

Because of the particular significance of this metabolic situation, there is a particular need to carry out a chip-based, timely analysis in this regard and be able to intervene punctually and thus more purposefully in the current bioprocess due to the result obtained quickly. This provides a loss of yield that would result from a glucose bottleneck that was not recognized or recognized too late.

It was therefore the task of identifying genes that can be brought as clearly as possible in organisms, in particular microorganisms with the stress signal of glucose 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 "glucose deficiency" in the course of a monitored bioprocess (glucose deficiency sensors) The problem arose in particular for those 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 at-line analysis one if necessary early intervention in order to optimize the relevant bioprocess for carbohydrate supply

Such a DNA-binding chip should be usable for several comparable processes and be adapted to specific applications with comparatively slight variations. Preferably, it should be based on bioprocesses based on 8ac / us species, in particular β-subtilis, B amyloliquefaciens, B lentus, B globigu, and especially focused on B licheniformis Bioprocesses focused on fermentations, in particular the technical production of products, in particular of overexpublished proteins

Furthermore, such a glucose-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 variety of genes from the biotechnologically important bacterium B licheniformis was examined with regard to their activability by the transfer of the relevant culture into a glucose deficient state (Example 1). It was surprisingly found that by far not all genes involved in the carbohydrate, In addition, it has surprisingly been observed that activation of such genes has not been previously readily associated with glucose metabolism, for example protease genes, or those that are still involved in proteins Furthermore, those genes were to be subtracted therefrom which clearly respond to more than just this one stress signal. The genes identified in this way should now, if appropriate regardless of their previously known function, be known as glucose metabolism genes According to the teaching of the present invention, genes are all the more suitable as indicators the more severely this response precipitates. According to the invention, therefore, such genes are selected as glucose deficiency indicators which show a clear indication signal that lies significantly above a certain threshold value the farther the result is, the more they are to be regarded as parts of the solutions of the task according to the invention, which explains a corresponding staggering of the preferred inventionapplications (see below).

Table 1 shows all the 268 genes of Bacillus licheniformis DSM 13 identified in Example 1, the induction of which was observed under glucose deficiency, with a factor of at least three being considered significant. Of these, all 85 genes are assimilated in table 2, their induction by glucose deficiency to any of the measured times at least the Factor 10 and where it could be concluded from parallel studies not shown here that they were comparatively specific to this signal. These are listed again in Table 3 for the strength of their observed maximal induction. Their DNA and amino acid sequences are listed in the Sequence Listing present

Application, wherein the odd-numbered numbers for DNA and the subsequent even-numbered are for each deduced amino acid sequences These sequences also refer to the respective SEQ ID numbers in Tables 2 and 3 These are the following genes, in order of strength the induced by glucose deficiency induction

(see Table 2 and 3)

Gene for a putative transketolase (EC 2 2 1 1, SEQ ID NO 11, 12),

- acoB (acetoin dehydrogenase EI component, TPP-dependent beta subunit, SEQ ID

NO 77, 78),

Gene for a putative malate synthase (EC 4 1 3 2, SEQ ID NO 107, 108), gene for a putative butyryl-CoA dehydrogenase (SEQ ID NO 101, 102), acoA (acetoin dehydrogenase E1) Component, TPP dependent alpha subunit, SEQ ID

NO 79, 80),

-malL (maltose-inducible alpha-glucosidase, SEQ ID NO 139, 140), -yfiA (unknown function, similar to unknown proteins, SEQ ID NO 67, 68), -yvdK (unknown function, similar to unknown proteins, SEQ ID NO 141, 142), -yvdJ (unknown function, SEQ ID NO 143, 144), - Wb / (2-amino-3-ketobutyrate-CoA-Liquases, SEQ ID NO 115, 116), -yoeB (unknown function, SEQ ID NO 19, 20), -ycgN (unknown function, similar to i-Pyrrolιne-5-carboxylate dehydrogenase, SEQ ID

NO 119, 120),

mmsA (methylmalonate-semialdehyde dehydrogenase, SEQ ID NO 97, 98), -ycgM (unknown function, similar to prodrug oxidase, SEQ ID NO 117, 118), - acoL (acetoin dehydrogenase-E3 component, dihydrolipoamide- Dehydrogenase, SEQ ID

NO 73, 74), - acoC (acetoin dehydrogenase E2 component, dihydrohpoamide acetyltransferase, SEQ ID

NO 75, 76), -yvdl (unknown function, similar to Maltodextπn transport system permease, SEQ ID

NO 145, 146),

Gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, SEQ ID NO 99, 100), -yusL (unknown function, similar to 3-hydroxyacyl-CoA dehydrogenase, SEQ ID NO 51, 52), -yvdG (unknown function, similar to maltose / maltodextrin binding protein, SEQ ID NO 149,

150) -ywjF (unknown function, similar to iron-sulfur binding reductase, SEQ ID NO 91,

92)

etfA (electron transfer flavoprotein, alpha subunit, SEQ ID NO 1, 2), etfB (electron transfer flavoprotein, beta subunit, SEQ ID NO 169, 170), glpD (glycerol-3-phosphate dehydrogenase, SEQ ID NO 127, 128), - gene for a putative enoyl-CoA hydratase (EC 4 2 17, SEQ ID NO 95, 96), - malA (6-phospho-alpha-glucosidase, SEQ ID NO 69, 70) .

-yus J (unknown function, similar to butyryl-CoA dehydrogenase, SEQ ID NO 55, 56), - putative membrane protein gene (SEQ ID NO 15, 16), - mmgA (acetyl-CoA-acetyltransferase, SEQ ID NO 89 , 90), - malP (PTS maltose-specific enzyme-IICB component, SEQ ID NO 65, 66), - gene for a putative transcriptional regulator (SEQ ID NO 17, 18), - gene for a putative isocitrate lyase ( EC 4 1 3 1, SEQ ID NO 105, 106), - mmgC (acyl-CoA dehydrogenase, SEQ ID NO 85, 86), - tdh (threonine-3-dehydrogenase, SEQ ID NO 113, 114), - mmgD ( Citrate synthase III, SEQ ID NO 41, 42), - a putative transketolase gene (EC 2 2 1 1, SEQ ID NO 13, 14), - mmgB (3-hydroxybutyryl-CoA dehydrogenase, SEQ ID NO 87, 88), -pgcM (beta-phosphoglucomutase / glucose-1-phosphate phosphodiprosase, SEQ ID NO 137,

138), -ysiA (unknown function, similar to a transcription regulator of the TetR / AcrR family,

SEQ ID NO 167, 168),

- acoR (transcriptional activator of the acetoin dehydrogenase operon, SEQ ID NO 71, 72), - acsA (acetyl-CoA synthetase, SEQ ID NO 135, 136), - acuA (acetoin dehydrogenase, SEQ ID NO 133, 134) .

-yusK (unknown function, similar to acetyl-CoA-C-acyltransferase, SEQ ID NO 53, 54), - aprE (Subtihsin-Carlsberg precursor, EC 3 4 21 62, SEQ ID NO 31, 32), gene for a putative secretion protein (SEQ ID NO 35, 36), -yvoA (unknown function, similar to a transcriptional regulator of the GntR family, SEQ ID

NO 81, 82),

acdA (acyl-CoA-dehydrogenase, SEQ ID NO 83, 84), bpr (Bacillopeptidase F, SEQ ID NO 3, 4), -yvqH (unknown function, similar to unknown proteins from S subtihs, SEQ ID NO 59,

60)

-ysbA (unknown function, SEQ ID NO 29, 30), gene for a putative enoyl (3-hydroxy-isobutyryl) coenzyme A-hydratase protein (EC 4 2 17,

SEQ ID NO 93, 94), -glpF (glycerol uptake facilitator, SEQ ID NO 131, 132),

-yvql (unknown function, SEQ ID NO 61, 62),

Gene for a putative hydroxybenzoate hydroxylase (SEQ ID NO 49, 50),

IOID (function in myo-inositol catabolism, SEQ ID NO 163, 164),

Gene for a putative formate dehydrogenase-alpha chain (EC 1 2 1 2, SEQ ID NO 9, 10),

Gene for a putative pectin methylesterase (SEQ ID NO 63, 64),

mmgE (unknown function, SEQ ID NO 43, 44),

Gene for a hypothetical protein (SEQ ID NO 123, 124),

-rsiX (negative regulator of Sgma-X-Aktιvιtat, SEQ ID NO 25, 26),

putative gene for a putative ABC transporter ATP restriction protein (SEQ ID NO 125, 126),

- / o / E (function in myo-inositol catabolism, SEQ ID NO 165, 166),

-licH (6-phospho-beta-glucosidase, SEQ ID NO 37, 38),

IOIC (function in myo-inositol catabolism, SEQ ID NO 161, 162),

s / ρ / X (RNA polymerase ECF-type Sgma factor, SEQ ID NO 27, 28),

-yqiQ (unknown function, similar to phosphoenolpyruvate mutase, SEQ ID NO 45, 46),

-ycgO (unknown function, similar to proline permease, SEQ ID NO 121, 122),

rocD (ornithine ammotransferase, SEQ ID NO 7, 8),

-licA (PTS-Lichenan specific enzyme IIA component (SEQ ID NO 57, 58),

Gene for a glucan-1,4-alpha-maltohydrolase (EC 3 2,133, SEQ ID NO 151, 152),

-yvyD (unknown function, similar to the gram-54 modulator factor of Gram-negative bacteria,

SEQ ID NO 5, 6),

-ispA (larger intracellular cut protease, SEQ ID NO 23, 24), -ywfL (unknown function, similar to unknown proteins, SEQ ID NO 47, 48), -yvdH (unknown function, similar to Maltodextπn transport system permease, SEQ ID

NO 147, 148),

Gene for a hypothetical protein (SEQ ID NO 103, 104), hxIA (3-hexulose-6-phosphate synthase, SEQ ID NO 155, 156), rbsK (ribokinase, SEQ ID NO 129, 130), yvdE (unknown function, similar to Lacl-type transcriptional regulator, SEQ ID

NO 153, 154), - gene for a putative methylmalonate-semialdehyde dehydrogenase [acyherend] (EC 1 2 1 27,

SEQ ID NO 159, 160),

Gene for a putative transcriptional regulator (SEQ ID NO 39, 40), -ybdN (unknown function, SEQ ID NO 33, 34), - frivK (fructose 1-phosphate kinase, SEQ ID NO 111, 112),

Gene for a tight homologue to Ald [L-alanine dehydrogenase] (SEQ ID NO 157, 158), dppE (dipeptide ABC transporter dipeptide binding protein [sporulation], SEQ ID NO 21, 22) -yxel (unknown function, similar to Penicillm amidase, SEQ ID NO 109, 110)

A solution of the problem is thus in a nucleic acid-binding chip, doped with probes for at least six of the following genes yxel, dppE, gene for a tight homolog to AId [L-alanine dehydrogenase] (homolog to SEQ ID NO 157) , fruK, ybdN, gene for a putative transcriptional regulator (homolog to SEQ ID NO 39), gene for a putative methylmalonate semialdehyde dehydrogenase [acyherend] (EC 1 2 1 27, homolog to SEQ ID NO 159), yvdE, rbsK, hxIA, gene for a hypothetical protein (homolog to SEQ ID NO 103), yvdti, ywfL, ispA, yvyD, gene for a glucan 1, 4-alpha-maltohydrolase (EC 3 2 1 133, homologue to SEQ ID NO 151) , hcA, rocD, ycgO, yqiQ, sigX, IOIC, licH, IOIE, putative gene for a putative ABC transporter ATP restriction protein (homologue to SEQ ID NO 125), rsiX, gene for a hypothetical protein (homologue to SEQ ID NO 123), mmgE, gene for a putative pectin methylesterase (homologue to SEQ ID NO 63), gene for a putative formate dehydrogenase-alpha-ket te (EC 1 2 1 2, homologue to SEQ ID NO 9), IOID, gene for a putative hydroxybenzoate hydroxylase (homolog to SEQ ID NO 49), yvql, glpF, gene for a putative enoyl (3-hydroxyιsobutyryl) coenzyme -A-hydratase protein (EC 4 2 17, homologue to SEQ ID NO 93), ysbA, yvqti, bpr, acdA, yvoA, gene for a putative secretion protein (homologue to SEQ ID NO 35), aprE, yusK, acuA , acsA, acoR, ysiA, pgcM, mmgB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 13), mmgD, tdh, mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1 , Homologue to SEQ ID NO 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl CoA hydratase (EC 4 2 1 17, homologue to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60 , Homolog to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, k bl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107) , acoB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11), wherein the total number of different specific for glucose deficiency 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. The sequence listing discloses these genes as obtainable from B licheniformis DSM 13. Most of these are also described from other species (see below) ) However, some are putative (presumable) genes or genes coding for putative (presumable) enzymes. According to the invention, these are as far as possible based on database comparisons as such with a putative (assumed) Function and additionally defined as a homologue to the found ß ichicheniformis genes For this purpose, two things must be explained

- On the one hand, it could turn out for one or the other by a biochemical analysis that this putative function does not correspond to the real function. Then the putative function is not held. Rather, these findings do not call the invention into question insofar as it merely relates to the invention the observation of increased transcription in connection with the glucose deficiency arrives so that the gene activity in question, regardless of the ultimate enzyme activity carried out can certainly serve as an indicator of glucose deficiency

On the other hand, in the absence of a gene name, no more suitable definition could be found than that about the gene itself. Homologues are therefore spoken of. It is to be assumed that homologous genes are activated in species other than B licheniformis under conditions of glucose deficiency In a species of interest for one of these genes, there are several homologs capable of transcoding in vivo, the phrase "homologue to SEQ ID NO" refers to the respective trailing genes of these various candidate genes

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

According to the invention, a nucleic acid-binding chip is to be understood as meaning 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

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 nucleic acid hybridization) derived therefrom molecule) with the probe presented on the chip Depending on the system for evaluating the signal released by the hybridization between the chips with an optical and with an electrical analysis system different invention invention, both systems are in principle applicable

Such chips are used in the following manner to control (monitor) the bioprocess under consideration. From the process, a sample is taken with the biological material to be analyzed at a certain time. From this, RNA is isolated by methods known per se, for example cell disruption and use of a denaturing buffer. in particular mRNA isolated This is itself marked or as starting molecule for a in the molecule introduced into the measurement (eg, cDNA obtained by reverse transcription) and the molecules obtained are advantageously passed over / through the chip in a buffer. Hybridization (sandwich labeling) of a prepared RNA or its derivative with the homologous (ie with respect to their sequence congruent) probe provided on the chip (target nucleic acid, for example target DNA or target Nukleinsaureanalog) 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 fluorescent marker, hybridization with a second probe or on a secondary detection reaction, such as via RT-PCR

Since in each case a plurality of molecules are bound to the chip by the same probe, the strength of the hybridization signal is over a certain range - possibly to be optimized in the individual case - proportional to the number of specific mRNA present in the sample at the time of sampling in this way the strength of the signal provides 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

When using a erfmdungsgemaßen chips considered (monitoned) organisms are in principle all plants, animals and microorganisms in question, especially those that are used commercially Thus, for example, from the application DE 19860313 A1 entitled "Method for the detection and characterization of active compounds against plants Pathogens "show that there are metabolic situations in plants, especially useful plants, which must be observed. Likewise, for example, livestock or laboratory animals can be observed Of not minor commercial interest are eukaryotic cell cultures, such as in the production of monoclonal antibodies, and in particular the fermentative production of food About the alcoholic fermentation operated by yeasts Bacteria are used in particular for the technical production of proteins or low-molecular-weight valuable materials (biotransformation), for example of vitamins or antibiotics

According to the invention, probes are to be understood as meaning all molecules which are capable of reacting (binding) with nucleic acids in each case to a large extent. This interaction is exploited according to the invention in order to form a signal which can be assigned to a significant extent within the context of a corresponding arrangement (chip) to obtain 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 possibly 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 sufficient affinity of the mRNAs to be detected for the presented probes and Perform calibration measurements to obtain reliable information on which signal strength corresponds to which concentration of specific mRNA. The identification of the 85 genes essential to the present invention is described in the examples of the present application. The sequences obtainable from Bacillus licheniformis are given in the Sequence Listing of the present application (SEQ ID Nos. 1 to 170), wherein the sequences with odd numbers are 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, for checking the gene function and may also serve to generate, for example, via jerk translation of the genetic code, similar nucleic acid-sensing probes

As shown in Example 1, numerous different gene transcripts, that is, mRNA molecules were examined, especially those of which participation in glucose metabolism was well known. These mRNA molecules were transduced at different times during the transition from B licheniformis DSM 13 to a glucose deficient state In Example 1 it is also described how the concentration increase of this mRNA in the cell interior of B licheniformis was determined experimentally. Alternative determination possibilities for this may be established in the prior art. The compilation in Table 1 (Example 2) is crucial to the understanding of the present invention shows the concentration changes associated with the transition for a total of 268 mRNAs. The following threshold values for the ratio of the RNA amount of the respective gene to the control value were regarded as significant. The genes according to the invention are considered to be induced RNA has a ratio> 3 (ie at least one tripling), a clear induction is present at a ratio of> 10, significantly repmpmiert are genes with an RNA ratio <0.3 (that is, a lowering to less than 30%) In the 268 genes listed in Table 1, at least one tripling was observed at any of the time points in question

Among these 268 genes, as evidenced by Example 3, there are 92 genes with at least 10-fold induction at any of the observed times under the conditions of the glucose deficiency described in Example 1. Of these, seven may not be considered specific, so that, surprisingly, only those shown in Table These 85 genes are considered according to the invention as representative indicators of a glucose deficient state Further information on these genes, for example on their function or deviating start codons Table 1 and the Sequence Listing are the respective German-speaking 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 DSM 13 have been determined from this microorganism and are virtually identical to those described in US Pat Publication "The Complete Genome Sequence of Bacillus licheniformis DSM13, to Organism with Great Industrial Potential" (2004) by B Veith et al. In J Mol Microbiol Biotechnol, vol. 7 (4), pages 204 to 211, and additionally under entry AE017333 (Bases 1 to 4,222,645) in the GenBank database (see above). The strain ß licheniformis DSM 13 is available from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 b, 38124 Braunschweig (http // www dsmz de He is the recipient of the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110 - 2209, USA (http // www atcc org) the accession number ATCC 14580

The genes from other organisms corresponding to the said 85 genes are to a large extent likewise 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 For example, the databases of the Institut Pasteur, 25.28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, which are available on the Internet at http // genolist pasteur fr / Colibπ / (for E. coli), or http // genossenst pasteur fr / SubtιLιst / (for B subtilis) are accessible (Stand 2 12 2004) Further suitable databases 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.genebio.com/sprot.html) or GenBank (National Center for Biotech Chnology 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 said proteins in S. licheniformis DSM 13. Most of these carry for other organisms similar names and abbreviations as those given in Tables 1 and 2 for ßlicheniformis, because these names stand for the respective function. In case of doubt, they identify themselves by their sequence, which from the respective organism those to those mentioned here Nachstahnhche (most strongly homologous) In the function assignment it depends above all on the similarity of the amino acid sequences to one another, because the amino acids represent the functional carriers of the protein and because of the degeneracy of the genetic code can code different nucleotide sequences for the same amino acid sequence Particularly high degrees of relationship exist between closely related species. In principle, it can be assumed that homologs can be found in most species for most of the abovementioned genes, including cyanobacteria, in eukaryotic cells such as fungi, or gram-negative species such as E. coli or Klebsiella This probability is even higher for Gram-positive bacteria, in particular of the genus Bacillus, because B licheniformis DSM 13, from which the sequences listed in the sequence listing originate, is such a Gram-positive bacterium. In addition, it can be assumed that in increasingly related organisms the homologous Genes are increasingly subject to the same or equivalent regulatory mechanisms, so these homologs should also show the same metabolic situation, especially a glucose deficiency Insofern Slicheniformis is a happy chosen example organism, because the commercially also particularly important Sp ezies B subtilis, B amylohquefaciens, B lentus, B globigu are also bacilli and thus gram-positive. This corresponds to the aspect of the task in question

It should be noted that to follow up the invention for a particular species not all of the 85 genes mentioned must be known, but only a few of them (see below) are sufficient to map the glucose metabolism and in particular to be able to detect the transition to a glucose deficiency state Number of probes the reliability of the statement about the glucose supply state 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 production of a corresponding chip, in order to produce a similar procedure as in Example 1 or also via a Northern Analysis to check whether the genes in question actually permit significant statements The less the species considered is related to B. licheniformis, the more likely to be shifts in the expression level caused by glucose deficiency, so that ( if not other than those listed below) subgroups of these 85 genes as particularly suitable and thus prove to be 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 comparing the DNA sequences known for the organism concerned with those indicated here Sequences These or parts thereof (see below) can then serve as probes or as templates 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 make use of the sequences disclosed in the sequence listing for the present application synthesize respective probes in order to use them to search for a gene library prepared for the desired organism (genomically or preferably on the basis of the cDNA) by generally customary methods for the homolog in question. Alternatively, it is also possible to use the DNA sequences given in the Sequence Listing. To synthesize sequences of oligonucleotides which serve as PCR primers to amplify the genes or probes thereof from a whole genomic DNA preparation or a cDNA preparation of the organism of interest. These or parts thereof (see below) can be used as probes according to the invention Nucleic acid-specific chips are used

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

Among the further glucose-specific-specific probes, for example, those which are induced by a Glucoeseuberschuß, possibly also others, which are not directly related to the glucose metabolism, but due to this inducibility can be defined as such Thus, such a chip also an evaluable and useful in the process considered information when the lack of glucose has been overcome, for example, by taking appropriate countermeasures

In addition, 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, multi-functional polypeptides, it may therefore be useful to use one and the same gene with two or more detect more different probes Thus, corresponding embodiments are possibly characterized by more than 85 probes, but respond to no more than these 85 genes

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

On the other hand, the core of the invention lies precisely in the specificity of the chip concerned, with which a specific metabolic situation should be detected. The production of a chip with more than 100 probes responding to different genes or even a chip which represents a large part of the genome of an organism. is not part of the invention described here in such a specific issue because of the associated effort Rather, both types of chips in an observed bioprocess sense next to each other Thus, the chips with numerous different gene probes or with a representative cross section of various possibly relevant situations, as provided by the application WO 2004/027092 A2, can provide a rough overview of the condition of the organism concerned, while a chip according to the invention for control, if there is cause for concern, the cells in question could enter a glucose 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

Thus, the genes listed in Table 3 are meant in reverse order. Thus, chips having probes for the gene yxel (coding for a protein of unknown function, similar to the penicillin amidase, SEQ ID NO 109, 110) are among the chips according to the invention in terms of gene selection Probe formation is least favored because it has been least induced among the 85 genes significantly transcribed by glucose deficiency. In contrast, those with a probe for the gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11) was most preferred in terms of gene selection because this showed over the starting level more than 123-fold induction and thus the strongest of all genes measured The signal associated with this should therefore be of all genes best suited to indicate the metabolic situation "glucose deficiency"

In a preferred embodiment, it is a nucleic acid-binding chip according to the invention, wherein at least one, more preferably two or three, of the probes are selected from the following 67 genes: ycgO, yqiQ, sigX, IOIC, licH, IOIE, putative gene for a putative ABC transporter ATP binding protein (homolog to SEQ ID NO 125), rsiX, gene for a hypothetical protein (homolog to SEQ ID NO 123), mmgE, gene for a putative pectin methylesterase (homolog to SEQ ID NO 63), gene for a putative formate dehydrogenase-alpha chain (EC 1 2 1 2, homologue to SEQ ID NO 9), IOID, gene for a putative hydroxybenzoate hydroxylase (homologue to SEQ ID NO 49), yvql, glpF , Gene for a putative enoyl (3-hydroxyιsobutyryl) coenzyme A hydratase protein (EC 4 2 17, homologue to SEQ ID NO 93), ysbA, yvqH, bpr, acdA, yvoA, gene for a putative secretion protein ( Homologue to SEQ ID NO 35), aprE, yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, gene for a putative transketolase (E. C 2 2 1 1, homologue to SEQ ID NO 13), mmgD, tdh, mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1, homologue to SEQ ID NO 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl CoA hydratase (EC 4 2 17, homologue to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, Homolog to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homologue to SEQ ID NO 101) , Gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107), a∞B, gene for a putative transketolase (EC 2 2 1 1, homolog to SEQ ID NO 11)

Because 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 12. Accordingly, preferred embodiments are the first 67 of the genes listed in Table 3 marked

Further preferred are such inventive nucleic acid-binding chips, wherein at least one, more preferably two or three of the probes from the following 34 genes is / are tdh, mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1, homolog to SEQ ID NO 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl CoA gene Hydratase (EC 4 2 1 17, homologue to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-Oxopropιonate-reductase (EC 1 1 1 60, homolog to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homologue to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homolog to SEQ ID NO 11)

For these genes showed in the examples carried out in the examples using ßlicheniformis DSM 13, shown in Examples 1 to 3 studies gene inductions that were increased by at least a factor of 30

Further preferred are such inventive nucleic acid-binding chips, wherein at least one, more preferably two or three of the probes from the following 10 genes is / are kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (Homologue to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homolog to SEQ ID NO 11) For these genes showed gene inductions, which were increased by at least a factor of 61, in the tests carried out in the examples on the basis of S licheniformis DSM 13 and described in Examples 1 to 3

Further preference is given to nucleic acid-binding chips according to the invention in which at least one, more preferably two or three, of the probes are selected from the following 4 genes: gene for a putative butyryl-CoA dehydrogenase (homologue to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homologue to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11)

For these showed in the examples carried out in the examples using B licheniformis DSM 13, shown in Examples 1 to 3 investigations gene inductions that were increased by at least a factor of 90

In preferred embodiments, nucleic acid-binding chips according to the invention with at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 of the present invention, that is doped in this sense probes doped

For the more of these probes respond to a corresponding signal, the more reliable is the associated statement about the instantaneous glucose supply or undersupply. Thus, it is also useful to combine the particularly informative and thus preferred embodiments of characterizing probes with apparently less meaningful ones in order to avoid false positives. It is also advantageous, according to the information in Table 2, to combine such probes which give signals of different strength at different times indicated there. Thus, it is possible to arrive at an estimate as to how long before the sampling of the glucose deficiency and whether it may be due to a particular environmental impact, under protocol of cultivation conditions

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), are counted only once within the meaning of this aspect of the invention, because they are equally responsive to the same signal in the best case and in principle were interchangeable In preferred embodiments of inventive nucleic acid-binding chips, the total number of all different probes is increasingly preferably not more than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10

This corresponds to the inventive idea outlined above, according to which a specific metabolic aspect is to be monitoπert with the chips described here, so that a larger number of probes than to detect this situation need not necessarily be applied to the respective chips of the situation remains unaffected that in individual cases, it may be useful to use more than one probe for the detection of 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 the said frame to chips for technical reasons only a few Be included in the scope of protection Bmdungsstellen

In preferred embodiments of nucleic acid-binding chips according to the invention, the probes referred to as being relevant to the invention are those which respond to the relevant or most highly homologous, in vivo transcribable genes from the organism selected for the bioprocess, preferably those derived from the respective ones highly homologous, in vivo transcribable genes are derived just this organism

For this purpose, it has already been stated above that the sequences of B licheniformis disclosed in the present application were obtained and, owing to the generally known relationships, in particular should be suitable 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 which have been defined only via their sequences. According to the invention, the genes closely related in the organism under consideration are used for deriving corresponding probes However, to ensure 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 cytoplasm Nuklemsaure 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 necessity to rely on the sequences given in this hybridization does not increase - if there are sequence differences - to use for the homologous genes from the species in question As explained, these can be known per se methods, in particular Genbank screening or PCR with primers oriented on the sequences disclosed herein (optionally in the form of so-called mismatch polymers 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

This is because the most commercially used organisms fall into these groups, in particular if the bioprocess to be observed is a fermentation. For example, cooking processes, for example for the production of wine or beer, or the biotechnological production of valuable substances such as proteins or low-molecular ones links

Depending on the nature of the desired product, various organisms are selected for a biotechnological process. For the purposes of the invention, these are to be understood as meaning not only the production strains but also all organisms preceding the production process, for example for the gene expression or the selection of suitable expression vectors. The need for a glucose limitation In this case, there is a partial existence during each subprocess

In preferred embodiments of nucleic acid chips according to the invention, the unicellular eukaryotes are protozoa or fungi, in particular yeast, more particularly sucrose or schizosaccharomyces

Because these are in addition to the production of alcoholic beverages and other foods obtained by cooking intensively used as host cells in particular for the gene products of eukaryotes. The latter is particularly advantageous if these gene products are to undergo special durchfuhrbare only by these strains modifications, such as Glykosyherungen of proteins

This object also includes chips according to the invention, which are directed to the monitoring of the course, in particular the growth of cell cultures of higher eukaryotes, such as rodents or humans They can be understood in some respects as at least largely single-celled eukaryotes, in particular in the Immunology have significant commercial importance, for example for the production of monoclonal antibodies

In preferred embodiments of inventive nucleic acid-binding chips, 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 S. 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 orientation on ß. licheniformis is explained by the fact that the sequences given in the sequence listing were obtained from this species and could be demonstrably linked to the transition into a glucose deficiency as described in Examples 2 and 3.

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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149 , 151, 153, 155, 157, 159, 161, 163, 165, 167 and 169.

Because these genes could, as described in Examples 2 and 3, in S. licheniformis be shown to be associated with the transition to a lack of glucose. In particular, if ß. licheniformis or other, especially related ßac / 7 / us species should 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 process-related, additionally expressed gene (s) , especially for one of them or this one

As noted above, the processes under observation serve a technical interest, often associated with other specific genes. For example, in the case where a protein is to be produced, the gene for that protein and, in the case of a low molecular weight compound It is also possible to affect other cell-specific genes, such as metabolic genes, which must be increasingly formed during the production of the product, for example a cell-derived oxidoreductase, if this is to be produced Product should be obtained from a reactant 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 This includes the transformation with the responsible for the actual product manufacturing genes oversight with selection markers or further adaptations of the metabolism, up to auxotrophies Such strains have a special requirement profile on the growth conditions and have z metabolic genes which are mutated compared to the wild-type genes Since chips according to the invention should advantageously be aimed at precisely these strains, especially the bioprocess considered , these strain-specific peculiarities should be taken into account and may be reflected in the choice of probes concerned

In preferred embodiments of such nucleic acid-binding chips according to the invention, the process-related additionally expressed gene 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 Soil contaminated with amylases, cellulases, lipases, hemicellulases and / or proteases. lysierbar, for the treatment of the materials in question, in particular by cellulases or to provide an oxidoreductase-based enzymatic bleaching system

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

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

This embodiment has the aim of improving the hybridization between the probe and the sample to be detected. This is especially true in the case where the content of the relevant mRNA is actually determined from the sample because it is single-stranded and in its sequence with the coding strand the DNA matches, should an optimal hybridization with the complementary, that is the codogenic strand done

In preferred embodiments of nucleic acids according to the invention of the chips, one, preferably several, of the probes which are relevant for the determination of the invention in the form of a DNA or of a nucleic acid analog, preferably of a nucleic acid analog, is / are made available

This requirement is aimed at improving the durability and multiple usability of the chips according to the invention. This need arises in particular during a single observed process, in the course of which continuous monitoring is desirable. The durability of chips according to the invention, in particular with respect to nucleic acid-hydrolyzing enzymes, is already demonstrated by the Providing the probes in the form of a DNA increased, since this is less susceptible to hydrolysis than about an RNA even more durable are Nuklemsaureanaloga, in which, for example, the phosphate of Zucker-phosphate backbone is replaced by a chemically different building block, which is not hydrolyzed by natural nucleases Such compounds are known in principle in the art and are commercially synthesized for desired, respectively to be given sequences of companies specialized in this on request The relevant probes are about to synthesize following 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 preferably comprise a plurality of the probes which are relevant for the invention 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

Hereby, the aspect is taken into account 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 considered here the first On the other hand, it should be noted that in particular eukaryotes introns occur, that is, the coding region is interrupted by sections that are not translated into mRNA Probes containing introns, therefore, were not allowed or In order 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, for the detection of an mRNA often no hybridization over the entire Seqzuenz- long required The specific probes ι d R need only to include a smaller of the transcribed into mRNA gene Advantageous for this is a selection of an area near the 5 'end the mRNA is located, as it is first transcribed into mRNA and thus is the first to be detected after activation of the gene. This precludes timely detection

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 relevant 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 the hybridization between the probes and the mRNA to be detected because mRNA molecules often have a secondary structure, which is based on hybridization of individual mRNA regions with their own other regions. or Stem Ioop Structures Such regions, however, tend to hybridize less readily to other nucleanic acid molecules, even if homologous. Such regions can be calculated quite accurately from computer programs directed thereto (see below). Thus, to realize this aspect, one should consider the gene whose Activity for an organism of interest, have it analyzed by such a program, and resort to sections for which a low level of mRNA secondary structures is predicted, to obtain a suitable - usually only a subset (see below) probe In preferred embodiments of nucleic acid-binding chips according to the invention, one, preferably several, of the probes called relevant to the invention have 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 to include only 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 experimentally if necessary in preliminary experiments be determined

The identification of suitable probes is known to those skilled in the art and is usually with the help of specialized software performed Examples of such software are the array program designer of the FA Premier Biosoft International, USA, and Vector NTI ^® Suite, V 7, available from InforMax , Ine, Bethesda, USA In addition to the secondary structures already mentioned, these software programs also take into account specified probe lengths and melting temperatures, for example

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 relevant probe called relevant to the invention

In the already mentioned article J Wang (Acc Chem Res, ISSN 0001-4842, Rec Sept 12, 2001, S AF) the advantages of an electrically evaluable system over an optical system are discussed. Further, various embodiments of such sensors developed in the prior art are discussed directed

Thus, at the present time, the time span from sampling to measuring the signal for optically analyzable chips is approximately 24 hours. Using an electrical system, the time required is currently less than 2 hours (see FIG. 1). In contrast, the number of simultaneously analyzable samples is electrical evaluable chips currently in the double-digit range, but a rapid development suggests that this magnitude can be exceeded in Briefly limiting the electronic evaluation units for the various signals

A method for mRNA quantification established in the prior art represents, for example, the RT-PCT. This is described in the article "Quantification of Bactenal mRNA by One-Step RT-PCR Using the LightCycler System" (2003) by S Tobisch, T Koburger, B Juergen, S Leja, M Hecker and T Schweder in BIOCHEMICA, Volume 3, pages 5 to 8 described in contrast possesses the Detection via electric chips another advantage, namely the higher reliability of the data, as they have compared to the RT-PCR significantly lower fluctuation ranges

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, the disclosure content of which 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 bound to magnetic carriers (beads) in known manner, which are located in chambers of the chips provided for this purpose. The specific hybridization of the corresponding mRNA to the respective mRNA The beads are held in this chamber by a magnet. After the hybridization of the RNA samples to the bead-bound DNA probes, a washing step is carried out to eliminate the non-beaded DNA probes. bound RNA, so that in the incubation only specific hybrids are still present, 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 with the alkaline phosphatase substrate para-aminophenol phosphate (pAPP) incubated 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 (eg, 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 can be varied both with regard to 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 measurement principle

One benefit of the present invention is that it has identified and made available to analysis via appropriately designed biochips glucose-specific and thus process-critical genes. The advantage of chips over conventional detection methods is, besides time saving and higher accuracy, that with the provision of multiple probes on a carrier at the same time in the same sample the activities of several different genes can be detected and can give a more solid and detailed picture in the application described here to a particular problem, for example, at the time when glucose deficiency has occurred

A separate subject of the invention is thus the simultaneous use of nucleic acid or nucleic acid analogue probes for at least six of the following genes yxe /, dppE, gene for a tight homologue to Ald [L-alanine dehydrogenase] (homologue to SEQ ID NO 157 ), fruK, ybdN, gene for a putative transcriptional regulator (homologue to SEQ ID NO 39), gene for a putative methylmalonate semialdehyde dehydrogenase [acyherend] (EC 1 2 1 27, homologue to SEQ ID NO 159), yvdE, rbsK , hxIA, gene for a hypothetical protein (homologue to SEQ ID NO 103), yvdH, ywfL, ispA, yvyD, gene for a glucan-1,4-alpha-maltohydrolase (EC 3 2,133, homologue to SEQ ID NO 151 ), licA, rocD, ycgO, yqiQ, sigX, IOIC, licH, IOIE, putative gene for a putative ABC transporter ATP restriction protein (homologue to SEQ ID NO 125), rsiX, gene for a hypothetical protein (homologue to SEQ ID NO 123), mmgE, gene for a putative pectin methyl esterase (homolog to SEQ ID NO 63), gene for a putative formate-De hydrogenase-alpha chain (EC 1 2 1 2, homologue to SEQ ID NO 9), IOID, gene for a putative hydroxybenzoate hydroxylase (homologue to SEQ ID NO 49), yvql, glpF, gene for a putative enoyl (3 Hydroxyιsobutyryl) coenzyme A hydratase Proteιn (EC 4 2 1 17, homologue to SEQ ID NO 93), ysbA, yvqH, bpr, acdA, yvoA, gene for a putative secretion protein (homologue to SEQ ID NO 35), aprE , yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 13), mmgD, tdh, mmgC, gene for putative isocitrate lyase (EC 4 1 3 1, homologue to SEQ ID NO 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4 2 17, homologue to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, homolog to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11) bound to a nucleic acid-binding chip, preferably to the same, for determining the physiological state of a biological process continuous organism

As discussed above, these genes are selected to provide an idea of the glucose metabolism situation of the subject organism because they are significantly induced in the transition from the gram-positive bacterium B hcheniformis to the glucose deficient state as described in Examples 1-3 comparable statement is also for to expect other organisms that have the homologous genes or proteins with substantially the same substance metabolism relevant properties

As already described in detail, such gene activities can be determined in principle in various ways, for example by Northern Hybπdisierung However, the analysis using a nucleic acid-binding chip, in particular one described above, but opens the possibility to determine in a very efficient manner simultaneously several gene activities and In addition, this in a very timely manner Thus, the metabolic changes of an organism that undergoes a biological process, observed promptly and possibly intervene regulatory

The above statements on nucleic acid-binding chips apply accordingly to the uses of the probes referred to herein

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

Accordingly, it is further preferred that such uses be used, the probes previously indicated as being according to the invention being used with increasing preference in the sequence previously indicated as preferred (inversely to the order in Table 3)

According to the above statements, the following uses of nucleic acid or nucleic acid analogue probes are increasingly preferred - use, wherein at least one, more preferably two or three of the nucleic acid or nucleic acid analog probes for genes from the following 67 genes specifically, ycgO, yqiQ, sigX, IOIC, licti, lolE, putative gene for a putative ABC transporter ATP binding protein (homologue to SEQ ID NO 125), rsiX, gene for a hypothetical protein (homolog to SEQ ID NO 123), mmgE, gene for a putative pectin methylesterase (homolog to SEQ ID NO 63), gene for a putative formate dehydrogenase-alpha chain (EC 1 2 1 2, homolog to SEQ ID NO 9), IOID, Gene for a putative hydroxybenzoate hydroxylase (homolog to SEQ ID NO 49), yvql, glpF, gene for a putative enoyl (3-hydroxyιsobutyryl) coenzyme A hydratase protein (EC 4 2 17, homologue to SEQ ID NO 93), ysbA, yvqH, bpr, acdA, yvoA, gene for a putative Seπ nprotease (homologue to SEQ ID NO 35), aprE, yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 13), mmgD, tdh, mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1, homolog to SEQ ID NO 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4 2 17, homologue to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, Gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1.1.1.60; homologue to SEQ ID NO: 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homologue to SEQ ID NO: 101), gene for a putative malate synthase (EC 4.1.3.2, homolog to SEQ ID NO: 107), acoB, gene for a putative Transketolase (EC 2.2.1.1, homologue to SEQ ID NO: 11);

of these, preferably for genes from the following 34 genes: tdh, mmgC, gene for a putative isocitrate lyase (EC 4.1.3.1, homologue to SEQ ID NO. 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO ), malP, mmgA, gene for a putative membrane protein (homologue to SEQ ID NO: 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4.2.1.17, homologue to SEQ ID NO: 95), glpD , etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1.1.1.60, homologue to SEQ ID NO: 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN , yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO.101), gene for a putative malate synthase (EC 4.1.3.2, homologue to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO 11);

- Of these, particularly preferred for genes from the following 10 genes: kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO. 101), gene for a putative malate synthase ( EC 4.1.3.2, homologue to SEQ ID NO: 107), acoB, gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO: 11);

Of these, very particular preference is given to genes from the following 4 genes: gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO.101), gene for a putative malate synthase (EC 4.1.3.2, homologue to SEQ ID NO. 107), acoB, gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO 11).

In accordance with the above, they are preferably uses according to the invention of simultaneously at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80 or 85 of said probes.

In accordance with what has been stated above, it is preferable to use according to the invention, wherein the total number of all different probes is increasingly preferably not more than 95, 90, 80, 70, 60, 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 glucose metabolism of the organism passing through the biological process, preferably for the detection of a glucose deficient state.

In accordance with what has been said above, it is furthermore preferable to use according to the invention, wherein at least one, increasingly preferably more, of the named probes are derived from the sequences listed in the sequence listing under the numbers SEQ ID Nos. 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 11, 113, 115, 117, 1, 19, 121, 123, 125, 127, 129, 131, 133 , 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167 and 169

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 methods look 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 under Considering the above-mentioned Verfahrensschπtte - such as sufficient incubation or washing off unspecifically binding nucleic acids - treated and finally fed to the detection device Such a process protocol is illustrated in principle with reference to the example of electrically evaluable chips in Figure 1

The statements made above regarding nucleic acid-binding chips apply correspondingly to the method of determining the physiological state of an organism passing through a biological process

Preferably, they are methods according to the invention wherein a change in the glucose metabolism of the organism passing through the biological process is determined, preferably a glucose deficient state

Because with respect to this field of application, the genes described in the examples and listed above have been selected. Their significant induction is accompanied by the occurrence of a glucose deficient state, at least in the case of B. licheniformis, so that they can be detected particularly reliably with the abovementioned methods, and not only in S. licheniformis but with increasingly better chances of success also in increasingly related species (see above) Moreover, this metabolic situation in the life cycle of many microorganisms is a critical time Thus, in the examples studied (see below) with the respective onset of glucose deficiency always a transition to the stationary Growth phases linked Processes that help to identify this point in time serve to delay this transition and, especially in the case of large-scale fermentations, extend the phase of producing a valuable substance Another possibility of the procedure in the cultivation of microorganisms is to provide next to the first (and thus better) used C source another. In the phase of the glucose deficiency observed first, the cells in question, if they are able to do so, adapt to the use of the second, for them less usable C-source. With this transition, a delay in growth is usually observed, which can be recognized early on a method according to the invention.

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, the unicellular eukaryotes being protozoa or fungi, in particular yeast, especially Sacharomyces or Schizosaccharomyces.

According to the above, those 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 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 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 the strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-I or Klebsiella planticola (Rf).

According to 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, 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167 or 169 are derived. Of these, in turn, those methods are preferred in which the probes mentioned here are used for the Gram-positive bacteria listed above, in particular licheniformis, since these sequences have been isolated from this very organism and thus can be applied most successfully to 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 points in time of the same process, preferably using a plurality of identically constructed nucleic acid-binding chips, particularly preferably the same nucleic acid-binding chip

In accordance with 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 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

In accordance with 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 the determination of 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 glucose metabolism of the organism passing through the biological process is determined, preferably a glucose 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, more particularly Sacharomyces or Schizosaccharomyces

According to the above, alternatively such uses according to the invention are 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 B 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 the strain 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 uses according to the invention are preferred, using those of said probes which are of the SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, listed in the Sequence Listing, 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167 or 169

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 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 fragments of the chips, more preferably of 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 such as those described in the handbook of Fntsch, Sambrook and Maniatis "Molecular cloning a laboratory manual", CoId Spring Harbor Laboratory Press, New York, 1989, or comparable works of enzymes and kits 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 likemformis DSM13 (available from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1 b, 38124 Braunschweig, Germany, http://www.dsmz.de) were incubated in glucose-fed, synthetic Belitsky minimal medium (0.28 mM final concentration). cultured at 37 ° C. under constant debris flow at 270 rpm. This medium has the following composition: (1) base medium (pH 7.5) 0.015M (NH ₄ J ₂ SO ₄ , 0.008M MgSO ₄ .7H ₂ O, 0.027M KCl, 0.007 M Na ₃ citrate × 2H ₂ O, 0.050 M Tns-HCl and 0.009 M glutamic acid, (2) supplements 0.2 M KH ₂ PO ₄ , 0.039 M L-tryptophan 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 basic 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 so that from an OD _50O of 0.8 to 1, 0 glucose deficiency occurs and thus the stationary phase is initiated Further sampling took place after 10, 20, 30, 60 and 120 min Samples immediately after collection were spiked with the same volume of ^{0 0} C cooled Kilhng buffer (20 mM NaN ₃ , 20 mM Tns-HCl, pH 7.5, 5 mM MgCl ₂ ). Immediately screen the cell pellet for 5 min at 4 ° C and 15 000 rpm, the supernatant was discarded and the pellet either frozen at -80 ° C or immediately worked up further

cell disruption

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

Total RNA isolation

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

RNA quality and quantity control

The purification of the RNA is followed by the quantitative and quantitative control. The Agilent Bioanalyzer 2100 apparatus (Agilent Technologies, Berlin, Germany) used for this purpose enables the analysis of RNA on the Lab-on-a-Chιp scale together with the "RNA 6000 Nano Kit "from Agilent, the total RNA is separated by gel electrophoresis and thus offers the possibility of examining the quality with regard to partial degradation and contamination. Here, nbosomal RNA (16 S and 23 S rRNA) is detected. If these are clear bands, It can be assumed 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 also determined

transcriptome

The transcriptome analyzes were carried out using genomic slicheniformis DSM13 DNA microarrays which had been prepared in a conventional manner (for example in accordance with WO 95/11995 A1) and can be evaluated via an optical system. Virtually every gene from B licheniformis was present on these DNA microarrays in duplicate, 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 reverse transcription into DNA, wherein one of the added Desoxyπbonukleotide carries a color marker. These labeled molecules are then with the known, in known places of the Hybridized probes and the respective strength of the at the relevant sites attributable to the fluorescent marker signal detected using two different fluorescent markers for a control and for a sample actually under investigation, which are brought simultaneously to hybridization and thus the binding to the submitted Probe compete, one arrives at different color values, which provide a measure of the ratio of the concentration of the control to the concentration of the sample

DUTP was chosen for this labeling, which was labeled with the fluorescent dye cyanine 3 or with cyanine 5. Thus, in each case 25 μg total RNA of the control (OD _50O 0.4) with the fluorescent dye cyanine 3 (Fa Amersham Biosciences Europe GmbH, Freiburg, Germany) and 25 .mu.g total RNA of the respective stress test (transient phase, 10, 20, 30, 60 and 120 min) with the fluorescent dye cyanine 5 (GE Healthcare, Freiburg, Germany) marked the competitive hybridization of the two samples on the conventional Total genome B // cs // brm / s DSM13 DNA microarray was carried out for at least 16 hours at 42 ° C

After washing the arrays to remove nonspecific binding, the optical readout of the arrays was performed using the ScanArray® Express Laser Scanner (PerkmElmer Life and Analytical Sciences, Rodgau-Jugesheim, Germany). All hybridizations were repeated using the samples with the other dye The quantitative evaluation of the arrays was carried out using the ScanArray® Express software (available from the company PerkmElmer Life and Analytical Sciences Rodgau-Jugesheim, Germany) according to the manufacturer's instructions 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, were according to the manufacturer's information known control DNA and so-called spikes in the form of oligos were applied to the array and hybridized with complementary sequences in the hybridization Thus, one could control the success of hybridization and incorporation of the dyes after scanning The controls should be present in both samples in the same amount and thus after appear yellow or have a ratio between both channels of 1, respectively. The spikes are specific to the respective sample and are applied in different dilutions, ie they appear red or green after scanning for the respective 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

Glucose-deficient genes

Table 1 below lists all the 268 genes of Bacillus hcheniformis DSM 13 determined in Example 1 whose induction (amounting to at least the factor 3) was observed under the conditions of the glucose deficiency described in Example 1. The first two columns show the respective name of the derived one The BLi number corresponds to the "locus_tag" of the 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.ncbiologie.org, Booth 2, 12, 2004), followed by the factors observed at the times indicated above to increase the concentration of the respective mRNAs associated therewith

Table 1 The 268 genes of Bacillus hcheniformis DSM13 determined in Example 1, their

(at least a factor of 3) induction was observed under glucose deficiency (for explanations see text)

Example 3

Genes specifically induced by glucose deficiency

As Table 1 shows, under the conditions of the glucose deficiency described in Example 1 at any of the measured times, a total of 92 of the Bacillus licheniformis DSM13 genes examined in Example 1 were induced by at least a factor of 10. However, the following seven genes were also identified by the Lack of at least one other compound (data not shown) and therefore can not be considered as specific signals for a glucose deficiency homolog to dhaS (BU03994), dhaS (BU02249), as S (BU03848), gene for a putative benzoate transport protein (BLi 03990), gene for a putative aromatic-specific dioxygenase (BLi 03991), gene for a putative decarboxylase / dehydratase (BLi 03993), gene for a hypothetical protein (BLi 00235)

Thus, there remained 85 genes that were induced at any of the measured times by a factor of at least 10 and that can be classified as relatively specific based on the studies of Example 1. They are summarized in Table 2 below. 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. Particulars of the respective sequences appearing as free text in the sequence listing are supplemented under the heading gene name / gene function

TABLE 2 The Bacillus licheniformis DSM13 genes determined in Example 1, whose specific induction caused by glucose deficiency at least amounted to at least one factor 10 at any of the measured times (for explanations see text)

In Table 3 below, the same 85 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 85 genes of Bacillus licheniformis DSM 13 determined in Example 1 whose specifically induced by glucose deficiency induction at any of the measured times was at least a factor of 10; in descending order of the measured maximum value (last column).

, 22 dipeptide ABC transporter (dipeptide-binding protein) dppE BLiOI 396 10.09 (sporulation)

9, 110 unknown; similar to penicillin amidase yxel BU04217 10,04

Example 4

Real-time RT-PCR quantification of selected genes

Real-Tιme-RT-PCR makes it 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 (Fa This method is based on the detection of the binding of the fluorescent dye to double-stranded DNA. The quantification of the specific mRNA molecules was carried out as described in Tobisch et al (2003, Quantification of Bacte- matic mRNA by One-Step RT-PCR Using the LightCycler System, US Pat. BIOCHEMICA, Volume 3, pages 5 to 8), with an external standard curve being generated for each mRNA to be investigated

For this purpose, dilutions of known concentrations of / n-wfro transcripts of the mRNA determined in Example 1 and Example 2 were measured with the aid of the LightCycler and then using the device-specific software created the standard curve For this purpose, specific Pπmer had to be selected in advance for each mRNA to be examined (using the software "Array Designer", available from PREMIER Biosoft International, Paolo Alto, USA), which enable a synthesis of in vitro transcπten and PCR amplification in LightCycler

After preparation of the external standard curve, it was possible to start the measurement in the LightCycler. Two different dilutions were used for the measurements for each sample to be analyzed. In the LightCycler run, the specific transcript was amphfied with the respective primers and the incorporation of the dye was measured

Using the LightCycler software as well as the script available on the website http // molbiol ru / ger / scrιpts / 01_07 html (2 12 2004), it was possible to determine the exact molecular number for selected transcripts with the values obtained in this way and their relations to one another For the selected transcripts, the induction values given in Tables 1 and 2 were confirmed

Description of the figures

Figure 1: Schematic representation of / W - // ^' ne monitoring 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

Nuclear acid-binding chip doped with probes for at least six of the following genes: yxel, dppE, gene for a close homolog to AId [L-alanine dehydrogenase] (homolog to SEQ ID NO 157), fruK, ybdN, gene for a putative Transcriptional regulator (homologue to SEQ ID NO 39), gene for a putative methylmalonate semialdehyde dehydrogenase [acylating] (EC 1 2 1 27, homologue to SEQ ID NO 159), yvdE, rbsK, hxIA, gene for a hypothetical protein (homolog to SEQ ID NO 103), yvdH, ywfL, ispA, yvyD, gene for a glucan-1,4-alpha-maltohydrolase (EC 3 2,133, homologue to SEQ ID NO 151), licA, rocD, ycgO, yqiQ, sigX, IOIC, licH, IOIE, putative gene for a putative ABC transporter ATP binding protein (homolog to SEQ ID NO 125), rsiX, gene for a hypothetical protein (homolog to SEQ ID NO 123), mmgE, gene for a putative pectin methyl esterase (homologue to SEQ ID NO 63), gene for a putative formate dehydrogenase alpha chain (EC 1 2 1 2, homologue to SEQ ID NO 9), IOID, gene for a e putative hydroxybenzoate hydroxylase (homologue to SEQ ID NO 49), yvql, glpF, gene for a putative enoyl (3-hydroxyιsobutyryl) coenzyme A hydratase protein (EC 4 2 17, homologue to SEQ ID NO 93) , ysbA, yvqH, bpr, acdA, yvoA, gene for a putative secretion protein (homologue to SEQ ID NO 35), aprE, yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 13), mmgD, tdh _% mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1, homologue to SEQ ID NO 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4 2 17, homologue to SEQ ID NO 95), glpD , etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, homolog to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN , yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative but yryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homolog to SEQ ID NO 11), wherein the total number of all different specific for glucose deficiency different probes is not more than 100

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

A nucleic acid binding chip according to claim 1 or 2, wherein at least one, more preferably two or three, of the probes are selected from the following 67 genes: ycgO, yqiQ, sigX, ioIC, licH, ioIE, putative gene for a putative ABC transporter -ATP- Binding protein (homolog to SEQ ID NO 125), rsiX, gene for a hypothetical protein (homologue to SEQ ID NO 123), mmgE, gene for a putative pectm methylesterase (homolog to SEQ ID NO 63), gene for a putative formate Dehydrogenase-alpha chain (EC 1 2 1 2, homologue to SEQ ID NO 9), IOID, gene for a putative hydroxybenzoate hydroxylase (homolog to SEQ ID NO 49), yvql, glpF, gene for a putative enoyl (3 Hydroxyιsobutyryl) - coenzyme A hydratase Proteιn (EC 4 2 1 17, homologue to SEQ ID NO 93), ysbA, yvqH, bpr, acdA, yvoA, gene for a putative Sennprotease (homologue to SEQ ID NO 35), aprE , yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 13), mmgD, tdh, mmgC, gene for putative isocitrate lyase (EC 4 1 3 1, homologue to SEQ ID NO. 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane proton (homolog to SEQ ID NO 15), yusJ, malA, gene for a putati enoyl-CoA hydratase (EC 4 2 1 17, homologue to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, homologue to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homologue to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11), preferably from the following 34 genes tdh, mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1, homologue to SEQ ID NO. 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4 2 17, homologue to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, homologue to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homologue to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11), particularly preferred from the following 10 genes kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107), acoS, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11), very particularly preferably from the following 4 genes gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homologue to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11) Nucleic acid-binding chip according to one of claims 1 to 3, doped with at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 of said probes

Nucleic acid-binding chip according to one of claims 1 to 4, wherein the total number of all different probes is increasingly preferably not more than 95, 90, 80, 70, 60, 50, 40, 30, 20 or 10

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

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

Nucleic acid-binding chip according to claim 7, wherein the unicellular eukaryotes are protozoa or fungi, including in particular yeast, especially Sacharomyces 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 the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtihs, B lichemformis, B amyloliquefaciens, B agaradherens, B stearothermophilus, B globign, or lentus, and especially slichemformis

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 more particularly derivatives of the strain Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf)

Nucleic acid-binding chip according to one of claims 1 to 10, wherein at least one, more preferably more of said probes are / are derived from the sequences, 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167 and 169 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 more of said probes single-stranded, are provided in the form of the codogenic strand.

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 six of the following 85 genes: yxel, dppE, gene for a tight homolog to AId [L-alanine dehydrogenase] (homologue to SEQ ID NO: 157), fruK , ybdN, gene for a putative transcriptional regulator (homologue to SEQ ID NO: 39), gene for a putative methylmalonate semialdehyde dehydrogenase [acylating] (EC 1.2.1.27, homologue to SEQ ID NO: 159), yvdE, rbsK, hxIA , Gene for a hypothetical protein (homolog to SEQ ID NO: 103), yvdH, ywfL, ispA, yvyD, gene for a glucan-1,4-alpha-maltohydrolase (EC 3.2.1.133, homologue to SEQ ID NO.115) , HcA, rocD, ycgO, yqiQ, sigX, iolC, HcH, iolE, putative gene for a putative ABC transporter ATP binding protein (homolog to SEQ ID NO: 125), rsiX, gene for a hypothetical protein (homologue to SEQ ID No. 123), mmgE, gene for a putative pectin methyl esterase (homologue to SEQ ID NO 63), gene for a putative formate dehydrogenase alpha chain (EC 1.2.1.2, H omolog to SEQ ID NO. 9), ioID, gene for a putative hydroxybenzoate hydroxylase (homolog to SEQ ID NO 49), yvqI, glpF, gene for a putative enoyl (3-hydroxyisobutyryl) coenzyme A hydratase protein (EC 4.2.1.17; Homologue to SEQ ID NO: 93), ysbA, yvqH, bpr, acdA, yvoA, gene for a putative serine protease (homologue to SEQ ID NO: 35), aprE, yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, Gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO 13), mmgD, tdh, mmgC, gene for a putative isocitrate lyase (EC 4.1.3.1, homologue to SEQ ID NO: 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO: 17), malP, mmgA, gene for a putative membrane protein (homologue to SEQ ID NO: 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4.2.1.17 Homologue to SEQ ID NO: 95), glpD, etfB, etfA, ywjF, yvdG, yυsL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1.1.1.60, homologue to SEQ ID NO: 99), yvdl, acoc, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, Gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO. 101), gene for a putative malate synthase (EC 4.1.3.2, homologue to SEQ ID NO: 107), acoB, gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO: 11), bound to a Nucleic acid-binding chip, preferably to the same, for determining the physiological state of an organism passing through a biological process. Use according to claim 20, wherein the total number of all glucose deficiency-specific different probes is not more than 100

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

Use according to any one of claims 20 to 22, wherein at least one, more preferably two or three, of the nucleic acid or nucleic acid analogue probes are specific for genes from the following 67 genes: ycgO, yqiQ, sigX, IOIC, licH, IOIE, putative gene for a putative ABC transporter ATP binding protein (homolog to SEQ ID NO 125), rsiX, gene for a hypothetical protein (homolog to SEQ ID NO 123), mmgE, gene for a putative pectin methylesterase (homolog to SEQ ID NO 63), gene for a putative formate dehydrogenase-alpha chain (EC 1 2 1 2, homologue to SEQ ID NO 9), IOID, gene for a putative hydroxybenzoate hydroxylase (homologue to SEQ ID NO 49), yvql, glpF, gene for a putative enoyl (3-hydroxyιsobutyryl) coenzyme A hydratase protein (EC 4 2 17, homologue to SEQ ID NO 93), ysbA, yvqH, bpr, acdA, yvoA, gene for a putative secretion protein (homologue to SEQ ID NO 35), aprE, yusK, acuA, acsA, acoR, ysiA, pgcM, mmgB, gene for a putative transketolase (EC 2 2 1 1 , Homologue to SEQ ID NO 13), mmgD, tdh, mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1, homolog to SEQ ID NO 105), gene for a putative transcriptional regulator (homolog to SEQ ID NO 17) , malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4 2 17, homologue to SEQ ID NO 95), glpD, etfB , etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, homolog to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB , kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homolog to SEQ ID NO 107 ), acoB, gene for a putative transketolase (EC 2 2 1 1, homologue to SEQ ID NO 11), preferably for genes from the following 34 genes tdh, mmgC, gene for a putative isocitrate lyase (EC 4 1 3 1, Homolog to SEQ ID NO. 105), gene for a putative transcript tion regulator (homolog to SEQ ID NO 17), malP, mmgA, gene for a putative membrane protein (homolog to SEQ ID NO 15), yusJ, malA, gene for a putative enoyl-CoA hydratase (EC 4 2 1 17, homolog to SEQ ID NO 95), glpD, etfB, etfA, ywjF, yvdG, yusL, gene for a putative 2-hydroxy-3-oxopropionate reductase (EC 1 1 1 60, homologue to SEQ ID NO 99), yvdl, acoC, acoL, ycgM, mmsA, ycgN, yoeB, kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO 101), gene for a putative malate synthase (EC 4 1 3 2, homologue to SEQ ID NO 107), acoB, gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO: 11); particularly preferred for genes from the following 10 genes: kbl, yvdJ, yvdK, yfiA, malL, acoA, gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO. 101), gene for a putative malate synthase (EC 4.1 Homologue to SEQ ID NO: 107), acoB, gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO: 11); very particularly preferred for genes from the following 4 genes: gene for a putative butyryl-CoA dehydrogenase (homolog to SEQ ID NO.101), gene for a putative malate synthase (EC 4.1.3.2, homolog to SEQ ID NO.107) , acoB, gene for a putative transketolase (EC 2.2.1.1, homologue to SEQ ID NO: 11).

24. Use according to one of claims 20 to 23 of simultaneously at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85 of said probes.

25. Use according to any one of claims 20 to 24, wherein the total number of all different probes is increasingly preferably not more than 95, 90, 80, 70, 60, 50, 40, 30, 20 or 10.

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

27. Use according to any one of claims 20 to 26, wherein at least one, increasingly preferably several of said probes 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167 and 169 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. A method according to claim 28, wherein a change in glucose metabolism of the organism passing through the biological process is determined, preferably a glucose deficient state

The method of claim 28 or 29, wherein the organism selected for the bioprocess is a member of a unicellular eukaryote, Gram-positive or Gram-negative bacteria

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

The method of claim 31, 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 globigu or B lentus, and especially B licheniformis

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

A method according to claim 33 or preferably 32, wherein those of said probes are used which are of the SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, listed in the sequence listing, 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167 or 169 are derived

Method according to one of claims 28 to 34, 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, particularly preferably the same nucleic acid-binding chip A 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 the production of a protein or a low-molecular chemical compound

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

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

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

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

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

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

Use according to claim 41, 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 subtihs, B licheniformis, B amylohquefaciens, B agaradherens, B stearothermophilus, B globigu or lentus, and especially S 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 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).

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, 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, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167 or 169 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.