SK14592000A3 - Nucleotide sequences coding for the lrp gene and fermentative method for producing l-aminoacids - Google Patents
Nucleotide sequences coding for the lrp gene and fermentative method for producing l-aminoacids Download PDFInfo
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- SK14592000A3 SK14592000A3 SK1459-2000A SK14592000A SK14592000A3 SK 14592000 A3 SK14592000 A3 SK 14592000A3 SK 14592000 A SK14592000 A SK 14592000A SK 14592000 A3 SK14592000 A3 SK 14592000A3
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
Abstract
Description
Vynález popisuje nukleotidové sekvencie kódujúce gén lrp. Ďalej vynález popisuje spôsoby fermentatívnej výroby aminokyselín, najmä potom lyzínu a izoleucínu za použitia koryneformných baktérií, v ktorých je ovplyvnená expresia génu lrp.The invention provides nucleotide sequences encoding the lrp gene. The invention further provides methods for the fermentative production of amino acids, particularly lysine and isoleucine, using coryneform bacteria in which expression of the lrp gene is affected.
Dotera-iši stav technikyBACKGROUND OF THE INVENTION
L-aminokyseliny sú významné suroviny, ktoré nachádzajú uplatnenie vo výžive ľudí aj zvierat, ako aj v humánnej medicíne a farmaceutickom priemysle.L-amino acids are important raw materials found in human and animal nutrition, as well as in human medicine and the pharmaceutical industry.
Je známe, že tieto aminokyseliny môžu byť vyrobené fermentáciou vhodných kmeňov baktérií, najmä Corynebacterium glutamicum. Na zlepšení spôsobu výroby sa sústavne pracuje vďaka významu, ktorý tieto aminokyseliny majú.It is known that these amino acids can be produced by fermentation of suitable strains of bacteria, in particular Corynebacterium glutamicum. Due to the importance of these amino acids, the process is continuously improved.
Zlepšenie spôsobu výroby týchto aminokyselín môže byť dosiahnuté prostredníctvom zmeny techniky fermentácie ako napríklad zmenou miešania a prevzdušňovania kyslíkom, či zmenou zloženia živného média ako napríklad zmenou koncentrácie cukru v priebehu fermentácie, či zmenou v spôsobe spracovania na výslednú formu napríklad chromatografickým čistením na iontomeniči, či zmenou výkonných vlastností samotného produkčného mikroorganizmu.Improvement of the process for producing these amino acids can be achieved by altering the fermentation technique such as by changing the mixing and aeration with oxygen, by changing the composition of the nutrient medium such as by changing the sugar concentration during fermentation, by changing the processing to the resulting form the performance properties of the production microorganism itself.
Ku zlepšeniu výkonnosti produkčných mikroorganizmov boli použité spôsoby mutagenézie, selekcie a vyhľadávania mutantných • · • ··· ·· · • · · · · • · · · · · t *< * * ( I a* · · í ·· ·· ·· ···» ·· ··· klonov. Týmto spôsobom boli získané kmene, ktoré sú odolné voči antimetabolitom ako napríklad voči analógu lyzínu S-(2-aminoetyl)-cysteinu alebo sú auxotrofné vzhľadom k regulačné dôležitým aminokyselinám a produkujú požadované L-aminokyseliny. Už niekoľko rokov sa používajú ku zlepšeniu kmeňov Corynebacterium sp. produkujúcich L-aminokyseliny rovnako spôsoby rekombinantnej DNA.Mutagenesis, selection and mutant screening methods were used to improve the performance of the producing microorganisms (I and I) (I and I). In this way strains are obtained which are resistant to antimetabolites such as the lysine analog of S- (2-aminoethyl) -cysteine or are auxotrophic with respect to regulatory important amino acids and produce the desired L Recombinant DNA methods have also been used to improve L-amino acid-producing strains of Corynebacterium sp.
Protein LRP (leucín-responzívny protein) bol najprv popísaný u baktérie Escherichia coli ako globálny regulátor, ktorý ovplyvňuje transkripciu celého radu génov, ktorých produkty sa zúčastňujú transportu, biosyntézy a odbúravania aminokyselín (Calvo a Matthews, Microbiological Reviews 58, strana 466 až 490, 1994).LRP (leucine-responsive protein) was first described in Escherichia coli as a global regulator that affects the transcription of a variety of genes whose products are involved in transport, biosynthesis, and amino acid degradation (Calvo and Matthews, Microbiological Reviews 58, pages 466 to 490, 1994).
V súčasnosti boli identifikované podobné gény tiež u ďaľších organizmov ako napríklad u Bradyrhizobium japonicum (King a 0' Brian, Journal of Bacteriology, 179, strana 1828 až 1831, 1997), Klebsiella aerogenes (Janes a Bender, Journal of Bacteriology, 181, strana 1054 až 1058, 1999), Sulfolobus acidocaldarius (Charlier a ďaľší, Gene 201, strana 63 až 68, 1997) a u Gram-pozitivnej baktérie Bacillus subtilis (Belitsky a ďaľší, Journal of Bacteriology, 179, strana 5448 až 5457, 1997).Recently, similar genes have also been identified in other organisms such as Bradyrhizobium japonicum (King and O'Brian, Journal of Bacteriology, 179, pp. 1828-1831, 1997), Klebsiella aerogenes (Janes and Bender, Journal of Bacteriology, 181, p. 1054-1058, 1999), Sulfolobus acidocaldarius (Charlier et al., Gene 201, pp. 63-68, 1997) and Gram-positive bacteria Bacillus subtilis (Belitsky et al., Journal of Bacteriology, 179, pp. 5448-554, 1997).
V baktériách E. coli reguluje protein LRP svoju vlastnú expresiu. Okrem toho ovplyvňuje LRP protein v E. coli expresiu mnohých gémov, a to buď negatívne alebo pozitívne. Obecne je pritom stimulovaná expresia produktov génov, ktoré sa zúčastňujú na úlohe biosyntetických pochodov.In E. coli, the LRP protein regulates its own expression. In addition, the LRP protein in E. coli affects the expression of many genes, either negative or positive. Generally, the expression of gene products involved in the role of biosynthetic processes is stimulated.
Naopak produkty génov, ktoré sa podieľajú na katabolizme sú spravidla regulované negatívne. V niektorých prípadoch sa dá zvýšiť účinok proteínu LRP prídavkom L-leucínu, pričom prídavok L-leucínu môže účinkovať tiež negatívne (Newman a ďaľší v knihe :Conversely, the products of genes involved in catabolism are generally negatively regulated. In some cases, the effect of the LRP protein may be enhanced by the addition of L-leucine, while the addition of L-leucine may also have a negative effect (Newman et al. In the book:
• · · · • · a · ·• · · · · · · · · · · · · · · · · · · · · · · ·
- 3 • · • ··· • a a a a aa · á • a ·· aa aaaa ·· ·- 3 • aa aa aa aa aa aaaa ·· ·
Neidhardt a ďaľší. Escherichia coli and Salmonella typhimurium : Cellular and molecular biology. American Society for Microbiology, Washington D.C., strana 1513 až 1525, 1996). Cez proteín LRP je v E. coli regulované veľké množstvo génov a operónov, ktoré hrajú centrálnu úlohu pri syntéze aminokyselín a ich katabolizme. V baktériách E. coli sú napríklad pomocou LRP negatívne regulované nasledujúce operóny : operón livJ, ktorý kóduje väzobný proteín z vysoko afinného komplexu pre príjem aminokyselín s rozvetveným reťazcom (Haney a ďaľší Journal of Bacteriology, 174, strana 108 až 115, 1992) a operón lysU, ktorý kóduje lyzín tRNA-syntetázu (Gazeau a ďaľší FEBS Letters, 300, strana 254 až 258, 1994).Neidhardt and others. Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. American Society for Microbiology, Washington, D.C., 1996, pages 1513-1525. A large number of genes and operons, which play a central role in amino acid synthesis and catabolism, are regulated in E. coli via the LRP protein. In E. coli, for example, the following operons are negatively regulated by LRP: the livJ operon, which encodes a binding protein from the high affinity branched chain amino acid complex (Haney et al. Journal of Bacteriology, 174, 108-115, 1992) and the operon lysU, which encodes a lysine tRNA synthetase (Gazeau et al. FEBS Letters, 300, 254-258, 1994).
Medzi doposiaľ známe gény pozitívne ovplyvňované proteínom Lrp u E. coli patria okrem iných : ilvIH, gltBDF a leuABCD (Lin a ďalší Journal of Bacteriology, 174, strana 1948 až 1955, 1992).Previously known genes positively influenced by the Lrp protein in E. coli include, but are not limited to: ilvIH, gltBDF and leuABCD (Lin et al. Journal of Bacteriology, 174, 1948-1955, 1992).
Posledný menovaný operón má zásadný význam pre biosyntézu leucínu a veľmi významný je aj pre biosyntézu lyzínu v Corynebacterium glutamicum. Dá sa predpokladať, že je určitý vzťah medzi auxotrofiou ohľadne leucínu a vysokou produkčnou schopnosťou lyzínu (Schrumpf a ďalší, Applied Microbiology and Biotechnology, 37, strana 566 až 571, 1992). Patek a ďaľší (Applied and Environmental Microbiology, 60, strana 133 až 140, 1994) dokázali, že inaktivácia operónu leuA v lyzín-produkujúcom kmeni C. glutamicum viedla ku zvýšeniu spotreby lyzínu. Tosaka a ďaľší (Agricultural Biological Chemistry, 43, strana 265 až 270, 1979) dokázali na jednom mutantnom kmeni Brevibacterium lactofermentum, že po prídavku od L-leucínu došlo ku zníženiu tvorby lyzínu pri súčasnom zvýšení tvorby treonínu.The latter operon is essential for leucine biosynthesis and is also very important for lysine biosynthesis in Corynebacterium glutamicum. It is believed that there is some relationship between auxotrophy regarding leucine and the high lysine production ability (Schrumpf et al., Applied Microbiology and Biotechnology, 37, 566-571, 1992). Patek et al. (Applied and Environmental Microbiology, 60, 133-140, 1994) demonstrated that inactivation of the leuA operon in a lysine-producing strain of C. glutamicum led to an increase in lysine consumption. Tosaka et al. (Agricultural Biological Chemistry, 43, 265-270, 1979) demonstrated on one mutant strain of Brevibacterium lactofermentum that the addition of L-leucine reduced lysine formation while increasing threonine production.
- 4 ··- 4 ··
Podstata vynálezuSUMMARY OF THE INVENTION
Vynález popisuje nový spôsob zlepšenej fermentatívnej výroby L-aminokyselín, najmä potom L-lyzínu, L-izoleucínu pomocou koryneformných baktérií.The invention describes a novel process for the improved fermentative production of L-amino acids, in particular L-lysine, L-isoleucine by means of coryneform bacteria.
I. Predmet vynálezu je polynukleotíd izolovaný z koryneformných baktérií, ktorý obsahuje polynukleotídovú sekvenciu, vybratú zo skupiny tvorenej :I. Object of the invention is a polynucleotide isolated from coryneform bacteria comprising a polynucleotide sequence selected from the group consisting of:
a) polynukleotídy, ktoré sú aspoň zo 70 % identické s polynukleotídom kódujúcim peptid, ktorého aminokyselinová sekvencia je uvedená v Sekvencii id. č. : 2.a) polynucleotides which are at least 70% identical to a polynucleotide encoding a peptide whose amino acid sequence is set forth in SEQ ID NO: 1; no. : 2.
b) polynukleotid, kódujúci peptid, ktorý obsahuje aminokyselinovú sekvenciu, ktorá je aspoň zo 70 % identická so sekvenciou aminokyselín podľa Sekvencie id. č. : 2.b) a polynucleotide encoding a peptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 1; no. : 2.
c) polynukleotíd, komplementárny k polynukleotídu podľa a) alebo b) a(c) a polynucleotide complementary to the polynucleotide of (a) or (b); and
d) polynukleotíd, obsahujúci aspoň 15 za sebou idúcich bází z polynukleotídovej sekvencie podľa bodov a), b) alebo c).d) a polynucleotide comprising at least 15 consecutive bases from the polynucleotide sequence of a), b) or c).
II. Vo výhodnom uskutočnení vynálezu je predmet vynálezu rovnako polynukleotíd podľa bodov a), b), c) alebo d), pričom sa jedná o replikovateľnú DNA obsahujúcu :II. In a preferred embodiment, the invention also provides the polynucleotide of (a), (b), (c) or (d), wherein the polynucleotide is a replicable DNA comprising:
(i) nukleotidovú sekvenciu podľa Sekvencie id.č.: 1, alebo (ii) aspoň jednu sekvenciu, ktorá zodpovedá sekvencii podľa bodu (i) vzhľadom k degenerácii genetického kódu alebo (iii) aspoň jednu sekvenciu, ktorá hybriduje so sekvenciou podľa bodu (i) alebo (ii) alebo s komplementárnymi sekvenciami, • · • ··· rt · * ··· · · ·· · • · · · ··· ·· ·· ·· ·· ···· ·· ·(i) the nucleotide sequence of SEQ ID NO: 1, or (ii) at least one sequence that corresponds to the sequence of (i) with respect to the degeneracy of the genetic code, or (iii) at least one sequence that hybridizes to the sequence of ( (i) or (ii) or with complementary sequences, • rt · * ··· · · ··· ···············
- 5 a poprípade (iv) funkčne neutrálna mutácia v sekvencii podľa bodu (i).And optionally (iv) a functionally neutral mutation in the sequence of (i).
Vynález ďalej popisuje polynukleotid podľa bodu II, obsahujúci nukleotidovú sekvenciu zodpovedajúcu Sekvencii id.č. : 1, polynukleotid podľa bodu II, kódujúci polypeptid, ktorého aminokyselinová sekvencia obsahuje Sekvenciu id.č. : 2.The invention further provides a polynucleotide according to item II comprising a nucleotide sequence corresponding to SEQ ID NO. 1, the polynucleotide of item II, encoding a polypeptide whose amino acid sequence comprises SEQ ID NO: 1; : 2.
Predmetom vynálezu sú rovnako polynukleotidy, ktoré podstatným spôsobom pozostávajú z polynukleotidovej sekvencie, ktorá sa dá získať hybridizačným skríningom zodpovedajúcej knižnice obsahujúcej celkový gén so sekvenciou podľa Sekvencie id.č. 1, za použitia hybridizačnej sondy, ktorá má sekvenciu podľa Sekvencie id.č.: 1 alebo aspoň jej fragment, ako aj izolácie uvedenej sekvencie DNA z knižnice.The invention also relates to polynucleotides which essentially consist of a polynucleotide sequence obtainable by hybridization screening of the corresponding library containing the total gene having the sequence of SEQ ID NO. 1, using a hybridization probe having the sequence of SEQ ID NO: 1 or at least a fragment thereof, as well as isolating said DNA sequence from the library.
Polynukleotidové sekvencie podľa vynálezu sú vhodné ako hybridizačné sondy pre RNA, cDNA a DNA, v prípade cDNA pre izolačné sekvencie o celkovej dĺžke (full-length) kódujúce proteín Lrp a pre izoláciu takých cDNA alebo génov, ktoré vykazujú vysokú sekvenčnú homológiu s génom lrp.Polynucleotide sequences of the invention are useful as hybridization probes for RNA, cDNA and DNA, in the case of cDNAs for full-length isolation sequences encoding the Lrp protein, and for isolating such cDNAs or genes that exhibit high sequence homology to the lrp gene.
Polynukleotidové sekvencie podľa vynálezu sú ďalej vhodné ako priméry prípravy DNA z génov kódujúcich proteín Lrp, pomocou polymerázovej reťazovej reakcie (PCR).The polynucleotide sequences of the invention are further suitable as primers for the preparation of DNA from genes encoding the Lrp protein by polymerase chain reaction (PCR).
Oligonukleotídy, ktorú slúžia ako sondy alebo priméry obsahujú aspoň 30, vo výhodnom uskutočnení aspoň 20, v celkom osobitne výhodnom uskutočnení aspoň 15 po sebe idúcich bází. Vhodné sú rovnako oligonukleotídy o dĺžke aspoň 40 alebo 50 párov bází.The oligonucleotides which serve as probes or primers comprise at least 30, preferably at least 20, in a particularly particularly preferred embodiment, at least 15 consecutive bases. Oligonucleotides of at least 40 or 50 base pairs in length are also suitable.
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- 6 V ďaľšom texte sa pod nasledujúcimi termínami chápe :- 6 In the following, the following terms are understood:
Izolovaný značí oddelený od svojho prirodzeného pozadia, respektíve okolia.Isolated means separated from its natural background or surroundings, respectively.
Polynukleotíd sa vzťahuje obecne na polyribonukleotídy a polydeoxyribonukleotídy, pričom môže ísť o nemodifikovanú RNA alebo DNA alebo modifikovanú RNA alebo DNA.A polynucleotide generally refers to polyribonucleotides and polydeoxyribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.
Pod pojmom polypeptíd sa chápe peptid alebo proteín, ktorý obsahuje dve alebo viacej aminokyselín viazaných peptídovými väzbami.A polypeptide is a peptide or protein that contains two or more amino acids linked by peptide bonds.
Polypeptídy podľa vynálezu zahŕňajú polypeptíd podľa Sekvencie id.č. : 2, a ďalej potom polypeptídy s biologickou aktivitou proteínu Lrp a rovnako také, ktoré sú aspoň zo 70 % identické s polypeptídom podľa Sekvencie id.č. : 2, vo výhodnom uskutočnení aspoň z 80 % identické a v najmä výhodnom uskutočnení aspoň z 90 % až 95 % identické s polypeptídom podľa Sekvencie id.č.: 2 a ktoré vykazujú uvedenú aktivitu.Polypeptides of the invention include the polypeptide of SEQ ID NO. 2, and polypeptides having Lrp protein biological activity as well as at least 70% identical to the polypeptide of SEQ ID NO: 2. 2, in a preferred embodiment at least 80% identical and in a particularly preferred embodiment at least 90% to 95% identical to the polypeptide of SEQ ID NO: 2 and which exhibit said activity.
Predmetom vynálezu je ďalej spôsob výroby aminokyselín, najmä L-lyzínu a L-izoleucínu, za použitia koryneformných baktérií, najmä potom takých, ktoré požadované aminokyseliny už produkujú, a v ktorých je nový gén lrp zosilnený alebo zoslabený v závislosti na požadovanej cieľovej substancii.The invention further provides a process for the production of amino acids, in particular L-lysine and L-isoleucine, using coryneform bacteria, in particular those already producing the desired amino acids, in which the new lrp gene is amplified or attenuated depending on the desired target substance.
Výrazom zosilnenie sa v tomto prípade chápe zvýšenie intracelulárnej aktivity jedného alebo niekoľkých enzýmov, ktoré sú v mikroorganizme kódované príslušnými DNA, napríklad tým, že sa zvýši počet kópií génu ( génov) na bunku, použije sa silný promotor, alebo sa použije gén kódujúci enzým so zvýšenou aktivitou, poprípade kombinácia týchto účinkov.Enhancement in this case means an increase in the intracellular activity of one or more enzymes that are encoded in the microorganism by the DNA of interest, for example by increasing the copy number of the gene (s) per cell, using a strong promoter, or using a gene encoding the enzyme. increased activity or a combination of these effects.
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Výrazom zoslabenie” sa v tomto prípade chápe zníženie alebo celkové zastavenie intracelulárnej aktivity jedného alebo niekoľkých enzýmov (proteínov) v mikroorganizme, ktoré sú kódované príslušnými DNA, napríklad tým, že sa použije slabý promotor, alebo sa použije gén poprípade alela, ktorá kóduje príslušný enzým s nižšou aktivitou alebo sa príslušný enzým (proteín) inaktivuje a poprípade kombinácie týchto účinkov.By "attenuation" in this case is meant the reduction or total arrest of the intracellular activity of one or more enzymes (proteins) in a microorganism that are encoded by the DNA of interest, for example by using a weak promoter or by using a gene or allele that encodes the enzyme with lower activity or the respective enzyme (protein) is inactivated and, if appropriate, a combination of these effects.
Mikroorganizmy podľa vynálezu môžu vytvárať L-lyzín z glukózy, sacharózy, laktózy, fruktózy, maltózy, melasy, škrobu, celulózy alebo z glycerínu a etanolu. Môže ísť o zástupcov koryneformných baktérií osobitne z rodu Corynebacterium. Z rodu Corynebacterium je potrebné menovať predovšetkým druh Corynebacterium glutamicum, ktorý je odborníkom známy pre svoju schopnosť produkovať L-aminokyseliny.The microorganisms of the invention may form L-lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerin and ethanol. They may be representatives of coryneform bacteria, in particular of the genus Corynebacterium. Of the genus Corynebacterium, mention should be made in particular of the species Corynebacterium glutamicum, which is known to those skilled in the art for its ability to produce L-amino acids.
Vhodnými kmeňmi baktérií rodu Corynebacterium, najmä druhu Corynebacterium glutamicum, sú napríklad tieto prirodzene sa vyskytujúce kmene :Suitable strains of bacteria of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are, for example, the following naturally occurring strains:
Corynebacterium glutamicum ATCC13032Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870Corynebacterium acetoacidophilum ATCC13870
Corynebacterium melassecola ATCC17965Corynebacterium melassecola ATCC17965
Corynebacterium thermoaminogenes FERM BP-1539Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 aBrevibacterium lactofermentum ATCC13869 a
Brevibacterium divaricatum ATCC14020 a od nich odvodené mutantné kmene produkujúce aminokyseliny, ako napríklad :Brevibacterium divaricatum ATCC14020 and mutant amino acid-producing strains derived therefrom, such as:
kmene produkujúce L-lyzínstrains producing L-lysine
Corynebacterium glutamicum FERM-P 1709 • · · ···· ··· • · ··· · · « t vCorynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712Brevibacterium lactofermentum FERM-P1712
Corynebacterium glutamicum FERM-P 6463Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 aCorynebacterium glutamicum FERM-P 6464 a
Corynebacterium glutamicum DSM5715 alebo kmene produkujúce L-izoleucín :Corynebacterium glutamicum DSM5715 or L-isoleucine-producing strains:
Corynebacterium glutamicum ATCC 14309Corynebacterium glutamicum ATCC 14309
Corynebacterium glutamicum ATCC 14310Corynebacterium glutamicum ATCC 14310
Corynebacterium glutamicum ATCC 14311Corynebacterium glutamicum ATCC 14311
Corynebacterium glutamicum ATCC 15168 aCorynebacterium glutamicum ATCC 15168 a
Corynebacterium ammoniagenes ATCC 6871Corynebacterium ammoniagenes ATCC 6871
Autorom vynálezu sa podarilo izolovať nový gén z baktérie C. glutamicum, ktorý kóduje proteín Lrp.We have succeeded in isolating a novel gene from C. glutamicum, which encodes the Lrp protein.
Na izoláciu tohto lrp-génu respektíve na izoláciu ľubovoľného iného génu z baktérií C. glutamicum je treba najprv vytvoriť génovú knižnicu tohto mikroorganizmu v baktériách E. coli. Vytvorenie takejto knižnice je popísané v obecne známych učebniciach a príručkách. Ako príklad možno uviesť učebnicu Winnacker : Gény a Klony : Úvod do genetických technológií (vydateľstvo Chemie, Weinheim, Nemecko, 1990) alebo príručku Sambrook a ďal’ší Nolecular Cloning : A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989).In order to isolate this lrp gene or to isolate any other gene from C. glutamicum, it is first necessary to create a gene library of the microorganism in E. coli. The creation of such a library is described in commonly known textbooks and manuals. Examples include the Winnacker textbook: Genes and Clones: An Introduction to Genetic Technologies (Chemie, Weinheim, Germany, 1990) or Sambrook and Nolecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989).
Príkladom jednej veľmi známej knižnice je E. coli K-12 kmeňa W3110 v X - vektoroch vytvorená a popísaná Koharom a ďalšími (viď Celí 50, strana 495 až 508 (1987). Bathe a ďaľší popisujú (viď Molecular and General Genetics, 252 : strana 255 až 265, 1996) DNA knižnicu baktérie C. glutamicum ATCC13032, vytvorenú za pomoci kosmidu SuperCos I (Wahl a ďal’ší 1987, Proceeding of the National Academy of Sciences USA, 84 : strana 2160 až 2164) v baktériách E. coli K-12 kmeňa NM554 (viď • · • ··· • · · ··· · · ·· · · · ··· ·· ·· ·· ·· ···· ·· « _ 9 Raleigh a ďalší 1988, Nucleic Acids Research 16 : strana 1563 až 1575). Bormann a ďalší (Molecular Microbiology 6(3), strana 317 až 326)) ďalej popisujú knižnicu baktérií C. glutamicum ATCC13032 vytvorenú za použitia kosmidu pHC79 (Hohn a Collins, Gene 11, strana 291 až 298 (1980)). K príprave DNA knižnice baktérie C. glutamicum v E. coli môžu byť použité tiež plazmidy ako napríklad pBR322 (Bolivar, Life Sciences, 25, strana 807 až 818 (1979)) alebo pUC9 (Norrander a ďaľší 1983, Gene 26 : strana 101 až 106). Ako hostiteľské sú vhodné najmä také kmene E. coli, ktoré sú reštrikčné a rekombinačne deficientné. Príklad takého kmeňa sú bunky kmeňa DHSeČMCR, viď Grant a ďaľší, Proceedings of the National Academy of Sciences USA, 87 (1990) strana 4645 až 4649.An example of one well-known library is the E. coli K-12 strain W3110 in X-vectors generated and described by Kohar et al. (See Cell 50, pp. 495-508 (1987). Bathe et al. (Molecular and General Genetics, 252): pages 255-265, 1996) C. glutamicum ATCC13032 DNA library, generated with the aid of SuperCos I cosmid (Wahl et al. 1987, Proceeding of the National Academy of Sciences, USA, 84: 2160-2164) in E. coli bacteria. K-12 strain NM554 (see 9 Raleigh et al. 1988) Nucleic Acids Research 16: 1563-1575. Bormann et al. (Molecular Microbiology 6 (3), 317-326)) further describe a C. glutamicum ATCC13032 library constructed using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). Plasmids such as pBR322 (Bolivar, Life Sciences, 25: 807-818 (1979)) or pUC9 (Norrander et al. 1983, Gene 26: 101-101) may also be used to prepare a DNA library of C. glutamicum in E. coli. 106). Especially suitable are E. coli strains which are restriction and recombinantly deficient. An example of such a strain is the cells of the DHSeCMCR strain, see Grant et al., Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649.
Dlhé fragmenty DNA klonované pomocou kosmidov môžu byť nasledovne použité pre subklonovanie do vektorov vhodných pre sekvenáciu a inzerty nasledovne sekvenované napríklad spôsobom podľa Sangera a ďaľších (Proceedings of the National Academy of Sciences of the United States of America, 74 : strana 5463 ažLong DNA fragments cloned by cosmids can then be used for subcloning into vectors suitable for sequencing and inserts subsequently sequenced, for example, by the method of Sanger et al. (74: 5463-8).
5467, 1977).5467, 1977).
Týmto spôsobom bola získaná nová sekvencia DNA kódujúca gén lrp z baktérií C. glutamicum. Táto sekvencia je súčasťou vynálezu a je uvedená ako Sekvencia id.č. sl. Ďalej bola od tejto sekvencie DNA vyššie popísanými spôsobmi odvodená sekvencia aminokyselín zodpovedajúceho proteínu. V sekvencii id.č.: 2 podľa vynálezu je uvedená práve sekvencia aminokyselín proteínu LRP.In this way, a new DNA sequence encoding the lrp gene from C. glutamicum was obtained. This sequence is part of the invention and is shown as SEQ ID NO. sl. Furthermore, the amino acid sequence of the corresponding protein was derived from this DNA sequence as described above. The amino acid sequence of the LRP protein is shown in SEQ ID NO: 2 according to the invention.
Kódujúce sekvencie DNA, ktoré v dôsledku degenerácie genetického kódu zodpovedajú Sekvencii id.č.: 1 sú rovnako súčasťou vynálezu. Rovnako sú súčasťou vynálezu sekvencie DNA, ktoré sa Sekvenciou id.č.: 1 hybridizujú. Odborníkom sú ďalej známe konzervatívne zámeny aminokyselín ako je napríklad zámena glycínu za alanín alebo zámena kyseliny aspargovej za kyselinu • · • · ·· ·· • · · • · ··· ·· ·· • · · · • · · ·· ···· • · ·· · glutámovú. Tieto zámeny aminokyselín sa mutations a vzhľadom k tomu, že nevedú aktivity proteínu nazývajú sa rovnako funkčne neutrálne. Ďalej je známe, že zmeny v N- alebo C-konci proteínu nemajú zásadný negatívny vplyv na funkciu proteínu, dokonca v niektorých prípadoch môže dôjst aj k jej stabilizácii.DNA coding sequences which, due to the degeneracy of the genetic code, correspond to SEQ ID NO: 1 are also part of the invention. Also included are DNA sequences which hybridize with SEQ ID NO: 1. Conservative amino acid substitutions, such as glycine to alanine or aspartic acid to acid, are also known to those skilled in the art. Glutamic. These amino acid exchanges are mutations and since they do not conduct protein activities they are also called functionally neutral. Furthermore, it is known that changes in the N- or C-terminus of a protein do not have a significant negative effect on the function of the protein, and in some cases it may also stabilize.
nazývajú sense k zásadnej zmenecalled a sense of profound change
Odborník môže nájsť údaje, ktoré to potvrdzujú okrem iného v práci Ben-Bassata a ďaľších v časopise Journal of Bacteriology 169 : strana 751 až 757 (1987), O' Regana a ďaľších v časopise Gene 77 : strana 237 až 251 (1989), Sahin-Totha a ďaľších v časopise Proteín Sciences 3 : strana 240 až 247 (1994), Hochuliho a ďaľších v Bio/Technology 6 : strana 1321 až 1325 (1988) a v známych učebniciach genetiky a molekulárnej biológie. Aminokyselinové sekvencie, ktoré sú v tomto zmysle analogické Sekvencií id.č.: 2 sú rovnako predmetom vynálezu.One skilled in the art may find data to confirm this, inter alia, in Ben-Bassat et al. In Journal of Bacteriology 169: 751-757 (1987), O'Ranana et al. In Gene 77: 237-251 (1989). Sahin-Totha et al. In Protein Sciences 3: 240-247 (1994), Hochuli et al. In Bio / Technology 6: 1321-1325 (1988) and in well-known textbooks of genetics and molecular biology. Amino acid sequences which are analogous to SEQ ID NO: 2 in this respect are also within the scope of the invention.
Rovnakým spôsobom sú predmetom vynálezu sekvencie DNA, ktoré sa Sekvenciou id.č.: 1 alebo jej časťou hybridizujú.In the same manner, the invention provides DNA sequences which are hybridized with SEQ ID NO: 1 or a portion thereof.
A konečne sú predmetom vynálezu sekvencie DNA amplifikované zo Sekvencie id.č.: možno za pomoci polymerázovej reťazovej reakcie (PCR) a špecifických primérov. Takéto oligonukleotídové priméry majú typickú dĺžku aspoň 15 párov bází.Finally, the invention provides DNA sequences amplified from SEQ ID NO: possibly using polymerase chain reaction (PCR) and specific primers. Such oligonucleotide primers are typically at least 15 base pairs in length.
Návody na identifikáciu DNA za pomoci hybridizácie môžu odborníci nájsť napríklad v príručke The DIG System Users Guide for Filter Hybridization” (Príručka užívateľa systému DIG pre hybridizáciu) od firmy Boehringer Mannheim GmbH (Mannheim, Nemecko, 1993) a ďalej v článku Liebl a ďal’ší (International Journal of Systematic Bacteriology (1991) 41 : strana 255 až 260). Návody na amplifikáciu DNA za pomoci polymerázovej reťazovej reakcie (PCR) nájde odborník napríklad v príručke • · • ·Techniques for identifying DNA using hybridization can be found, for example, in The DIG System Users Guide for Filter Hybridization by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in the article by Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). Instructions for amplifying DNA using polymerase chain reaction (PCR) can be found by one skilled in the art, for example, in the • • • ·
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Gait : Oligonucleotide synthesis : a practical approach (Syntéza oligonukleotidov : praktický prístup, IRL Press,Gait: Oligonucleotide synthesis: a practical approach (IRL Press,
Oxford, Spojené kráľovstvo 1984) a v knihe Newton a Graham: PCR (Akademické vydavateľstvo Spektrum, Heidelberg, Nemecko, 1994).Oxford, United Kingdom 1984) and in Newton and Graham: PCR (Spektrum Academic Publishing, Heidelberg, Germany, 1994).
Pôvodci vynálezu odhalili dôležitosť génu lrp pre produkciu aminokyselín, najmä L-lyzínu a L-izoleucínu. Vynález ďalej popisuje skutočnosť, že dodatočne zvýšená expresia jedného alebo niekolkých enzýmov stávajúcich biosyntetických dráh, glykolýzy, cyklu kyseliny citrónovej, anaplerotickej (doplňovacej) látkovej výmeny alebo exportu aminokyselín, či už jednotlivo alebo v kombinácii, poskytujú ďaľšie výhody pre produkciu aminokyselín, osobitne L-lyzínu a L-izoleucínu.The inventors have discovered the importance of the lrp gene for the production of amino acids, in particular L-lysine and L-isoleucine. The invention further describes the fact that additionally increased expression of one or more enzymes of existing biosynthetic pathways, glycolysis, citric acid cycle, anaplerotic (supplemental) metabolism or amino acid export, either singly or in combination, provide additional benefits for amino acid production, especially L- lysine and L-isoleucine.
Produkcia L-lyzínu môže byť podľa vynálezu zvýšená napríklad takto :According to the invention, L-lysine production can be increased, for example, as follows:
- súčasným zvýšením expresie génu dapA (EP-B 0 197 335) kódujúceho dihydrodipikolinát syntázu, alebo- by simultaneously increasing expression of the dapA gene (EP-B 0 197 335) encoding dihydrodipicolinate synthase, or
- súčasným zvýšením expresie génu gap (Schwinde a ďal’ší, Journal of Bacteriology 175 : strana 3905 až 3908 (1993), kódujúceho glyceraldehyd-3-fosfát dehydrogenázu, alebo- by simultaneously increasing expression of the gap gene (Schwinde et al., Journal of Bacteriology 175: pp. 3905 to 3908 (1993), encoding glyceraldehyde-3-phosphate dehydrogenase, or
- súčasným zvýšením expresie génu mgo (Molenaar a ďalší European Journal of Biochemistry 254, strana 395 až 403 (1998), kódujúceho malát : chinón oxidoreduktázu, alebo- by simultaneously increasing expression of the mgo gene (Molenaar et al., European Journal of Biochemistry 254, pages 395-403 (1998), coding for malate: quinone oxidoreductase, or
- súčasným zvýšením expresie génu pyc (DE-A-19 831 609), kódujúceho pyruvát karboxylázu alebo- by simultaneously increasing expression of the pyc gene (DE-A-19 831 609) encoding pyruvate carboxylase, or
- súčasným zvýšením expresie génu LysE (DE-A-195 48 222), ktorý kóduje proteín podieľajúci sa na exporte lyzínu.by simultaneously increasing expression of the LysE gene (DE-A-195 48 222), which encodes a protein involved in lysine export.
«·· · · · · · · ·· • · ··· · · « · · · ······· ···· · ···· ··· ··· ·· ·· ·· ···· ·· ··· · · · «« «« · · · · · · · · · · · · · · · · · · · · ···· ·· ··
- 12 Produkcia L-izoleucínu môže byť podľa vynálezu zvýšená napríklad takto :The production of L-isoleucine according to the invention can be increased, for example, as follows:
- súčasným zvýšením expresie génu ilvA kódujúceho treoníndehydratázu (Mockel a ďalší Journal of Bacteriology (1992) strana 8065 až 8072)) alebo zvýšením expresie génu ilvA (Fbr), ktorý kóduje treonin dehydratázu odolnú proti spätneväzobnej regulácii (Mockel a ďalší (1994), Molecular Microbiology 13 : strana 833 až 842), aleboby simultaneously increasing expression of the ilvA gene encoding threonine dehydratase (Mockel et al. Journal of Bacteriology (1992) pages 8065-8072)) or by increasing expression of the ilvA gene (Fbr), which encodes feedback-resistant threonine dehydratase (Mockel et al. (1994), Molecular Microbiology 13: 833-842), or
- súčasným zvýšením expresie génu gap (Schwinde a ďalší (Journal of Bacteriology 175 : strana 3905 až 3908 (1993)), kódujúceho glyceraldehyd-3-fosfát dehydrogenázu alebo- by simultaneously increasing the expression of the gap gene (Schwinde et al. (Journal of Bacteriology 175: 3905 to 3908 (1993)), encoding glyceraldehyde-3-phosphate dehydrogenase, or
- súčasným zvýšením expresie génu mgo (Molenaar a ďalší (European Journal of Biochemistry 254, strana 395 až 403 (1988)), kódujúceho malát : chinón oxidoreduktázu, alebo- by simultaneously increasing expression of the mgo gene (Molenaar et al. (European Journal of Biochemistry 254, pages 395-403 (1988)), coding for malate: quinone oxidoreductase, or
- súčasným zvýšením expresie génu pyc (DE-A-19 831 609), kódujúceho pyruvát karboxylázu.by simultaneously increasing expression of the pyc gene (DE-A-19 831 609) encoding pyruvate carboxylase.
Ďalej môže byť výhodné pre produkciu aminokyselín podľa vynálezu, najmä potom pre produkciu L-lyzínu a L-izoleucínu, inhibovať nežiadúce vedľajšie reakcie (viď Nakayama : Breeding of amino Acid Producing Micro-organisms (Pestovanie mikroorganizmov produkujúcich aminokyseliny) v knihe : Overproduction of Microbial Products, (Produkcia mikrobiálnych produktov) editorov Krumphanzla, Sikyty a Vanka, Academic Press, Londýn, Spojené kráľovstvo, 1982.Furthermore, it may be advantageous for the production of amino acids of the invention, particularly for the production of L-lysine and L-isoleucine, to inhibit unwanted side reactions (see Nakayama: Breeding of Amino Acid Producing Micro-organisms) in the book: Overproduction of Microbial Products, (Producing Microbial Products) by Editors Krumphanzl, Sikyty and Vanka, Academic Press, London, United Kingdom, 1982.
Mikroorganizmy skonštruované podľa vynálezu môžu byť za účelom produkcie aminokyselín, najmä potom L-lyzínu a L-izoleucínu kultivované kontinuálnym spôsobom, alebo spôsobmi diskontinuálnymi, či už vsádkovou kultiváciou alebo prietokovou kultiváciou.The microorganisms constructed according to the invention can be cultured in a continuous manner or batchwise, either batch or flow-through, to produce amino acids, in particular L-lysine and L-isoleucine.
- 13 • · · ···· ···· • · ··· · · · · · · • · · · · · · · ·· · · • · · · · · · ··· ·· ·· ·· ···« ·· ···- 13 · · 13 13 · 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 ·· ··· «·· ···
Prehľad známych spôsobov kultivácie Chmiel : Bioprozesstechnik 1.Overview of known Chmiel cultivation methods: Bioprozesstechnik 1.
Bioverfahrenstechnik (Úvod do Fischer, Stuttgart, 1991)Bioverfahrenstechnik (Introduction to Fischer, Stuttgart, 1991)
Bioreaktoren und Periphere periférne zariadenie, vydavateľstvo Braunschweig/Wiesbaden, 1994).Bioreaktoren und Periphere Peripherals, Braunschweig Publishing House / Wiesbaden, 1994).
je uvedený v učebnici Einfuhrung in die biotechník, vydavateľstvo Gustáv alebo v učebnici Storhas :is given in the Einfuhrung in die biotechnik, Gustáv publishing house or in the Storhas textbook:
Einrichtungen (Bioreaktory aEinrichtungen (Bioreactors and
Vieweg Verlag,Vieweg Verlag,
Použité kultivačné médium musí patričným spôsobom vyhovovať nárokom príslušného mikroorganizmu. Popisy rôznych známych kultivačných médií pre mikroorganizmy sú uvedené v príručke Manual of Methods for General Bacteriology (Metodický manuál pre obecnú bakteriológiu) vydaný Americkou bakteriologickou spoločnosťou (American Society for Bacteriology), Washington D.C., USA, v roku 1981. Ako zdroje uhlíku môžu byť použité cukry a uhľohydráty ako napríklad glukóza, sacharóza, laktóza, fruktóza, maltóza, melasa, škrob a celulóza; oleje a tuky ako napríklad sójový olej, slnečnicový olej, arašídový olej a kokosový tuk; mastné kyseliny ako napríklad kyselina palmitová, kyselina steárová a kyselina linolová, alkoholy ako napríklad glycerín a etanol a organické kyseliny ako napríklad kyselina octová. Tieto látky môžu byť použité buď jednotlivo alebo v zmesi. Ako zdroje dusíku môžu byť použité zlúčeniny obsahujúce organický dusík ako napríklad peptón, extrakt z droždia, extrakt z mäsa, extrakt zo sladu, kukuričný extrakt, sójová múka a močovina alebo anorganické zlúčeniny ako napríklad síran amonný, chlorid amonný, fosforečnan amonný, uhličitan amonný a dusičnan amonný. Tieto zdroje dusíku môžu byť použité buď jednotlivo alebo v zmesi. Ako zdroje fosforu možno použiť hydrogénfosforečnan alebo dihydrogénfosforečnan draselný či zodpovedajúce sodné soli.The culture medium used must suitably meet the requirements of the microorganism concerned. Descriptions of various known culture media for microorganisms are given in the Manual of Methods for General Bacteriology published by the American Society for Bacteriology, Washington DC, USA, 1981. Carbon sources may be used. sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose; oils and fats such as soybean oil, sunflower oil, peanut oil and coconut fat; fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerin and ethanol, and organic acids such as acetic acid. These may be used either singly or as a mixture. As nitrogen sources, organic nitrogen containing compounds such as peptone, yeast extract, meat extract, malt extract, corn extract, soy flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium carbonate can be used. ammonium nitrate. These nitrogen sources can be used either singly or in a mixture. Potassium phosphate or potassium dihydrogen phosphate or the corresponding sodium salts can be used as phosphorus sources.
Kultivačné médium musí ďalej obsahovať soli kovov, ktoré sú nevyhnutné pre rast, ako napríklad síran horečnatý alebo síran železnatý. A konečne môže byť kultivačné médium okrem vyššie uvedených látok obohatené o esenciálne rastové látky ako ·· • · · • · ··· • · · • · · ·· · · ·· ·· ·· • · · · · · · • · · · a • · · · · ·· ···· ·· ·The culture medium must also contain metal salts which are necessary for growth, such as magnesium sulfate or ferrous sulfate. Finally, in addition to the above-mentioned substances, the culture medium can be enriched with essential growth substances such as: · · · And · · · · ··········
- 14 napríklad aminokyseliny a vitamíny. Pre zvýšenie tvorby aminokyselín možno kultivačné médium ešte obohatit o jej prekurzory. Uvedené zložky môžu byt pridané do média jednorázovo na začiatku, alebo vhodným spôsobom v priebehu kultivácie.For example amino acids and vitamins. To increase amino acid production, the culture medium can be enriched with precursors. Said components may be added to the medium at one time at the beginning or in a suitable manner during the cultivation.
Pre kontrolu pH v priebehu kultivácie sú vhodné zásadité zlúčeniny ako napríklad hydroxid sodný, hydroxid draselný, amonika alebo kyselé zlúčeniny ako napríklad kyselina fosforečná alebo kyselina sírová. Penenie kultivačnej zmesi možno kontrolovať prídavkom činidiel potlačujúcich tvorbu peny ako sú napríklad polyglykolestery mastných kyselín. Stabilitu plazmidov možno udržať prostredníctvom vhodného selekčného činidla napríklad antibiotika. Vhodné aerobné podmienky možno udržovať privádzaním kyslíku alebo kyslík - obsahujúce zmesi plynov, napríklad vzduchu, do kultivačného média. Vhodná teplota na pestovanie baktérií sa normálne pohybuje od 20° do 50° C a výhodne od 25° C do 45° C. Doba kultivácie by mala byť taká, aby bolo dosiahnuté maximum tvorby aminokyselín. Toto maximum je zvyčajne dosiahnuté počas 10 až 160 hodín.Basic compounds such as sodium hydroxide, potassium hydroxide, ammonium or acidic compounds such as phosphoric acid or sulfuric acid are suitable for controlling the pH during cultivation. The foaming of the culture mixture can be controlled by the addition of suds suppressors such as polyglycol esters of fatty acids. Stability of the plasmids can be maintained by a suitable selection agent, for example, an antibiotic. Suitable aerobic conditions can be maintained by introducing oxygen or oxygen-containing gas mixtures, e.g. air, into the culture medium. A suitable temperature for the cultivation of bacteria is normally from 20 ° to 50 ° C and preferably from 25 ° C to 45 ° C. The cultivation time should be such that the maximum amino acid formation is achieved. This maximum is usually achieved within 10 to 160 hours.
Analýza produkovaných aminokyselín sa môže uskutočňovať za pomoci iontomeničovej chromatografie na anexu s nasledovnou derivatizáciou za pomoci ninhydrínu, tak ako je popísané v práci Spackmana a ďalších (Analytical Chemistry, 30, (1958), strana 1190), alebo sa môže uskutočňovať za pomoci HPLC s reverznou fázou, viď Lindroth a ďaľší. (Analytical Chemistry (1979) 51 : strana 1167 až 1174).Analysis of the amino acids produced can be performed by anion exchange chromatography followed by derivatization with ninhydrin, as described by Spackman et al. (Analytical Chemistry, 30, (1958), page 1190), or can be performed by HPLC using reverse phase, see Lindroth and others. (Analytical Chemistry (1979) 51: 1167-1174).
Spôsoby podľa vynálezu slúžia k ferementatívnej produkcii aminokyselín, osobitne L-lyzínu a L-izoleucínu.The methods according to the invention serve for the fermentative production of amino acids, in particular L-lysine and L-isoleucine.
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Vynález bude bližšie popísaný v nasledujúcich príkladoch.The invention will be described in more detail in the following examples.
·· ·· • · · • · ··· • · · · • · · · ·· ·· ·· ·· • · · · • · · • · · ·· ···· • · · ·· ·················································· · ·
Príklad 1 :Example 1:
Príprava kosmidovej génomovej knižnice baktérie Corynebacterium glutamicum ATCC 13032Preparation of Corynebacterium glutamicum cosmid genomic library ATCC 13032
Chromozomálna DNA baktérie Corynebacterium glutamicum ATCC 13032 bola najprv izolovaná a čiastočne naštiepená (viď Tauch a ďalší (1995), Plasmid 33 : strana 168 až 179) reštrikčným enzýmom Sau3AI (Amersham Pharmacia, Freiburg, Nemecko, katalógové č. 27-0913-02). Izolované DNA fragmenty boli defosforylované alkalickou fosfatázou z morských garnátov (shrimp alkaline phosphatase, Roche Molecular Biochemicals, Mannheim, Nemecko, katalógové č. 1758250). DNA kosmidového vektoru SuperCos I (viď Wahl a ďalší, (1987) Proceedings of the National Academy of Sciences USA 84 :strana 2160 až 2164) získaného od firmy Stratagene (La Jolla, USA, SuperCos I Cosmid Vector Kit, katalógové č. 251301) bola naštiepená reštrikčným enzýmom Xbal (Amersham Pharmacia, Freiburg, Nemecko, katalógové č. 27-0948-02) a rovnako defosforylovaná alkalickou fosfatázou. Potom bola kosmidová DNA naštiepená reštrikčným enzýmom BamHI Pharmacia, Freiburg, Nemecko, katalógové č. Takto získané fragmenty kosmidovej DNA boli zmiešané s fragmentárni génomovej DNA Corynebacterium glutamicum ATCC13032 a potom spojené pomocou T4-DNA-ligázy (Amersham Pharmacia, Freiburg, Nemecko, katalógové č. 27-0870-04).The chromosomal DNA of Corynebacterium glutamicum ATCC 13032 was first isolated and partially digested (see Tauch et al. (1995), Plasmid 33: 168-179) with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Catalog No. 27-0913-02). . Isolated DNA fragments were dephosphorylated with shrimp alkaline phosphatase, Roche Molecular Biochemicals, Mannheim, Germany, Catalog No. 1758250. SuperCos I cosmid vector DNA (see Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164) obtained from Stratagene (La Jolla, USA, SuperCos I Cosmid Vector Kit, Catalog No. 251301) was digested with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Catalog No. 27-0948-02) and also dephosphorylated with alkaline phosphatase. The cosmid DNA was then digested with the restriction enzyme BamHI Pharmacia, Freiburg, Germany, catalog no. The cosmid DNA fragments thus obtained were mixed with fragmentary genomic DNA of Corynebacterium glutamicum ATCC13032 and then ligated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Catalog No. 27-0870-04).
(Amersham 27-086804).(Amersham 27-086804).
Ligačná zmes bola potom zabalená do fágových častíc za pomoci pakážovacieho extraktu Gigapack II XL (Stratagene, La Jolla, USA, Gigapack II XL Packing Extract, katalógové č. 200217). Tieto fágové častice boli použité pre infekciu E. coli kmeňa NM554 (viď Raleigh a ďalší, 1988, Nucleic Acid Res. 16 : strana 1563 až 1575). Bunky boli resuspendované v 10 mM MgSO^ a zmiešané s alikvótom fágovej suspenzie. Infekcia a titrácia kosmidovej knižnice bola uskutočnená ako v knihe Sambrook a ďalší (1989), Molecular Cloning : A laboratory Manual, Cold Spring Harbor), pričom bunky boli vysiate na LB-agar (viď ·· ·· ·· ·» • · · · · · · • · ··· · · · • · · · · · · · · ·· ·· ·· ···· ·· ·· ·The ligation mixture was then packaged in phage particles using Gigapack II XL packaging extract (Stratagene, La Jolla, USA, Gigapack II XL Packing Extract, Catalog No. 200217). These phage particles were used to infect E. coli strain NM554 (see Raleigh et al., 1988, Nucleic Acid Res. 16: 1563-1575). Cells were resuspended in 10 mM MgSO 4 and mixed with an aliquot of phage suspension. Infection and titration of the cosmid library were performed as in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), and cells were seeded on LB-agar (see. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·
Lennox, 1955, Virology, 1 : strana 190) s prídavkom 100 ;ug/ml ampicilínu. Po inkubácii cez noc pri teplote 37° C boli vybraté jednotlivé rekombinantné klony.Lennox, 1955, Virology, 1: 190) with the addition of 100 µg / ml ampicillin. After incubation overnight at 37 ° C, individual recombinant clones were selected.
Príklad 2 :Example 2:
Izolácia a sekvenovanie génu lrpIsolation and sequencing of the lrp gene
Najprv bola vyizolovaná kosmidová DNA jednej dobre oddelenej kolónie za pomoci Qiaprep Spin Miniprep Kitu (kat.č. 27106, Qiagen, Hilden, Nemecko) podľa inštrukcií výrobcu a tá bola potom čiastočne (Amersham Pharmacia, 270913-02). Získané reštrikčným enzýmom Sau3AI Nemecko, katalógové č.First, cosmid DNA of one well separated colony was isolated using Qiaprep Spin Miniprep Kit (Cat. No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and then partially (Amersham Pharmacia, 270913-02). Obtained by the restriction enzyme Sau3AI Germany, catalog no.
DNA boli defosforylované naštiepená Freiburg, fragmenty alkalickou fosfatázou (Roche Molecular Biochemicals, Mannheim, Nemecko, katalógové č. 1758250). Po elektroforetickej separácii nasledovala izolácia fragmentov kosmidu o veľkosti medzi 1500 a 2000 bp za pomoci kitu QiaExII Gel Extraction Kit (katalógové č. 20021, Qiagen, Hilden, Germany).The DNAs were dephosphorylated digested with Freiburg, alkaline phosphatase fragments (Roche Molecular Biochemicals, Mannheim, Germany, Catalog No. 1758250). Electrophoretic separation was followed by isolation of cosmid fragments between 1500 and 2000 bp in size using the QiaExII Gel Extraction Kit (Catalog No. 20021, Qiagen, Hilden, Germany).
Potom bol reštrikčným enzýmom BamHI (Amersham Pharmacia, Freiburg, Nemecko, katalógové č. : 27-0868-04) naštiepený sekvenačný vektor pZero-1 od firmy Invitrogen (Groningen, Holandsko, katalógové č. K2500-01). Ligácia fragmentov kosmidu do sekvenačného vektoru pZero-1 bola uskutočnená podľa príručky Sambrook a ďaľší (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), pričom bola zmes DNA s T4-ligázou (Pharmacia Biotech, Freiburg, Nemecko) inkubovaná cez noc. Ligačnou zmesou boli potom elektroporáciou (Tauch a ďaľší 1994, FEMS Microbiol Letters, 123 : strana 343 až 347) transformované baktérie E. coli kmeňa DH5o(MCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87 : strana 4645 až 4649). Transformované baktérie boli vysiate a LB-agar (Lennox, 1955, Virology, 1 : strana 190) s prídavkom 50 jug/ml zeocínu. Izolácia plazmidov z rekombinantných klonov bola uskutočnená za pomoci zariadenia Biorobot 9600 (katalógové číslo 900200,The restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, catalog # 27-0868-04) was then digested with the pZero-1 sequencing vector from Invitrogen (Groningen, The Netherlands, catalog # K2500-01). Ligation of the cosmid fragments into the pZero-1 sequencing vector was performed according to Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), incubating the DNA with T4-ligase DNA (Pharmacia Biotech, Freiburg, Germany) overnight. . The ligation mixture was then transformed with E. coli strain DH 50 (MCR, Grant, 1990, Proceedings of the National Academy of Sciences, USA, 87: 4645-45) by electroporation (Tauch et al. 1994, FEMS Microbiol Letters, 123: 343-347). The transformed bacteria were seeded and LB-agar (Lennox, 1955, Virology, 1: 190) with the addition of 50 µg / ml zeocin The isolation of plasmids from recombinant clones was performed using a Biorobot 9600 (catalog number 900200,
Qiagen, Hilden, Nemecko). Dideoxy-terminačné sekvenovanie plazmidov (Sanger a ďalší, 1977, Proceeding of the National Academies of Sciences U.S.A., 74 : strana 5463 až 5467) s modifikáciami podľa Zimmermanna a ďaľších (1990, Nucleic Acids Research, 18 : strana 1067) bolo uskutočnené s využitím sekvenačného kitu RR dRhodamin Terminátor Cycle Sequencing Kit od firmy PE Applied Biosystems (katalógové číslo 403044, Weiterstadt, Nemecko). Elektroforetická separácia a analýza sekvenačnej reakcie bola uskutočnená na gélu Rotiphorese NF Acrylamid/Bisacrylamid (29 : 1) (katalógové číslo A124.1, Roth, Karlsruhe, Nemecko) v prístroji ABI Prism 377 od firmy PE Applied Biosystems (Weiterstadt, Nemecko).Qiagen, Hilden, Germany). Dideoxy-terminating sequencing of plasmids (Sanger et al., 1977, Proceeding of the National Academic Sciences of the United States, 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067) was performed using RR dRhodamin Cycle Sequencing Kit from PE Applied Biosystems (Catalog No. 403044, Weiterstadt, Germany). Electrophoretic separation and sequencing reaction analysis was performed on a Rotiphorese NF Acrylamide / Bisacrylamide (29: 1) gel (catalog number A124.1, Roth, Karlsruhe, Germany) in an ABI Prism 377 from PE Applied Biosystems (Weiterstadt, Germany).
Získané hrubé sekvenačné dáta boli spracované za pomoci programového balíku Staden (1986, Nucleic Acids Research, 14 : strana 217 až 231) verzia 97-0. Jednotlivé sekvencie derivátov plazmidu pZerol boli zostavené do súvislého kontigu. Počítačová analýza kódujúcich oblastí bola uskutočnená programom XNIP (Staden, 1986, Nucleic Acids Research, 14 : strana 217 až 231). Ďaľšie analýzy boli uskutočnené programom BLAST (Altschul a ďaľší 1997, Nucleic Acids Research, 25 : strana 3389 až 3402), proti ne-redundantnej databázi NCBI uloženej v Národnej lekárskej knižnici USA (National Library of Medicíne).The raw sequencing data obtained was processed using the Staden (1986, Nucleic Acids Research, 14: 217-231) software package version 97-0. The individual sequences of the derivatives of plasmid pZerol were assembled into a contiguous contig. Computer analysis of the coding regions was performed by the XNIP program (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analyzes were performed with the BLAST program (Altschul et al. 1997, Nucleic Acids Research, 25: 3389-3402), against a non-redundant NCBI database stored in the National Library of Medicine.
Takto bola získaná nukleotídová sekvencia, ktorá je uvedená vo vynáleze ako Sekvencia id.č. : 1. Analýza tejto nukleotídovej sekvencie odhalila jeden otvorený čítací rámec o velkosti 462 párov bází, ktorý bol označený ako gén lrp. Tento gén kóduje polypeptid o veľkosti 154 aminokyselín.In this way, the nucleotide sequence shown in SEQ ID NO. 1. Analysis of this nucleotide sequence revealed one 462 base pair open reading frame, which was designated as the lrp gene. This gene encodes a 154 amino acid polypeptide.
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