SK14582000A3 - Nucleotide sequences codig for the eno gene - Google Patents
Nucleotide sequences codig for the eno gene Download PDFInfo
- Publication number
- SK14582000A3 SK14582000A3 SK1458-2000A SK14582000A SK14582000A3 SK 14582000 A3 SK14582000 A3 SK 14582000A3 SK 14582000 A SK14582000 A SK 14582000A SK 14582000 A3 SK14582000 A3 SK 14582000A3
- Authority
- SK
- Slovakia
- Prior art keywords
- polynucleotide
- sequence
- gene
- amino acid
- lysine
- Prior art date
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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Abstract
Description
Nové- Nukleotidové sekvencie kódujúce gén enoNovel- Nucleotide sequences encoding the eno gene
Oblasť technikyTechnical field
Predmetom vynálezu sú nukleotidové sekvencie kódujúce gén eno a spôsob fermentačnej výroby aminokyselín, najmä L-lyzínu, použitím koryneformných baktérií, v ktorých sa zosilňuje gén eno.The present invention provides nucleotide sequences encoding the eno gene and a method for fermentative production of amino acids, particularly L-lysine, using coryneform bacteria in which the eno gene is amplified.
Doterajší stav technikyBACKGROUND OF THE INVENTION
Aminokyseliny, najmä L-lyzín, sa používajú v humánnej medicíne a vo farmaceutickom priemysle, ale najmä vo výžive zvierat.Amino acids, especially L-lysine, are used in human medicine and in the pharmaceutical industry, but especially in animal nutrition.
Je známe, že aminokyseliny sa vyrábajú fermentáciou kmeňov koryneformných baktérií, najmä Corynebacterium glutamicum. Kvôli velkému významu sa stále pracuje na zlepšení spôsobov výroby. Zlepšenia spôsobov sa môžu dokonca týkať fermentačno-technologických opatrení, ako napríklad miešania a zásobovania kyslíkom, alebo zloženia živných médií, ako napríklad koncentrácie cukru počas fermentácie, alebo spracovania na produktovú formu, napríklad ionexovou chromatografiou, alebo vlastných úžitkových vlastností samotného mikroorganizmu.It is known that amino acids are produced by fermenting strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great importance, improvements are still being made to the production methods. Process improvements may even relate to fermentation technology measures such as mixing and oxygen supply, or nutrient composition such as sugar concentration during fermentation, or processing into a product form, for example by ion exchange chromatography, or the intrinsic utility of the microorganism itself.
Na zlepšenie úžitkových vlastností týchto mikroorganizmov sa používajú metódy mutagenézy, selekcie a volby mutantov. Týmto spôsobom sa získajú kmene, ktoré sú rezistentné voči antimetabolitom, ako je napríklad analogón lyzínu S-(2·· ·«·· • · · · · · · ···· · ·· ···· ··Mutagenesis, selection and mutant selection methods are used to improve the utility properties of these microorganisms. In this way, strains that are resistant to antimetabolites, such as the lysine analogue S- (2), are obtained.
-aminoetyl)cystein, alebo sú auxotrofné vzhladom na regulačné významné aminokyseliny a produkujú L-lyzin.-aminoethyl) cysteine, or are auxotrophic with respect to regulatory significant amino acids and produce L-lysine.
Už niekoľko rokov sa taktiež používajú metódy technológie rekombinantných DNA na zlepšenie kmeňov Corynebacteri um produkujúcich aminokyseliny tak, že jednotlivé gény pre biosyntézu aminokyselín sa amplifikujú a skúma sa účinok na produkciu aminokyselín. Prehľadný článok k tomu sa nachádza medzi iným v Kinoshita („Glutamic Acid Bacteria, v: Biology of Industrial Microorganisms, Demain and Solomon (vyd.),Recombinant DNA technology techniques have also been used for several years to improve amino acid producing Corynebacteri um strains by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production. A review of this can be found, inter alia, in Kinoshita (Glutamic Acid Bacteria, in: Biology of Industrial Microorganisms, Demain and Solomon (Ed.),
Benjamín Cummings, Londýn, Veľká Británia, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991), Eggeling (Amino Acids 6:261-272 (1994)), Jetten a Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) a Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).Benjamin Cummings, London, United Kingdom, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991), Eggeling (Amino Acids 6: 261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15 , 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
Vynálezcovia si stanovili za úlohu poskytnúť nové opatrenia na zlepšenú fermentačnú výrobu aminokyselín, najmä L-lyzínu.The inventors have set out to provide new measures for improved fermentative production of amino acids, in particular L-lysine.
Aminokyseliny, najmä L-lyzín, sa používajú v humánnej medicíne, vo farmaceutickom priemysle a najmä vo výžive zvierat. Existuje preto všeobecný záujem o poskytnutie nových, zlepšených spôsobov výroby aminokyselín, najmä L-lyzínu.Amino acids, especially L-lysine, are used in human medicine, in the pharmaceutical industry and in particular in animal nutrition. There is therefore a general interest in providing new, improved methods for producing amino acids, particularly L-lysine.
Keď sa v nasledovnom texte uvedie L-lyzín alebo lyzín, mieni sa tým nielen zásada, ale aj soli, ako napríklad monohydrochlorid lyzínu alebo sulfát lyzínu.When L-lysine or lysine is mentioned in the following, it is meant not only the base but also salts such as lysine monohydrochloride or lysine sulfate.
Podstata vynálezu • · ···· • · · · · · · ··· · ······ ··The essence of the invention · ···· · · · · · · ··· · ·······
Predmetom vynálezu je izolovaný polynukleotid z koryneformných baktérií, obsahujúci polynukleotidovú sekvenciu vybranú zo skupiny zahŕňajúcejThe present invention provides an isolated polynucleotide from coryneform bacteria comprising a polynucleotide sequence selected from the group consisting of:
a) polynukleotid, ktorý je aspoň na 70 % identický s polynukleotidom, ktorý kóduje polypeptid, ktorý obsahuje aminokyselinovú sekvenciu SEQ ID No. 2,a) a polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO. 2
b) polynukleotid, ktorý kóduje polypeptid, ktorý obsahuje amínokyselinovú sekvenciu, ktorá je aspoň na 70 % identická s aminokyselinovou sekvenciou SEQ ID No. 2,b) a polynucleotide that encodes a polypeptide that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO. 2
c) polynukleotid, ktorý je komplementárny k polynukleotidom a) alebo b), ac) a polynucleotide that is complementary to polynucleotides a) or b), and
d) polynukleotid obsahujúci aspoň 15 za sebou nasledujúcich báz polynukleotidovej sekvencie a), b) alebo c).d) a polynucleotide comprising at least 15 consecutive bases of the polynucleotide sequence a), b) or c).
Predmetom vynálezu je aj polynukleotid podľa nároku 1, pričom sa prednostne jedná o replikovateľnú DNA obsahujúcu:The present invention also provides a polynucleotide according to claim 1, wherein the polynucleotide is preferably a replicable DNA comprising:
(i) nukleotidovú sekvenciu znázornenú v SEQ-ID-No. 1, alebo (ii) aspoň jednu sekvenciu, ktorá zodpovedá sekvencií (i) v oblasti degenerácie genetického kódu, alebo (iii) aspoň jednu sekvenciu, ktorá sa hybridizuje so sekvenciou komplementárnou k sekvencií (i) alebo (ii), a poprípade (iv) funkčne neutrálne mutácie so zmyslom v (i).(i) the nucleotide sequence shown in SEQ-ID-No. Or (ii) at least one sequence that corresponds to sequence (i) in the region of degeneracy of the genetic code, or (iii) at least one sequence that hybridizes to a sequence complementary to sequence (i) or (ii), and optionally (iv) functionally neutral mutations with sense in (i).
·· ···· ·· • · • · · · · · · ···· · ·· ···· ·· ·································
Ďalším predmetom je polynukleotid podľa nároku 4 obsahujúci nukleotidovú sekvenciu znázornenú v SEQ ID No. 1, polynukleotid podľa nároku 6, ktorý kóduje polypeptid, ktorý obsahuje aminokyselinovú sekvenciu znázornenú v SEQ ID No.Another object is a polynucleotide according to claim 4 comprising the nucleotide sequence shown in SEQ ID NO. 1, the polynucleotide of claim 6, which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO.
2, vektor obsahujúci polypeptid podľa nároku 1, najmä kyvadlový vektor alebo plazmidový vektor a koryneformné baktérie slúžiace ako hostiteľské bunky, ktoré obsahujú tento vektor.2, a vector comprising the polypeptide of claim 1, in particular a shuttle vector or plasmid vector, and coryneform bacteria serving as host cells comprising the vector.
Predmetom vynálezu sú aj polynukleotidy, ktoré sa skladajú v podstate z polynukleotidovej sekvencie, ktoré možno získať skríningom prostredníctvom hybridizácie príslušnej génovej banky, ktorá obsahuje úplný gén s polynukleotidovou sekvenciou zodpovedajúcou SEQ ID No. 1, pomocou sondy, ktorá obsahuje sekvenciu uvedeného polynukleotidu podía SEQ ID No.The present invention also provides polynucleotides consisting essentially of a polynucleotide sequence obtainable by screening by hybridization of an appropriate gene bank containing the complete gene with a polynucleotide sequence corresponding to SEQ ID NO. 1, using a probe comprising the sequence of said polynucleotide of SEQ ID NO.
alebo jej fragment, a izoláciou uvedenej sekvencie DNA.or a fragment thereof, and isolating said DNA sequence.
Polynukleotidové sekvencie podľa vynálezu sú vhodné ako hybridizačné sondy pre RNA, cDNA a DNA, aby sa izolovali cDNA s celou dĺžkou, ktoré kódujú enolázu, a aby sa izolovali také cDNA alebo gény, ktoré vykazujú veľkú podobnosť sekvencie s génom pre enolázu.The polynucleotide sequences of the invention are useful as hybridization probes for RNA, cDNA and DNA to isolate full length cDNAs that encode enolase and to isolate such cDNAs or genes that exhibit great sequence similarity to the enolase gene.
Polynukleotidové sekvencie podľa vynálezu sú ďalej vhodné ako priméry na produkciu DNA génov, ktoré kódujú enolázu, polymerázovou reťazovou reakciou (PCR).The polynucleotide sequences of the invention are further suitable as primers for the production of DNA genes that encode enolase by a polymerase chain reaction (PCR).
·· ···· • · · · · · · ···· · ·· ···· ·· ······································
Také oligonukleotidy slúžiace ako sondy alebo priméry obsahujú aspoň 30, prednostne aspoň 20, celkom obzvlášť prednostne aspoň 15 za sebou nasledujúcich báz. Vhodné sú taktiež oligonukleotidy s dĺžkou aspoň 40 alebo 50 párov báz.Such oligonucleotides serving as probes or primers comprise at least 30, preferably at least 20, very particularly preferably at least 15 consecutive bases. Oligonucleotides of at least 40 or 50 base pairs in length are also suitable.
„Izolovaný znamená oddelený zo svojho prirodzeného prostredia.“Isolated means separated from its natural environment.
„Polynukleotid sa vzťahuje všeobecne na polyribonukleotidy a polydeoxyribonukleotidy, pričom sa môže jednať o nemodifikované RNA alebo DNA alebo modifikované 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 „polypeptidy sa rozumejú peptidy alebo proteíny, ktoré obsahujú dve alebo viac aminokyselín viazaných peptidovými väzbami.The term "polypeptides" refers to peptides or proteins that contain two or more amino acids linked by peptide bonds.
Polypeptidy podľa vynálezu zahŕňajú polypeptid podlá SEQ ID No. 2, najmä s biologickou aktivitou enolázy a aj také, ktoré sú aspoň na 70 % identické s polypeptidom podľa SEQ ID No. 2, prednostne aspoň na 80 % a obzvlášť také, ktoré vykazujú identitu s polypeptidom podlá SEQ ID No. 2 aspoň na 90 % až 95 % a uvedenú aktivitu.Polypeptides of the invention include the polypeptide of SEQ ID NO. 2, in particular with the biological activity of enolase, as well as those which are at least 70% identical to the polypeptide of SEQ ID NO. 2, preferably at least 80%, and particularly those that exhibit identity to the polypeptide of SEQ ID NO. 2 to at least 90% to 95% and said activity.
Vynález sa ďalej týka spôsobu fermentačnej výroby aminokyselín, najmä lyzínu, použitím koryneformných baktérií, ktoré najmä už produkujú aminokyseliny a v ktorých sa zosilňujú, najmä nadmerne exprimujú nukleotidové sekvencie kódujúce gén eno.The invention further relates to a method for the fermentative production of amino acids, in particular lysine, using coryneform bacteria which in particular already produce amino acids and in which they amplify, in particular overexpress, the nucleotide sequences encoding the eno gene.
Pojem „zosilnenie opisuje v tejto súvislosti zvýšenie intracelulárnej aktivity jedného alebo viacerých enzýmov v • · • · · · · · · ···· · ·· ···· ·· · mikroorganizme, ktoré sú kódované príslušnou DNA, tým, že sa napríklad zvýši počet kópií génu, poprípade génov, použije sa silný promótor alebo sa použije gén, ktorý kóduje príslušný enzým s vysokou aktivitou a tieto opatrenia sa poprípade kombinujú.The term "enhancer" in this context describes the enhancement of the intracellular activity of one or more enzymes in a microorganism that is encoded by the DNA of interest by for example, it will increase the copy number of the gene or genes, use a strong promoter, or use a gene that encodes a particular high-activity enzyme and, if necessary, combine these measures.
Mikroorganizmy, ktoré sú predmetom predloženého vynálezu, môžu produkovať L-aminokyseliny, najmä lyzín, z glukózy, sacharózy, laktózy, fruktózy, maltózy, melasy, škrobu, celulózy alebo z glycerolu a etanolu. Môže sa jednať o zástupcov koryneformných baktérií, najmä rodu Corynebacterium. Pri rode Corynebacterium treba uviesť najmä druh Corynebacterium glutamicum, ktorý je v odbornom svete známy pre svoju schopnosť produkovať L-aminokyseliny.The microorganisms of the present invention can produce L-amino acids, in particular lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may be representatives of coryneform bacteria, in particular of the genus Corynebacterium. In the genus Corynebacterium, mention should be made in particular of the species Corynebacterium glutamicum, which is known in the art for its ability to produce L-amino acids.
Vhodnými kmeňmi rodu Corynebacterium, najmä druhu Corynebacterium glutamicum, sú napríklad známe kmene divého typuSuitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are for example known wild-type strains.
Corynebacterium Corynebacterium Corynebacteri um Corynebacterium Corynebacterium Brevibacterium Brevibacterium Brevibacterium glutamicum ATCC13032, acetoglutamicum ATCC15806, acetoacidophilum ATCC13870, thermoaminogenes FERM BP-1539, melassecola ATCC17965, flavum ATCC14067, lactofermentum ATCC13869 a divaricatum ATCC14020 a z nich vytvorené mutanty, poprípade kmene, produkujúce L-lyzín, ako napríkladCorynebacterium Corynebacterium Corynebacterium um Corynebacterium Corynebacterium, Brevibacterium Brevibacterium Brevibacterium glutamicum ATCC13032, acetoglutamicum ATCC15806, acetoacidophilum ATCC13870, thermoaminogenes FERM BP-1539, melassecola ATCC17965 flavum ATCC14067, lactofermentum ATCC13869 and divaricatum ATCC14020 and are producing mutants or strains producing L-lysine, such as e.g.
Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708,Corynebacterium glutamicum FERM-P 1709, Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712, ·· ····Brevibacterium lactofermentum FERM-P 1712, ·· ····
• · • · • · • · • · · · · ·· ···· • · · • · ·· ·· · · · · · · · · · · · · · · · · · · · · · · · ·
Corynebacterium glutamicum FERM-P 6463,Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 aCorynebacterium glutamicum FERM-P 6464 a
Corynebacterium glutamicum DSM5715.Corynebacterium glutamicum DSM5715.
Vynálezcom sa podarilo izolovať nový gén eno z C. glutamicum, kódujúci enzým enolázu (EC 4.2.1.11).The inventors succeeded in isolating a new eno gene from C. glutamicum, encoding the enzyme enolase (EC 4.2.1.11).
Na izoláciu génu eno alebo aj iných génov z C. glutamicum sa najskôr založí génová banka tohto mikroorganizmu v E. coli. Založenie génových bánk je opísané vo všeobecne známych učebniciach a príručkách. Ako príklad možno uviesť učebnicu od Winnackera: Gene und Klone, Eine Einfuhrung in die Gentechnologie (Verlag Chemie, Weinheim, Nemecko, 1990) alebo príručku od Sambrooka et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press,To isolate the eno gene or other genes from C. glutamicum, a gene bank of this microorganism is first established in E. coli. The establishment of gene banks is described in commonly known textbooks and manuals. Examples include Winnacker's textbook: Gene und Klone, Eine Einfuhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual from Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press,
1989). Veľmi známou génovou bankou je génová banka kmeňa W3110 E. coli K-12, ktorú založili Kohara et al. (Celí 50, 495 - 508 (1987)) v λ-vektoroch. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) opisujú génovú banku C. glutamicum ATCC13032, ktorá bola založená pomocou kozmidového vektora SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) v kmeni NM554 E. coli K12 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bôrmann et al. (Molecular Microbiology 6(3), 317-326)) zasa opisujú génovú banku C. glutamicum ATCC13032 s použitím kozmidu pHC79 (Hohn a Collins, Gene 11, 291-298 (1980)). Na vytvorenie génovej banky C. glutamicum v E. coli sa môžu použiť aj plazmidy, ako napríklad pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979) alebo pUC9 (Viera et al., 1982, Gene, 19:259-268). Ako hostitelia sú vhodné najmä také kmene E. coli, ktoré sú reštrikčné a rekombinačne defektné. Príkladom toho je kmeň DH5amcr, ktorý opísal Grant et al. (Proceedings of the National Academy of ·· ···· • · • · · · · · · ···· · ·· ···· ·· ·1989). A well known gene bank is the gene bank of strain W3110 of E. coli K-12, founded by Kohara et al. (Cell 50, 495-508 (1987)) in λ-vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a C. glutamicum gene bank ATCC13032, which was established using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160). -2164) in the NM554 strain of E. coli K12 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Bormann et al. (Molecular Microbiology 6 (3), 317-326)) in turn describe a C. glutamicum gene bank ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). Plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979) or pUC9 (Viera et al., 1982, Gene, 19: 259-) may also be used to generate a C. glutamicum gene bank in E. coli. Especially suitable as hosts are those E. coli strains that are restriction and recombinantly defective, as exemplified by the DH5amcr strain described by Grant et al. (Proceedings of the National Academy of Sciences). · · · · · · · · · · · · · · · · · ·
Sciences USA, 87 (1990) 4645-4649). Dlhé fragmenty DNA klonované pomocou kozmidov sa potom zasa môžu subklonovať do bežných vektorov vhodných na sekvenovanie a potom sekvenovať, ako sa opisuje napríklad v Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America 74:5463-5467 (1977)).Sciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned by cosmids can then be subcloned into conventional vectors suitable for sequencing and then sequenced as described, for example, in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America 74: 5463-5467 (1977)).
Týmto spôsobom sa získala nová sekvencia DNA z C. glutamicum, kódujúca gén eno, ktorá je ako SEQ ID NO 1 súčasťou predloženého vynálezu. Ďalej sa z predloženej sekvencie DNA odvodila aminokyselinové sekvencia príslušného proteínu pomocou vyššie opísaných metód. V SEQ ID NO 2 je znázornená vyplývajúca aminokyselinové sekvencia génového produktu eno.In this way, a new DNA sequence from C. glutamicum encoding the eno gene was obtained, which is part of the present invention as SEQ ID NO 1. Further, the amino acid sequence of the respective protein was derived from the present DNA sequence using the methods described above. SEQ ID NO 2 shows the resulting amino acid sequence of the eno gene product.
Kódujúce sekvencie DNA, ktoré vyplývajú z SEQ ID NO 1 v dôsledku degenerovatelnosti genetického kódu, sú taktiež súčasťou vynálezu. Rovnako sú súčasťou vynálezu sekvencie DNA, ktoré sa hybridizujú s SEQ ID NO 1 alebo časťami SEQ ID NO 1. V odbornom svete sú ďalej konzervatívne výmeny aminokyselín, ako napríklad výmena glycínu za alanín alebo kyseliny asparágovej za kyselinu glutámovú v proteínoch, známe ako „mutácie so zmyslom (sense mutations), ktoré nevedú k žiadnej zásadnej zmene aktivity proteínu, t. zn. sú funkčne neutrálne. Ďalej je známe, že zmeny na N-konci a/alebo C-konci proteínu nemôžu jeho funkciu podstatne poškodzovať alebo ju môžu dokonca stabilizovať. Údaje k tomu nájde odborník medzi iným v Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), v O'Regan et al. (Gene 77:237251 (1989)), v Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), v Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) a v známych učebniciach genetiky a molekulovej bio9 ·· ···· ·· ·· ··The coding DNA sequences which result from SEQ ID NO 1 due to the degeneracy of the genetic code are also part of the invention. DNA sequences that hybridize to SEQ ID NO 1 or portions of SEQ ID NO 1 are also part of the invention. Furthermore, conservative amino acid exchanges, such as glycine to alanine or aspartic acid to glutamic acid in proteins, are known as "mutations" sense mutations that do not result in any major change in protein activity, i. no. are functionally neutral. It is further known that changes at the N-terminus and / or C-terminus of a protein cannot significantly impair its function or even stabilize it. Data to this can be found by one skilled in the art, among others, in Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), in O'Regan et al. (Gene 77: 237251 (1989)), in Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)), in Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and in well-known textbooks of genetics and molecular bio9.
9 9 9 9 9 9 9 9 9 • · · · 9 · 9 • · 9. · 9 9 9 9 9 • · 9 9 9 9 99 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9
9999 9 ·· ···· ·· · lógie. Aminokyselinové sekvencie, ktoré vyplývajú príslušným spôsobom z SEQ ID NO 2, sú taktiež súčasťou vynálezu.9999 9 ·· ······ Amino acid sequences which result, respectively, from SEQ ID NO 2 are also part of the invention.
Súčasťou vynálezu sú rovnako aj sekvencie DNA, ktoré sa hybridizujú s SEQ ID NO 1 alebo časťami SEQ ID NO 1. Napokon sú súčasťou vynálezu sekvencie DNA, ktoré sa vytvárajú polymerázovou reťazovou reakciou (PCR) použitím primérov, ktoré vyplývajú z SEQ ID NO 1. Také oligonukleotidy majú typickým spôsobom dĺžku aspoň 15 párov báz.DNA sequences that hybridize to SEQ ID NO 1 or portions of SEQ ID NO 1 are also part of the invention. Finally, the invention provides DNA sequences that are generated by a polymerase chain reaction (PCR) using primers resulting from SEQ ID NO 1. Such oligonucleotides are typically at least 15 base pairs in length.
Návody na identifikáciu sekvencii DNA pomocou hybridizácie nájde odborník medzi iným v príručke „The DIG System Users Guide for Filter Hybridization od firmy Boehringer Mannheim GmbH (Mannheim, Nemecko, 1993) a v Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). Návody na amplifikáciu sekvencii DNA pomocou polymerázovej reťazovej reakcie (PCR) nájde odborník medzi iným v príručke Gait: Oligonukleotide synthesis: a practical approach (IRL Press, Oxford, Veľká Británia, 1984) a v Newton a Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Nemecko, 1994).Guidelines for identifying DNA sequences by hybridization can be found by one skilled in the art, inter alia, in " The DIG System Users Guide for Filter Hybridization by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). Instructions for amplifying DNA sequences by polymerase chain reaction (PCR) can be found by one skilled in the art, among others, in the Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
Vynálezcovia zistili, že koryneformné baktérie po nadmernej expresii génu eno produkujú zlepšeným spôsobom aminokyseliny, najmä L-lyzín.The inventors have found that coryneform bacteria produce amino acids, especially L-lysine, in an improved manner after overexpression of the eno gene.
Na dosiahnutie nadmernej expresie sa môže zvýšiť počet kópií príslušných génov alebo sa môže mutovať promotorová a regulačná oblasť alebo miesto naviazania ribozómov, ktoré sa nachádzajú proti smeru štruktúrneho génu. Rovnakým spôsobom pôsobia expresívne kazety, ktoré sa vkladajú proti smeru štruktúrneho génu. Pomocou indukovatelných promótorov možno dodatočne zvýšiť expresiu v priebehu fermentačnej produkcie ·· ···· • · • · ···· ·To achieve overexpression, the copy number of the respective genes may be increased or the promoter and regulatory region or ribosome binding site mutating upstream of the structural gene may be mutated. Expression cassettes which are inserted upstream of the structural gene act in the same way. Inducible promoters can additionally increase expression during fermentation production.
·. · • · • · ·· ·····. · · · · · · · ·
L-lyzínu. Opatreniami na predĺženie trvanlivosti m-RNA sa expresia taktiež zlepšuje. Ďalej sa enzýmová aktivita taktiež zosilňuje zabránením odbúravania enzýmového proteínu. Gény alebo génové konštrukty môžu buď existovať v plazmidoch s rozdielnym počtom kópií, alebo môžu byť integrované a amplifikované v chromozóme. Ďalej sa môže alternatívne dosiahnuť nadmerná expresia príslušných génov zmenou zloženia médií a zmenou uskutočnenia kultivácie.L-lysine. Expression measures also improve m-RNA durability measures. Further, the enzyme activity is also enhanced by preventing the degradation of the enzyme protein. The genes or gene constructs may either exist in plasmids with different copy numbers or may be integrated and amplified in the chromosome. Further, overexpression of the respective genes may alternatively be achieved by changing the composition of the media and changing the culture.
Návody na to nájde odborník medzi iným v Martin et al. (Bio/Technology 5, 137-146 (1987)), v Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya a Morinaga (Bio/Technology 6, 428-430 (1988)), v Eikmanns et al. (Gene 102, 93-98 (1991)), v európskom patentovom spise EPS 0 472 869, v US patente 4,601,893, vo Schwarzer a Puhler (Bio/Technology 9, 84-87 (1991)), v Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), v LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), v patentovej prihláške WO 96/15246, v Malumbres et al. (Gene 134, 15 - 24 (1993)), v japonskom zverejnenom spise JP-A-10-229891, v Jensen a Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), v Makrides (Microbiological Reviews 60:512-538 (1996)) a v známych učebniciach genetiky a molekulovej biológie .Instructions for this can be found by one skilled in the art in, inter alia, Martin et al. (Bio / Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), European Patent Specification EPS 0 472 869, US Patent 4,601,893, Schwarzer and Puhler (Bio / Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), Japanese JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60: 512 -538 (1996)) and in well-known textbooks of genetics and molecular biology.
Gén eno podía vynálezu sa napríklad nadmerne exprimoval pomocou plazmidov. Ako plazmidy sú vhodné také, ktoré sa replikujú v koryneformných baktériách. Početné známe plazmidové vektory, ako napríklad pZl (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl Eikmanns et al. (Gene 102, 93-98 (1991) alebo pHS2-l (Sonnen et al., Gene 107:69-74 (1991)) sa zakladajú na kryptických plazmidoch pHM1519, pBLl alebo pGAl. Rovnako sa môžu použiť ·· ···· ·· ·· • · · · · · · • · · · · · · • · ·. ·· ···· • · · · · · e ···· · ·· ···· ·· iné plazmidové vektory, ako napríklad tie, ktoré sú založené na pCG4 (US-A 4,489,160) alebo pNG2 (Serwold-Davis et al.,For example, the eno gene of the invention was overexpressed with plasmids. Suitable plasmids are those which replicate in coryneform bacteria. Numerous known plasmid vectors, such as pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl Eikmanns et al. (Gene 102, 93-98 (1991) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. They may also be used. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · plasmid vectors such as those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al.,
FEMS Microbiology Letters 66, 119-124 (1990)) alebo pAGl (US-A 5,158,891).FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891).
Dodatočne môže byť pre produkciu L-aminokyselín, najmä L-lyzínu, výhodné okrem génu eno nadmerne exprimovať jeden alebo viac enzýmov príslušnej biosyntetickej dráhy, glykolýzy, anaplerotiky, cyklu kyseliny citrónovej alebo prenosu aminokyselín.Additionally, for the production of L-amino acids, especially L-lysine, it may be advantageous in addition to the gene to overexpress one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, citric acid cycle or amino acid transfer.
Takto sa napríklad môže na produkciu L-lyzínu nadmerne exprimovať • súčasne gén dapA kódujúci dihydrodipikolinátsyntázu (EP-B 0 197 335) alebo • súčasne gén gap kódujúci glyceraldehyd-3-fosfátdehydrogenázu (Eikmanns (1992). Journal of Bacteriology 174:Thus, for example, for the production of L-lysine, the dapA gene encoding dihydrodipicolinate synthase (EP-B 0 197 335) may be overexpressed or the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992). Journal of Bacteriology 174:
6076-6086) alebo • súčasne gén tpi kódujúci triózofosfátizomerázu (Eikmanns (1992). Journal of Bacteriology 174:6076-6086) or simultaneously the tpi gene coding for triosophosphate isomerase (Eikmanns (1992). Journal of Bacteriology 174:
6076-6086) alebo • súčasne gén pgk kódujúci 3-fosfoglycerátkinázu (Eikmanns (1992). Journal of Bacteriology 174:6076-6086) or simultaneously the pgk gene encoding 3-phosphoglycerate kinase (Eikmanns (1992). Journal of Bacteriology 174:
6076-6086) alebo • súčasne gén pyc kódujúci pyruvátkarboxylázu (Eikmanns (1992). Journal of Bacteriology 174: 6076-6086) alebo ·· ···· ·· ·· ·· • · · · · · · * t · · · ·· · · · · · · • · · · · ·· ···· ·· · • · · • · • · • e ···· · • súčasne gén lysE kódujúci prenos lyzínu (DE-A-195 48 222) .6076-6086) or at the same time the pyc gene encoding pyruvate carboxylase (Eikmanns (1992). Journal of Bacteriology 174: 6076-6086), or Simultaneously, the lysE gene coding for lysine transfer (DE-A-195) 48 222).
Ďalej môže byť pre produkciu L-aminokyselín, najmä L-lyzínu, výhodné okrem nadmernej expresie génu eno vylúčiť nežiaduce vedľajšie reakcie (Nakayama: „Breeding of Amino Acid Producing Micro-organisms, v: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (vyd.), Academic Press, Londýn, Veľká Británia, 1982).Further, for the production of L-amino acids, especially L-lysine, it may be advantageous to avoid undesirable side reactions in addition to the overexpression of the enen gene (Nakayama: "Breeding of Amino Acid Producing Micro-organisms" in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek) eds., Academic Press, London, UK, 1982).
Mikroorganizmy vyrobené podľa vynálezu sa môžu na účely produkcie aminokyselín, najmä L-lyzínu, kultivovať kontinuálne alebo diskontinuálne spôsobom batch (vsádzková kultivácia) alebo spôsobom fed batch (prítokový systém) alebo spôsobom repeated fed batch (opakovaný prítokový systém). Zhrnutie známych kultivačných metód je opísané v učebnici od Chmiela (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustáv Fischer Verlag, Stuttgart, 1991) alebo v učebnici od Storhasa (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994).The microorganisms produced according to the invention can be cultivated continuously or discontinuously in batch or batch fed or repeated fed batch processes for the production of amino acids, in particular L-lysine. A summary of known cultivation methods is described in a textbook by Hop (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in a textbook from Storhas (Bioreactor and Periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).
Použité kultivačné médium musí vhodným spôsobom vyhovovať nárokom daných kmeňov. Opisy kultivačných médií rozličných mikroorganizmov sa nachádzajú v príručke „Manual of Methods for General Bacteriology od American Society for Bacteriology (Washington D.C., USA, 1981). Ako zdroje uhlíka sa môžu používať cukry a sacharidy, ako je napríklad glukóza, sacharóza, laktóza, fruktóza, maltóza, melasa, škrob a celulóza; oleje a tuky, ako napríklad sójový olej, slnečnicový olej, podzemnicový olej a kokosový olej; mastné kyseliny, ako je kyselina palmitová, kyselina steárová, kyselina linolová; alkoholy, ako je napríklad glycerol a etanol; a organické kyseliny, ako je napríklad kyselina octová. Tieto ·· ···· • · • · • · ··· ···· • · · · · e · ··· · ·· ···· ·· · látky sa môžu používať ako jednotlivé zložky alebo ako zmes.The culture medium used must suitably suit the requirements of the strains. Descriptions of culture media of various microorganisms can be found in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington, D.C., USA, 1981). As carbon sources, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose can be used; oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil; fatty acids such as palmitic acid, stearic acid, linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used as individual constituents or as a mixture. .
Ako zdroje dusíka sa môžu používať organické zlúčeniny obsahujúce dusík, napríklad peptóny, kvasnicový extrakt, mäsový extrakt, sladový extrakt, máčacia voda, sójová múka a močovina, alebo anorganické zlúčeniny, ako je napríklad síran amónny, chlorid amónny, fosforečnan amónny, uhličitan amónny a dusičnan amónny. Zdroje dusíka sa môžu používať jednotlivo alebo ako zmes. Ako zdroje fosforu sa môžu používať kyselina fosforečná, dihydrogénfosforečnan draselný alebo hydrogenfosforečnan draselný alebo príslušné sodné soli. Kultivačné médium musí ďalej obsahovať soli kovov, ako napríklad síran horečnatý alebo síran železa, ktoré sú potrebné na rast.Nitrogen-containing organic compounds such as peptones, yeast extract, meat extract, malt extract, soaking water, soy flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used singly or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or potassium hydrogen phosphate or the corresponding sodium salts can be used as phosphorus sources. The culture medium must further comprise metal salts such as magnesium sulfate or iron sulfate, which are needed for growth.
Napokon sa môžu dodatočne k vyššie uvedeným látkam používať esenciálne rastové látky, ako sú aminokyseliny a vitamíny.Finally, essential growth agents such as amino acids and vitamins may be used in addition to the above-mentioned substances.
Ku kultivačnému médiu sa môžu okrem toho pridávať vhodné prekurzory. Uvedené používané suroviny sa môžu ku kultúre pridávať vo forme jednorazovej vsádzky alebo sa môžu vhodným spôsobom pridávať počas kultivácie.In addition, suitable precursors may be added to the culture medium. The raw materials used can be added to the culture as a single batch or added in a suitable manner during the cultivation.
Na kontrolu pH kultúry sa vhodne používajú zásadité zlúčeniny, ako je hydroxid sodný, hydroxid draselný, amoniak, poprípade amoniaková voda, alebo kyslé zlúčeniny, ako je kyselina fosforečná alebo kyselina sírová. Na kontrolu tvorby peny sa môžu používať odpeňovadlá, ako napríklad polyglykolestery mastných kyselín. Na udržiavanie stability plazmidov sa môžu k médiu pridávať vhodné selektívne pôsobiace látky, napríklad antibiotiká. Aby sa udržiavali aeróbne podmienky, zavádza sa do kultúry kyslík alebo zmesi plynov obsahujúce kyslík, napríklad vzduch. Teplota kultúry je zvyčajne približne 20 °C až 45 °C a najmä približne 25 °C až 40 °C. Kultúra sa udržiava tak dlho, kým sa nevytvorí maximum žiadanej L-lyzínu. Tento ciel sa zvyčajne dosiahne za 10 hodín až 160 hodín.Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and optionally ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid are suitably used to control the pH of the culture. Antifoams, such as polyglycol esters of fatty acids, can be used to control foaming. Suitable selective agents, for example antibiotics, may be added to the medium to maintain the stability of the plasmids. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, for example air, are introduced into the culture. The temperature of the culture is usually about 20 ° C to 45 ° C, and especially about 25 ° C to 40 ° C. The culture is maintained until the maximum of the desired L-lysine is formed. This target is usually achieved in 10 hours to 160 hours.
·· ···· ·· ·· ·· • · · · · • · · · ··· ······················
Analýza L-lyzínu sa môže uskutočňovať anexovou chromatografiou s následnou ninhydrínovou derivatizáciou tak, ako sa opisuje v Spackman et al. (Analytical Chemistry,L-lysine analysis can be performed by anion exchange chromatography followed by ninhydrin derivatization as described by Spackman et al. (Analytical Chemistry,
30, (1958) 1190).30, (1958) 1190).
Spôsob podľa vynálezu slúži na fermentačnú výrobu aminokyselín, najmä L-lyzínu.The process according to the invention serves for the fermentative production of amino acids, in particular L-lysine.
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Predložený vynález sa v nasledovnom texte bližšie vysvetľuje na základe príkladov uskutočnenia.The present invention is explained in more detail below with reference to exemplary embodiments.
Príklad 1Example 1
Vytvorenie genómovej kozmidovej génovej banky z Corynebacterium glutamicum ATCC 13032Generation of genomic cosmid gene bank from Corynebacterium glutamicum ATCC 13032
Chromozómová DNA z Corynebacterium glutamicum ATCC 13032 sa izolovala tak, ako sa opisuje v Tauch et al., (1995, Plasmid 33: 168-179), a čiastočne štiepila reštrikčným enzýmom Sau3AI (Amersham Pharmacia, Freiburg, Nemecko, opis produktu Sau3AI, Code no. 27-0913-02). Fragmenty DNA sa defosforylovali pomocou alkalickej fosfatázy z garnátov (shrimp) (Roche Molecular Biochemicals, Mannheim, Nemecko, opis produktu SAP, Code no. 1758250) . DNA kozmidového vektora SuperCosl (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84:2160-2164), získaná od firmy Stratagene (La Jolla, USA, opis produktu SuperCosl Cosmid Vektor Kit, Code no. 251301), sa štiepila reštrikčným enzýmom Xbal (Amersham Pharmacia, Freiburg, Nemecko, opis produktu Xbal, Code no. 27-0948-02) a taktiež defosforylovala alkalickou fosfatázou z garnátov (shrimp). Následne sa ··· ·· · ···· ·· • · 9 9 9 9 9 • · · · · · · ···· · ·· ···· ·· kozmidová DNA štiepila reštrikčným enzýmom BamHI (Amersham Pharmacia, Freiburg, Nemecko, opis produktu BamHI, Code no. 27-0868-04). Týmto spôsobom spracovaná kozmidová DNA sa zmiešala so spracovanou DNA ATCC13032 a násada sa spracovala T4-DNA-ligázou (Amersham Pharmacia, Freiburg, Nemecko, opis produktu T4-DNA-ligáza, Code no. 27-0870-04) . Ligačná zmes sa potom pomocou extraktu Gigapack II XL Packing (Stratagene, La Jolla, USA, opis produktu Gigapack II XL Packing Extract, Code no. 200217) vbalila do fágov. Na infekciu kmeňa E. coli NM554 (Raleigh et al. 1988, Nucleic Acid Research 16:1563-1575) sa bunky zachytili v lOmM MgSOí a zmiešali s alikvotom suspenzie fágov. Infekcia a titrácia kozmidovej banky sa uskutočnila tak, ako sa opisuje v Sambrook et al. (1989, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor), pričom sa bunky rozotreli na agar LB (Lennox, 1955, Virology, 1:190) so 100 μg/ml ampicilínu. Po inkubácii cez noc pri 37 °C sa selektovali jednotlivé rekombinantné klony.Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described in Tauch et al., (1995, Plasmid 33: 168-179), and partially digested with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, Code No. 27-0913-02). DNA fragments were dephosphorylated using shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, SAP product description, Code no. 1758250). SuperCosl cosmid vector DNA (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164), obtained from Stratagene (La Jolla, USA, description of SuperCosl Cosmid Vector Kit, Code no. 251301), was digested with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, Code no. 27-0948-02) and also dephosphorylated with shrimp alkaline phosphatase. Subsequently, the cosmid DNA was digested with the restriction enzyme BamHI (Amersham Pharmacia), 9 9 9 9 9, and 9 9 9 9 9. , Freiburg, Germany, product description BamHI, Code no. 27-0868-04). The cosmid DNA treated in this way was mixed with the treated ATCC13032 DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, Code no. 27-0870-04). The ligation mixture was then packaged into phages using Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). For infection with E. coli strain NM554 (Raleigh et al. 1988, Nucleic Acid Research 16: 1563-1575), cells were captured in 10 mM MgSO 4 and mixed with an aliquot of phage suspension. Infection and titration of the cosmid bank were performed as described in Sambrook et al. (1989, Molecular Cloning: and Laboratory Manual, Cold Spring Harbor), where cells were spread on LB agar (Lennox, 1955, Virology, 1: 190) with 100 µg / ml ampicillin. After incubation overnight at 37 ° C, individual recombinant clones were selected.
Príklad 2Example 2
Izolácia a sekvenovanie génu enoEno gene isolation and sequencing
Kozmidová DNA jednotlivej kolónie sa izolovala pomocou Qiaprep Spin Miniprep Kit (produkt č. 27106, Qiagen, Hilden, Nemecko) podía návodov výrobcu a čiastočne štiepila reštrikčným enzýmom Sau3AI (Amersham Pharmacia, Freiburg, Nemecko, opis produktu Sau3AI, produkt č. 27-0913-02). Fragmenty DNA sa defosforylovali pomocou alkalickej fosfatázy z garnátov (shrimp) (Roche Molecular Biochemicals, Mannheim, Nemecko, opis produktu SAP, produkt č. 1758250). Po oddelení gélovou elektroforézou sa uskutočnila izolácia kozmidových plazmidov s rozsahom veľkostí 1 500 až 2 000 bp pomocou QiaExII gel Extraction Kit (produkt č. 20021, ·· ···· • a · aaaa ·· a a a · a a · a a a a · a a aaaa a aa aaaa aaSingle colony cosmid DNA was isolated using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and partially digested with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913). -02). DNA fragments were dephosphorylated using shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, SAP Product Description, Product No. 1758250). After gel electrophoresis, cosmid plasmids ranging in size from 1,500 to 2,000 bp were isolated using the QiaExII gel Extraction Kit (Product No. 20021, ·· ····) aaaaa ·· aaa · aa · aaaa · aa aaaa and aa aaaa aa
Qiagen, Hilden, Nemecko). DNA sekvenčného vektora pZero-1, získaná od firmy Invitrogen (Groningen, Holandsko, opis produktu Zero Background Cloning Kit, produkt č. K2500-01), sa štiepila reštrikčným enzýmom BamHI (Amersham Pharmacia, Freiburg, Nemecko, opis produktu BamHI, produkt č. 27-086804). Ligácia kozmidových fragmentov do sekvenčného vektora pZero-1 sa uskutoénila tak, ako sa opisuje v Sambrook et al. (Molecular Cloning: A laboratory Manual, Cold Spring Harbor) , pričom zmes DNA s T4-ligázou (Pharmacia Biotech, Freiburg, Nemecko) sa inkubovala cez noc. Táto ligačná zmes sa následne elektroporovala (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) do kmeňa E. coli DH5aMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87: 4645-4649) a rozotrela na agar LB (Lennox, 1955, Virology, 1:190) s 50 pg/ml zeocínu. Plazmidová preparácia rekombinantných klonov sa uskutočňovala pomocou Biorobot 9600 (product č. 900200, Qiagen, Hilden, Nemecko). Sekvenovanie sa uskutočňovalo dideoxymetódou ukončenia reťazca od Sanger et al. (1997, Proceedings of the National of Sciences U.S.A., 74:5463-5467) s modifikáciami podía Zimmermann et al. (1990, Nucleic Acids Research, 18:1067). Použil sa „RR dRhodamin Terminátor Cycle Sequencing Kit od PE Applied Biosystems (produkt č. 403044, Weiterstadt, Nemecko). Oddelenie gélovou elektroforézou a analýza sekvenačnej reakcie sa uskutočňovala v géle „Rotiphorese NF Acrylamid/Bisacrylamid (29:1) (produkt č. A124.1, Roth, Karslruhe, Nemecko) so sekvenčným prístrojom „ABI prism 377 od PE Applied Biosystems (Weiterstadt, Nemecko).Qiagen, Hilden, Germany). The DNA sequence vector pZero-1, obtained from Invitrogen (Groningen, The Netherlands, product description Zero Background Cloning Kit, product # K2500-01), was digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, product no. 27-086804). Ligation of cosmid fragments into the pZero-1 sequence vector was performed as described in Sambrook et al. (Molecular Cloning: A laboratory manual, Cold Spring Harbor), where the DNA mixture with T4-ligase (Pharmacia Biotech, Freiburg, Germany) was incubated overnight. This ligation mixture was subsequently electroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123: 343-7) into E. coli strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649) and was spread on LB agar (Lennox, 1955, Virology, 1: 190) with 50 µg / ml zeocin. Plasmid preparation of recombinant clones was performed with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Sequencing was performed by the dideoxy chain termination method of Sanger et al. (1997, Proceedings of the National of Sciences, U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). The RR dRhodamine Terminator Cycle Sequencing Kit from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) was used. Gel electrophoresis and sequencing analysis were carried out in a Rotiphorese NF Acrylamide / Bisacrylamide (29: 1) gel (Product No. A124.1, Roth, Karslruhe, Germany) with a sequencing apparatus "ABI prism 377 from PE Applied Biosystems (Weiterstadt, Germany).
Získané nespracované údaje o sekvenciách sa následne spracovali procesorom použitím programového balíka Staden (1986, Nucleic Acids Research, 14:217-231) Version 97-0. Jednotlivé sekvencie derivátov pZerol sa zostavili do • · ···· ·· ·· • · · · » » » • · · ·· ···· jedného súvislého celku. Počítačová analýza kódujúcich oblastí sa uskutočnila pomocou programu XNIP (Staden, 1986, Nucleic Acids Research, 14:217-231). Ďalšie analýzy sa uskutočnili pomocou „BLAST search programs (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402) proti neredundantnej databanke od „National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).The obtained raw sequence data was then processed by the processor using the Staden (1986, Nucleic Acids Research, 14: 217-231) Version 97-0 software package. The individual sequences of the pZerol derivatives were assembled into one contiguous whole. Computer analysis of coding regions was performed using the XNIP program (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analyzes were performed using BLAST search programs (Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402) against a non-redundant database from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).
Získaná nukleotidová sekvencia je znázornená v SEQ ID NO 1. Analýza nukleotidovej sekvencie poskytla otvorený čítací raster s 1275 pármi báz, ktorý sa označil ako gén eno. Gén eno kóduje polypeptid so 425 aminokyselinami.The nucleotide sequence obtained is shown in SEQ ID NO 1. Nucleotide sequence analysis yielded an open reading frame of 1275 base pairs, which was termed the eno gene. The eno gene encodes a 425 amino acid polypeptide.
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| AU2001287682A1 (en) * | 2000-09-12 | 2002-03-26 | Degussa A.G. | Nucleotide sequences coding for the ftsx gene |
| DE10046625A1 (en) * | 2000-09-20 | 2002-04-11 | Degussa | New nucleotide sequences coding for the ndkA gene |
| DE10145043A1 (en) * | 2001-09-13 | 2003-04-03 | Degussa | Process and manufacture of fine chemicals |
| DE10210527A1 (en) | 2002-03-09 | 2003-09-18 | Degussa | Alleles of the aceA gene from coryneform bacteria |
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| US20070092951A1 (en) | 2005-03-24 | 2007-04-26 | Degussa Ag | Alleles of the zwf gene from coryneform bacteria |
| CN103725702A (en) * | 2013-01-31 | 2014-04-16 | 北京师范大学 | Application of ENO2 gene to regulation and control of plant growth and development |
| CA3007635A1 (en) | 2015-12-07 | 2017-06-15 | Zymergen Inc. | Promoters from corynebacterium glutamicum |
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| US9988624B2 (en) | 2015-12-07 | 2018-06-05 | Zymergen Inc. | Microbial strain improvement by a HTP genomic engineering platform |
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| WO2018005655A2 (en) | 2016-06-30 | 2018-01-04 | Zymergen Inc. | Methods for generating a bacterial hemoglobin library and uses thereof |
| KR20200026881A (en) | 2017-06-07 | 2020-03-11 | 지머젠 인코포레이티드 | Uses thereof to modulate promoter and accessory gene expression from Corynebacterium glutamicum |
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