Abstract:
The present invention provides arrays of single- or doublestranded desoxyribonucleic acid (DNA) probes immobilized on solid supports and for using those probe arrays to detect specific nucleic acid sequences contained in a target nucleic acid in a sample, especially a method to monitore a fermentation process.

Description:
[0001]    The present invention provides arrays of single- or doublestranded desoxyribonucleic acid (DNA) probes immobilized on solid supports and for using those probe arrays to detect specific nucleic acid sequences contained in a target nucleic acid in a sample, especially a method to monitore a fermentation process.  
         FIELD OF THE INVENTION  
         [0002]    DNA probes have long been used to detect complementary nucleic acid sequences in a nucleic acid of interest (the “target” nucleic acid).  
           [0003]    In general the DNA probe is tethered, i.e. by covalent attachment, to a solid support, and arrays of DNA probes immobilized on solid supports have been used to detect specific nucleic acid sequences in a target nucleic acid (see, e.g., PCT WO 89/10977 or 89/11548). Methods for making high density arrays of DNA probes on silica chips and for using these probe arrays are provided in U.S. Pat. No. 5,837,832 and EP Patent No. 0 373 203 or EP Patent No. 0 386 229.  
           [0004]    The so called “DNA-chips” offer great promise for a wide variety of applications. New methods and applications are required to realize this promise, and the present invention helps meet that need.  
         SUMMARY OF THE INVENTION  
         [0005]    The genome-wide transcriptional monitoring of organisms by the DNA-chip technology opens a new level of complexity in the functional analysis of living organisms. We have used DNA-chips for the analysis of gene expression patterns in the compound producing microorganism  Corynebacterium glutamicum  and  Escherichia coli . Based on the available sequence information DNA-fragments of the bacterium are immobilized on a solid support. Transcription profiles of the organisms are analyzed under various fermentational conditions by DNA-Microarray experiments. The obtained data are verified by Northern-Blot analysis, real time RT-PCR or two-dimensional gel electrophoresis.  
           [0006]    Different to the classical applications of the DNA-Microarray technology, e.g. in the biomedical research, the invention provides DNA-chips to be used for the monitoring of process related target genes in the production of fermentative available compounds.  
           [0007]    The invention provides an analysis system for the detection of microbial gene expression patterns in large-scale industrial fermentations. The information obtained from these patterns can be used for controlling of the fermentation process.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0008]    We claim an array of DNA probes immobilized on a solid support, said array having at least 10 probes and no more than 200.000 different DNA probes 15 to 4.000 nucleotides in length occupying separate known sites in said array, said DNA probes comprising at least one probe that is exactly complementary to selected reference sequences of a compound producing microorganism.  
           [0009]    In a preferred embodiment of the invention, said DNA probes are nucleic acids covering a genomic region of a compound producing microorganism, e.g. obtained from a genomic shotgun library.  
           [0010]    In another preferred embodiment of the invention, said DNA probes are nucleic acids, e.g. obtained from a polymerase chain reaction, covering an whole genetic element, an internal fragment of a genetic element or the genetic element and additionally flanking regions of it.  
           [0011]    In a preferred embodiment of the invention, said DNA-probes are single-stranded nucleic acids, e.g. obtained from an on chip synthesis or an attachment of presynthesized oligonucleotides complementary to nucleic acids of a compound producing microorganism.  
           [0012]    In a preferred embodiment of the invention, said reference sequence is a single-stranded nucleic acid and probes complementary to the single-stranded nucleic acid or to a DNA or RNA copy (cDNA/cRNA) of the single-stranded nucleic acid of said reference are in said array. The reference sequence is a polynucleotide sequence from a compound producing strain, especially a  Corynebacterium glutamicum  strain or an  Escherichia coli  strain.  
           [0013]    Another embodiment of the invention is a method of analyzing a polynucleotide sequence of a compound producing microorganism, by the use of an array of DNA probes immobilized on a solid support, the different DNA&#39;s occupying separate cells of the array, which method comprises labeling the polynucleotide sequence or fragments thereof, applying the polynucleotide sequence or fragments thereof under hybridization conditions to the array, and observing the location of the label on the surface associated with particular members of the set of DNA.  
           [0014]    The DNA-chips as mentioned above can be used to study and detect different RNA sequences or fragments thereof. Therefore the polynucleotide sequence or fragments thereof or a copy of the polynucleotide sequence or fragments thereof are applied to the DNA-chip under hybridization conditions.  
           [0015]    The sequences of the compound producing Corynebacterium or  Escherichia coli  can be found in different databases, e.g.:  
           [0016]    The NCBI is the National Center for Biotechnology Information. It is the database of the National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894, USA. (http://www.ncbi.nlm.nih.gov/)  
           [0017]    Swissprot and Trembl entries can be accessed from the Swiss Institute of Bioinformatics, CMU—Rue Michel-Servet 1, 1211 Genève 4, Switzerland.(http://www.expasy.ch/)  
           [0018]    PIR is the Protein Information Resource Database of the National Biomedical Research Foundation, 3900 Reservoir Rd., NW. Washington, D.C. 20007, USA. (http://www-nbrf.georgetown.edu/pirwww/pirhome.shtml)  
           [0019]    Selected reference sequences are especially:  
                                       NAME   ACCESSION No.   DATABASE                   16s rDNA   X84257   NCBI       aceA   X75504   NCBI       aceB   L27123   NCBI       acn   AB025424   NCBI       aroB   AF124600   NCBI       aroC   AF124600   NCBI       aroE   AF124518   NCBI       aroK   AF124600   NCBI       asd   X57226   NCBI       cat   AJ132968   NCBI       citE   AJ133719   NCBI       clgIIR   U13922   NCBI       cop1   X66078   NCBI       phage 304L int   Y18058   NCBI       csp2   X69103   NCBI       cydA   AB035086   NCBI       cydB   AB035086   NCBI       dapA   E16749   NCBI       dapB   E16752   NCBI       dapD   AJ004934   NCBI       dapE   X81379   NCBI       dciAE   AF038651   NCBI       ddh   Y00151   NCBI       DNA-Sequence   E16888   NCBI       DNA-Sequence   E16889   NCBI       DNA-Sequence   E16890   NCBI       DNA-Sequence   E16891   NCBI       DNA-Sequence   E16892   NCBI       DNA-Sequence   E16893   NCBI       DNA-Sequence   E16894   NCBI       DNA-Sequence   E16895   NCBI       DNA-Sequence   E16896   NCBI       dtsR1   AB018530   NCBI       efP   X99289   NCBI       fda   X17313   NCBI       ftsQ   E17182   NCBI       ftsY   AJ010319   NCBI       gap   X59403   NCBI       gdhA   X59404   NCBI       glna   AF005635   NCBI       glnB   AJ010319   NCBI       glt   X66112   NCBI       glyA   E12594   NCBI       gnd   E13660   NCBI       grcC   AF130462   NCBI       hisE   AF086704   NCBI       hom   E14598   NCBI       icd   X71489   NCBI       inhA   AF145898   NCBI       leuA   X70959   NCBI       leuB   Y09578   NCBI       lmrB   AF237667   NCBI       lpd   Y16642   NCBI       ltsA + ORF1   AB029550   NCBI       lysA   E16358   NCBI       lysC   E16745, E16746   NCBI       lysE   X96471   NCBI       lysG   X96471   NCBI       lysI   X60312   NCBI       malE   AF234535   NCBI       mgo   AJ224946   NCBI       murF   E14256   NCBI       murI   AB020624   NCBI       ndh   AJ238250   NCBI       nrdE   AF112535   NCBI       nrdF   AF112536   NCBI       nrdH   AF112535   NCBI       nrdI   AF112535   NCBI       nusG   AF130462   NCBI       obg   U31224   NCBI       odhA   E14601   NCBI       ORF4   X95649   NCBI       panB   X96580   NCBI       panC   X96580   NCBI       panD   AF116184   NCBI       pepQ   AF124600   NCBI       porA   AJ238703   NCBI       proP   Y12537   NCBI       ptsM   L18874   NCBI       pyc   Y09548   NCBI       pyk   L27126   NCBI       recA   X75085   NCBI       rel   Y18059   NCBI       rep   AB003157   NCBI       rplA   AF130462   NCBI       rplK   AF130462   NCBI       secA   D17428   NCBI       secE   D45020, AF130462   NCBI       secG   AJ007732   NCBI       Seq 1 Patent EP0563527   A78798   NCBI       Seq 1 Patent WO9519442   A45577   NCBI       Seq 11 Patent WO9519442   A45587   NCBI       Seq 2 Patent EP0563527   A78797   NCBI       Seq 2 Patent WO9723597   A93933   NCBI       Seq 3 Patent EP0563527   A78796   NCBI       Seq 3 Patent WO9519442   A45579   NCBI       Seq 5 Patent WO9519442   A45581   NCBI       Seq 7 Patent WO9519442   A45583   NCBI       Seq 9 Patent WO9519442   A45585   NCBI       soxA   AJ007732   NCBI       thrB   Y00546   NCBI       tkt   AB023377   NCBI       tnp   AF189147   NCBI       tpi   X59403   NCBI       tRNA-Thr   AF130462   NCBI       tRNA-Trp   AF130462   NCBI       ureA   AJ251883   NCBI       —   PIR: I40724   PIR       —   PIR: S18758   PIR       —   PIR: S52753   PIR       —   PIR: S60064   PIR       argS   PIR: A49936   PIR       aro   PIR: I40837   PIR       aroP   PIR: S52754   PIR       aspA   PIR: JC4101   PIR       atpD   PIR: I40716   PIR       bioA   PIR: I40336   PIR       bioB   PIR: JC5084   PIR       bioD   PIR: I40337   PIR       cglIIR   PIR: B55225   PIR       cglIR   PIR: A55225   PIR       dtsR   PIR: JC4991   PIR       dtxR   PIR: I40339   PIR       galE   PIR: JC5168   PIR       gdh   PIR: S32227   PIR       hisA   PIR: JE0213   PIR       hisF   PIR: JE0214   PIR       ilvA   PIR: A47044   PIR       ilvB   PIR: A48648   PIR       ilvC   PIR: C48648   PIR       pgk   PIR: B43260   PIR       pheA   PIR: A26044   PIR       proA   PIR: S49980   PIR       secY   PIR: I40340   PIR       thiX   PIR: I40714   PIR       thrA   PIR: DEFKHG   PIR       trpA   PIR: G24723   PIR       trpB   PIR: F24723   PIR       trpC   PIR: E24723   PIR       trpE   PIR: B24723   PIR       trpG   PIR: C24723   PIR       —   YFDA_CORGL   Swissprot       —   YPRB_CORGL   Swissprot       ackA   ACKA_CORGL   Swissprot       amt   AMT_CORGL   Swissprot       argB   ARGB_CORGL   Swissprot       argD   ARGD_CORGL   Swissprot       argJ   ARGJ_CORGL   Swissprot       betP   BETP_CORGL   Swissprot       brnQ   BRNQ_CORGL   Swissprot       clpB   CLPB_CORGL   Swissprot       efp   EFP_BRELA   Swissprot       ftsZ   FTSZ_BRELA   Swissprot       gluA   GLUA_CORGL   Swissprot       gluB   GLUB_CORGL   Swissprot       gluC   GLUC_CORGL   Swissprot       gluD   GLUD_CORGL   Swissprot       proB   PROB_CORGL   Swissprot       proC   PROC_CORGL   Swissprot       thtR   THTR_CORGL   Swissprot       trpD   TRPD_CORGL   Swissprot       tuf   EFTU_CORGL   Swissprot       unkdh   YPRA_CORGL   Swissprot       ypt5   YFZ1_CORGL   Swissprot       —   AB009078_1   Trembl       —   CGFDA_2   Trembl       —   CGLYSEG_3   Trembl       accBC   CGU35023_2   Trembl       aecD   CGCSLYS_1   Trembl       amtP   CAJ10319_2   Trembl       amtR   CGL133719_2   Trembl       apt   AF038651_2   Trembl       argC   AF049897_1   Trembl       argF   AF031518_1   Trembl       argG   AF030520_1   Trembl       argH   AF048764_1   Trembl       argR   AF041436_1   Trembl       aroA   AF114233_1   Trembl       aroD   AF036932_1   Trembl       cglIM   CG13922_1   Trembl       cmr   CG43535_1   Trembl       dtsR2   AB018531_2   Trembl       ectP   CGECTP_1   Trembl       ftSW   BLA242646_2   Trembl       glnD   CAJ10319_4   Trembl       gltB   AB024708_1   Trembl       gltD   AB024708_2   Trembl       hisG   AF050166_1   Trembl       hisH   AF060558_1   Trembl       ilvD   CGL012293_1   Trembl       impA   AF045998_1   Trembl       metA   AF052652_1   Trembl       metB   AF126953_1   Trembl       murC   AB015023_1   Trembl       murG   BLA242646_3   Trembl       ocd   CGL007732_4   Trembl       ppc   A09073_1   Trembl       ppx   C031224_1   Trembl       pta   CGPTAACKA_1   Trembl       putP   CGPUTP_1   Trembl       rel   AF038651_3   Trembl       sigA   BLSIGAGN_1   Trembl       sigB   BLSIGBGN_2   Trembl       srp   CAJ10319_5   Trembl       ureB   AB029154_3   Trembl       urec   AB029154_4   Trembl       ured   AB029154_8   Trembl       ureE   AB029154_5   Trembl       uref   AB029154_6   Trembl       ureg   AB029154_7   Trembl       ureR   AB029154_1   Trembl       wag31   BLA242594_1   Trembl       xylB   CGPAN_3   Trembl       yfiH   BLFTSZ_4   Trembl       yhbw   CGCSLYS_3   Trembl       yjcc   CGL133719_1   Trembl       accDA   DE: 19924365.4   Patent application       acp   DE: 10023400.3   Patent application       brnE   DE: 19951708.8   Patent application       brnF   DE: 19951708.8   Patent application       cdsA   DE: 10021828.8   Patent application       cls   DE: 10021826.1   Patent application       cma   DE: 10021832.6   Patent application       dapC   DE: 10014546.9   Patent application       dapF   DE: 19943587.1   Patent application       eno   DE: 19947791.4   Patent application       fadD15   DE: 10021831.8   Patent application       glk   DE: 19958159.2   Patent application       gpm   DE: 19953160.6   Patent application       lrp   DE: 19947792.2   Patent application       opcA   US: 09/531,267   Patent application       pfk   DE: 19956131.1   Patent application       pfkA   DE: 19956133.8   Patent application       pgi   US: 09/396,478   Patent application       pgsA2   DE: 10021829.6   Patent application       poxB   DE: 19959327.2   Patent application       ptsH   DE: 10001101.2   Patent application       sdhA   DE: 19959650.6   Patent application       sdhB   DE: 19959650.6   Patent application       sdhC   DE: 19959650.6   Patent application       sod   US: 09/373,731   Patent application       sucC   DE: 19956686.0   Patent application       sucD   DE: 19956686.0   Patent application       tal   US: 60/142,915   Patent application       thrE   DE: 19941478.5   Patent application       zwa1   DE: 19959328.0   Patent application       zwa2   DE: 19959327.2   Patent application       zwf   JP: A-092246.61   Patent application                  
 
           [0020]    In a preferred embodiment of the invention, such arrays can be used for monitoring the transcriptional status of cells on a genomic scale during a fermentation.  
           [0021]    In another preferred embodiment of the invention, such arrays can be used for monitoring the transcriptional status of a diagnostic subset of genes during a fermentation.  
           [0022]    The arrays according to the invention are preferably used in a method of monitoring a fermentation process by analyzing polynucleotide sequences or fragments thereof of a compound producing microorganism, by the use of an array of DNA probes comprising at least a set that is exactly complementary to select reference sequences of the compound producing microorganism immobilized on a solid support, the different probe DNA&#39;s occupying separate cells of the array, which method comprises labeling the reference polynucleotide sequence or fragments thereof, applying the polynucleotide sequence or fragments thereof under hybridization conditions to the array, and observing the location and the intensity of the label on the surfaces associated with particular members of the probe DNA&#39;s.  
           [0023]    In a preferred embodiment the polynucleotide sequence of  Corynebacterium glutamicum  strain separated from a fermentation broth is analyzed.  
           [0024]    In another embodiment the polynucleotide sequence of an  Escherichia coli  strain separated from a fermentation broth is analyzed.  
           [0025]    The array is used to monitore the process related target genes of compound producing microorganisms in the fermentation process.  
           [0026]    In a preferred embodiment the fermentation process of compound production is monitored by said method including the following steps:  
           [0027]    fermentation of the bacteria producing L-amino acid(s), vitamins, metabolites, antioxidants, cellular or secreted proteins, pigments, nucleotides, sugars or peptides  
           [0028]    isolation of the microorganism cells during the fermentation and preparation of the cellular ribonucleic acid (RNA)  
           [0029]    labeling of the isolated RNA with a known technique like a direct labeling method or an incorporation of labeled nucleotides during by generation of a copy of the isolated RNA, e.g. to cDNA/cRNA.  
           [0030]    subsequent hybridisation of the labeled RNA/cDNA/cRNA to an array of single or double stranded nucleic acid probes for the detection of transcripts of coryneform or coliform bacteria  
           [0031]    detection of the hybridization pattern of the signals by known methods  
           [0032]    comparison of obtained hybridization patterns  
           [0033]    usage of the obtained results for improving processes and productivity.  
       
    
    
     EXAMPLES  
     Example 1  
       [0034]    Manufacture of Arrays  
         [0035]    The primers for the PCR amplification of the probe DNA is chosen using the Primer3 software with the default settings. The only exemption is the product size, which settings are set to 200-3000 base pairs with an optimum product size of 500 base pairs (Steve Rozen, Helen J. Skaletsky (1998) Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html.) On account of the sequences of the probe genes known from databases, as an example the following oligonucleotides are selected for the polymerase chain reaction of the aceA gene:  
         [0036]    aceA1:  
                                   aceA1:           5′ ccacacctaccctgaccagt 3′                       aceA2:           5′ ggctcgagaccattcttgac 3′          
 
         [0037]    The chosen primers are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reactions for all genes is carried out according to the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) using Taq polymerase from Boehringer Mannheim (Germany, Product Description Taq DNA Polymerase, Product No. 1 146 165). Amongst other sources, one skilled in the art will find further instructions for the amplification of DNA sequences with the aid of polymerase chain reaction (PCR) in the Handbooks by Gait: Oligonucleotides synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and by Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).  
         [0038]    Chromosomal DNA as template for the PCR reaction is isolated from the strain ATCC 13032 by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). With the aid of the polymerase chain reaction the primers permit the amplification of internal fragment of the selected genes that can be used as a hybridization probe which is immobilized on a microarray. The thus amplified products are tested electrophoretically in a 1.0% agarose gel.  
         [0039]    The PCR products are desalted and purified using Multiscreen PCR plates (Cat. No. MANU 030 10, Millipore Corporation, Bedford, Mass., USA) according to the manufacturers instructions. These probe DNA&#39;s are mixed with spotting buffer and printed onto ArrayLink hydrophob microarray substrates (GeneScan Europe AG, Freiburg, Germany) using a Microgrid Microarray Spotter (Biorobotics, Cambridge, UK). The microarrays are produced following the manufacturers instructions.  
       Example 2  
       [0040]    L-amino Acids Fermentation  
         [0041]    For production of L-lysine the  C. glutamicum  strains ATCC13032, DSM5715 and ATCC21513 are cultivated in a nutrient medium suitable for the production of L-lysine and the L-lysine content in the culture supernatant is determined. The strains ATCC13032 and ATCC21513 can be obtained from the American Type Culture Collection (Manassas, Va., USA), the strain DSM5715 is described in EP-B-0435132.  
         [0042]    For the purpose of L-Lysine production the strain is first of all incubated for 24 hours at 33° C. on an agar plate (brain-heart agar, starting from this agar plate culture a preculture is inoculated (10 ml of medium in a 100 ml Erlenmeyer flask). The full medium CgIII is used as medium for the preculture.  
                                             medium Cg III                                    NaCl   2.5 g/l           Bacto-Peptone    10 g/l           Bacto-Yeast Extract    10 g/l           Glucose (autoclaved     2% (w/v)           separately)           The pH value is   The preculture is incubated for 16           adjusted to pH 7.4   hours at 33° C. at 240 rpm on a shaker               table. From this preculture a main               culture is inoculated so that the               initial OD (660 nm) of the main               culture is 0.1 OD. The medium MM is               used for the main culture.                      
 
         [0043]    [0043]                                             Medium MM                                    CSL (Corn Steep Liquor)     5 g/l           MOPS    20 g/l           Glucose (autoclaved separately)    50 g/l           Salts:           (NH4) 2SO4)    25 g/l           KH2PO4   0.1 g/l           MgSO4.7H2O   1.0 g/l           CaCl2.2H2O    10 mg/l           FeSO4.7H2O    10 mg/l           MnSO4.H2O   5.0 mg/l           Biotin (sterile filtered)   0.3 mg/l           Thiamine.HCl (sterile filtered)   0.2 mg/l           Homoserine (sterile filtered)   0.1 g/l           Leucine (sterile filtered)   0.1 g/l           CaCO3    25 g/l               CSL, MOPS and the salt               solution are adjusted with               ammonia water to pH 7 and               autoclaved. The sterile               substrate solutions and               vitamin solutions as well as               the dry autoclaved CaCO 3  are               then added.                        
         [0044]    Cultivation is carried out in a 10 ml volume in a 100 ml Erlenmeyer flask equipped with baffles. The cultivation is carried out at 33° C. and 80% atmospheric humidity.  
         [0045]    After 48 hours the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of L-lysine formed is determined by ion exchange chromatography and post-column derivatisation with ninhydrin detection using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany).  
         [0046]    The results of the experiment are shown in Table 1.  
                                                 TABLE 1                                   Strain   OD (660)   L-lysine-HCl g/l                                        ATCC13032   12.8   0.1           DSM5715   8.2   12.8           ATCC21513   8.4   13.4                      
 
       Example 3  
       [0047]    Isolation and Labeling of RNA from  C. glutamicum    
         [0048]    From the  C. glutamicum  cultures described in Example 2, total RNA is isolated after 12, 24, 36 and 48 hours. Therefore an appropriate volume, e.g. 5 ml of such a culture is mixed with the some volume of ice cold 20 mM NaN 3  (Catalog number 1.06688.0100, Merck, Darmstadt, Germany). The cells are harvested by centrifugation for 10 minutes at 10000×g. The RNA extraction is done using a Ribolyser machine (Catalog number HB6000-120, Hybaid, Heidelberg, Germany) an the Hybaid RiboLyser™ Blue Kit (Catalog number RY61100 Hybaid, Heidelberg, Germany). This crude RNA-preparation is further purified with the SNAP total RNA isolation kit from Invitrogen Corporation (Carlsbad, Calif., USA; Cat. No. K1950-05). By this treatment DNA contaminations in the RNA preparation are removed by digestion with DNAseI followed by RNA purification on silica membrane spin columns, done according to the manufacturers instructions. 50-100 μg of this RNA preparation is used for one labeling procedure.  
         [0049]    Total bacterial RNA is labeled by generation of a single stranded copy DNA (cDNA). For the labeling 100 μg total RNA are mixed with 10 μg of oligonucleotide primers as starting point for the reverse transcription. These primers consist of an equimolar mixture of random hexamers and random octamers. The random primers are synthesized by MWG (Ebersberg, Germany). The incorporation of the fluorescent dyes and the purification of the labeled cDNA is done using the Atlas™ Glass Fluorescent Labeling Kit (Cat. No. K1037-1, Clontech, Heidelberg, Germany) following the manufacturers instructions.  
         [0050]    Using the described protocol, the cDNA of the no L-lysine producing strain ATCC13032 is labeled with the fluorescent dye Cy3. The cDNA of the L-lysine producing strain ATCC21513 is labeled with the fluorescent dye Cy5.  
       Example 4  
       [0051]    Comparative Analysis of the Transcriptional Status of the Strains ATCC13032 and ATCC21513  
         [0052]    As described in Example 3, for each strain the total cellular RNA is isolated and labeled at different time points during the fermentation. For the analysis of differences in the transcriptional status, the labeled cDNA of both strains is hybridized competitively for each time point on the arrays described in example 1. For the experienced user beneath other sources, the principles and further technical and methodological details are described in the book of M. Schena, (DNA Microarrays, Editor: M. Schena, Oxford University Press, 1999)  
         [0053]    The hybridization is done using the Atlas™ Glass Hybridization Chamber and GlassHyb Solution (Catalog numbers 7899-1 and 8016-1 Clontech, Heidelberg, Germany). The slides are scanned using a Scanarray 4000 confocal microarray scanner (GSI-Lumonics, Billerica, Mass., USA) following the manufacturers instructions. The acquired images are further analyzed using the QuantArray Software, provided together with the scanner.  
         [0054]    The fluorescence intensity for each spot and each fluorescence dye is calculated separately. The results of each data point of the single experiments are plotted against each other. Signals that give a data point that is more than a factor of 1.5 away from the correlation line are regulated differentially in the two strains.  
         [0055]    Examples of genes that are upregulated or downregulated at one or more time points and the maximum fold change difference in the expression level in the L-lysine producing strain ATCC21513 compared to the no L-lysine producing strain ATCC13032 are listed in Table 2:  
                                                         TABLE 2                                   Genes higher expressed in       Genes lower expressed in               ATCC21513 compared       ATCC21513 compared               to ATCC13032       to ATCC13032                                            gap   3 fold   pgi   4 fold           lysC   4 fold   fda   2 fold           dapA   4 fold   pyk   5 fold           lysE   2 fold   glt   3 fold           sucC   4 fold   icd   2 fold           dapC   3 fold   rel   2 fold           ptsM   5 fold   ilvC   2 fold                      
 
       Example 5  
       [0056]    Monitoring a Fermentation by Comparison of Gene Expression Patterns  
         [0057]    The gene expression patterns described in the Examples 2-4 can be used to monitore a fermentation process.  
         [0058]    Therefore the RNA of cells from a good fermentation, i.e. with the expected L-lysine productivity, is prepared and labeled as described in Example 3. The hybridization pattern of the labeled cDNA resulting from one or more combined RNA preparations from a good fermentation is compared with the hybridization pattern of a cDNA resulting from an other fermentation that is to be monitored. The hybridization patterns are basically obtained as described in Example 4. In order to achieve shorter analysis times the amount of cDNA can be increased and the hybridization time can be decreased.  
         [0059]    The sample that is monitored is taken at about the same time point and the same optical density as the sample from the good reference fermentation that is used as reference sample.  
         [0060]    The expression data are analyzed by a scatter plot analysis. Signals that give a data point that is more than a factor of about 1.5-2.0 away from the correlation line are regulated differentially in the two fermentations. Such differences in gene expression indicate a problem with the fermentation efficiency in respect to product formation or biomass formation.  
         [0061]    If more than &gt;0-6%, preferable &gt;0-3% of the genes are located more than the factor of 2 away from the correlation line, the fermentation is good.  
         [0062]    If more than 3-15%, preferable 3-8% of the genes are located more than the factor of 2 away from the correlation line, the fermentation might give low product, biomass or sugar conversion yields.  
         [0063]    If more than 15% of the genes are located more than the factor of 2 away from the correlation line, the fermentation will give low product yields.  
         [0064]    Within these gene expression patterns that can be correlated to the fermentation yield, there are also single genes that can be used to monitor a fermentation. Changes in the individual gene expression level of these genes indicate a problem in the fermentation process. An example is the glt gene, whose expression is about 3-fold decreased in a L-Lysin producing strain compared to a wild type as shown in example 4. If this ration is increased to more than 5-fold weaker expression, the L-Lysine yield obtained as described in Example 2 will decrease for about 5% from 13.4 g/l to 13.1 g/l. Probes for such genes can be immobilized on diagnostic DNA-arrays and be used for monitoring a fermentation process.  
       Example 6  
       [0065]    Improving a Fermentation by Inactivation of the pgi Gene  
         [0066]    The genes described in Example 4 are differentially regulated in a L-lysine producing  C. glutamicum  strain. In order to show the positive effect of this differential regulation on L-lysine production, as an example the pgi gene is inactivated in the L-lysine producing strain DSM5715.  
         [0067]    Therefore an integration vector for the integration mutagenesis of the pgi gene is constructed.  
         [0068]    Chromosomal DNA is isolated from the strain ATCC 13032 by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On account of the sequence of the pgi gene for  C. glutamicum , the following oligonucleotides are selected for the polymerase chain reaction:  
                                   pgi-int1:           5′ GACCTCGTTTCTGTGTTGG 3′                       pgi-int2:           5′ TGACTTGCCATTTGATTCC 3′          
 
         [0069]    The represented primers are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction is carried out according to the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) using Taq polymerase from Boehringer Mannheim (Germany, Product Description Taq DNA Polymerase, Product No. 1 146 165). With the aid of the polymerase chain reaction the primers permit the amplification of a 516 bp large internal fragment of the pgi gene. The thus amplified product is tested electrophoretically in a 0.8% agarose gel.  
         [0070]    The amplified DNA fragment is ligated into the vector pCR2.1-TOPO (Mead at al. (1991) Bio/Technology 9:657-663) using the TOPO TA Cloning Kit from Invitrogen Corporation (Carlsbad, Calif., USA; Cat. No. K4500-01).  
         [0071]    The  E. coli  strain TOP10 is then electroporated with the ligation batch (Hanahan, In: DNA cloning. A practical approach. Vol. I. IRL-Press, Oxford, Washington D.C., USA, 1985). Plasmid-carrying cells are selected by plating out the transformation batch onto LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) that has been supplemented with 50 mg/l of kanamycin. Plasmid DNA is isolated from a transformant using the QIAprep Spin Miniprep Kit from Qiagen and is checked by restriction with the restriction enzyme EcoRI followed by agarose gel electrophoresis (0.8%). The plasmid is named pCR2.1pgiint.  
         [0072]    The vector pCR2.1pgiint is electroporated into  Corynebacterium glutamicum  DSM 5715 according to the electroporation method of Tauch et. al.(FEMS Microbiological Letters, 123:343-347 (1994)). The strain DSM 5715 is an AEC-resistant L-lysine producer. The vector pCR2.1pgiint cannot replicate independently in DSM5715 and thus only remains in the cell if it has integrated into the chromosome of DSM 5715. The selection of clones with pCR2.1pgiint integrated into the chromosome is made by plating out the electroporation batch onto LB agar (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) that has been supplemented with 15 mg/l of kanamycin.  
         [0073]    In order to demonstrate the integration the pgiint fragment is labeled using the Dig Hybridisation Kit from Boehringer according to the method described in “The DIG System User&#39;s Guide for Filter Hybridization” published by Boehringer Mannheim GmbH (Mannheim, Germany, 1993). Chromosomal DNA of a potential integrant is isolated according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and is in each case cleaved with the restriction enzymes SacI, EcoRI and HindIII. The resultant fragments are separated by means of agarose gel electrophoresis and hybridized at 68° C. using the Dig Hybridisation Kit from Boehringer. The plasmid pCR2.1pgiint has inserted itself into the chromosome of DSM5715 within the chromosomal pgi gene. The strain is designated DSM5715::pCR2.1pgiint.  
         [0074]    The  C. glutamicum  strain DSM5715::pCR2.1pgiint is cultivated in a nutrient medium suitable for the production of L-lysine and the L-lysine content in the culture supernatant is determined.  
         [0075]    For this purpose the strain is first of all incubated for 24 hours at 33° C. on an agar plate with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg/l). Starting from this agar plate culture a preculture is inoculated (10 ml of medium in a 100 ml Erlenmeyer flask). The rich medium CgIII described in Example 2 is used as medium for the preculture.  
         [0076]    Cultivation is carried out in MM-Medium described in Example 2 with 10 ml volume in a 100 ml Erlenmeyer flask equipped with baffles. Kanamycin is added (25 mg/l). The cultivation is carried out at 33° C. and 80% atmospheric humidity.  
         [0077]    After 48 hours the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of L-lysine formed is determined by ion exchange chromatography and post-column derivatisation with ninhydrin detection using an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany).  
         [0078]    The results of the experiment are shown in Table 3.  
                               TABLE 3                                   Strain   OD (660)   L-lysine-HCl g/l                           DSM5715   8.2   13.7           DSM5715::pCR2.lpgiint   7.9   18.8                      
 
       Example 7  
       [0079]    Improving a Fermentation by Overexpression of the Gap Gene  
         [0080]    The genes described in Example 4 are differentially regulated in a L-lysine producing  C. glutamicum  strain. In order to show the positive effect of this differential regulation on L-lysine production, as an example the gap gene is overexpressed in the L-lysine producing strain DSM5715.  
         [0081]    Therefore the gap gene is cloned in the vector pJC1. Chromosomal DNA from  Corynebacterium glutamicum  ATCC 13032 is isolated as described in example 5. A DNA fragment bearing the gap gene is amplified by polymerase chain reaction. The following primers are used for this purpose:  
                                   gapA1           5′-TGCTCTAGATTGAAGCCAGTGTGAGTTGC-3′                       gapA2           5′-TGCTCTAGAGATGACACATCACCGTGAGC-3′          
 
         [0082]    The primers illustrated are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction is carried out by the standard PCR method of Innis et al.(PCR protocol. A guide to methods and applications, 1990, Academic Press). The primers enabled amplification to be effected of a DNA fragment with a size of about 1520 bp and bearing the gap gene of  Corynebacterium glutamicum.    
         [0083]    After separation by gel electrophoresis, the PCR fragment is isolated from the agarose gel using a QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).  
         [0084]    The  E. coli - C. glutamicum  shuttle vector pJC1 (Cremer et al., 1990, Molecular and General Genetics 220: 478-480) is used as a vector. This plasmid is completely cleaved with the restriction enzyme BamHI, is treated with Klenow polymerase (Roche Diagnostics GmbH, Mannheim, Germany) and is subsequently dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, Product No. 1758250).  
         [0085]    The gap fragment obtained in this manner is mixed with the prepared vector pJC1 and is ligated with the aid of a SureClone Ligation Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer&#39;s instructions. The ligation batch is transformed in the  E. coli  strain DH5 (Hanahan, in: DNA cloning. A practical approach. Vol. I. IRL Press, Oxford, Washington D.C., USA). Plasmid-bearing cells are selected by plating out the transformation batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/l kanamycin. After incubation overnight at 37° C., recombinant individual clones are selected. Plasmid DNA is isolated from a transformant using a Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer&#39;s instructions and is cleaved with the restriction enzyme XbaI in order to investigate the plasmid by subsequent agarose gel electrophoresis. The plasmid obtained is designated as pJC1gap.  
         [0086]    The  C. glutamicum  strains ATCC13032 and DSM5715 are transformed with the plasmid pjc1gap using the electrophoration method described by Liebl et al. (FEMS Microbiology Letters, 53:299-303 (1989)). The transformants are selected on LBHIS agar consisting of 18.5 g/l brain-heart infusion bouillon, 0.5 M sorbitol, 5 g/l bacteriological trypton, 2.5 g/l bacteriological yeast extract, 5 g/l NaCl and 18 g/l bacteriological agar which is supplemented with 25 mg/l kanamycin. Incubation is effected for 2 days at 33° C.  
         [0087]    Plasmid DNA is isolated from each transformant by the usual methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), is cut with the restriction endonuclease XbaI and the plasmid is investigated by subsequent agarose gel electrophoresis. The strain obtained is designated as DSM5715/pJC1gap.  
         [0088]    The  C. glutamicum  strains DSM5715 and DSM5715/pJC1gap are cultivated as described in Example 2.  
         [0089]    After 48 hours, the OD and the L-lysine content in the culture supernatant is determined as described in Example 2  
         [0090]    The results of the experiment are given in Table 4.  
                               TABLE 4                                   Strain   OD (660 nm)   L-lysine-HCl (g/l)                           DSM5715   8.1   13.6           DSM5715//pJC1gap   7.6   14.4                      
 
         [0091]    [0091] 
     
       
       
         1 
         
           
             6  
           
           
             1  
             20  
             DNA  
             Corynebacterium glutamicum  
             
               primer aceA1  
             
           
            1 

ccacacctac cctgaccagt                                                 20 

 
           
             2  
             20  
             DNA  
             Corynebacterium glutamicum  
             
               primer aceA2  
             
           
            2 

ggctcgagac cattcttgac                                                 20 

 
           
             3  
             19  
             DNA  
             Corynebacterium glutamicum  
             
               primer pgi-int1  
             
           
            3 

gacctcgttt ctgtgttgg                                                  19 

 
           
             4  
             19  
             DNA  
             Corynebacterium glutamicum  
             
               primer pgi-int2  
             
           
            4 

tgacttgcca tttgattcc                                                  19 

 
           
             5  
             29  
             DNA  
             Corynebacterium glutamicum  
             
               primer gapA1  
             
           
            5 

tgctctagat tgaagccagt gtgagttgc                                       29 

 
           
             6  
             29  
             DNA  
             Corynebacterium glutamicum  
             
               primer gapA2  
             
           
            6 

tgctctagag atgacacatc accgtgagc                                       29