HRP960053A2 - Microbiological preparation of 5-cetogluconate - Google Patents
Microbiological preparation of 5-cetogluconate Download PDFInfo
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- HRP960053A2 HRP960053A2 HR19503946.7-41A HRP960053A HRP960053A2 HR P960053 A2 HRP960053 A2 HR P960053A2 HR P960053 A HRP960053 A HR P960053A HR P960053 A2 HRP960053 A2 HR P960053A2
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- gene
- gluconate
- oxidoreductase
- ketogluconate
- gluconobacter
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- 230000002906 microbiologic effect Effects 0.000 title claims description 5
- 238000002360 preparation method Methods 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 64
- 229940050410 gluconate Drugs 0.000 claims abstract description 37
- 241000589232 Gluconobacter oxydans Species 0.000 claims abstract description 24
- IZSRJDGCGRAUAR-MROZADKFSA-M 5-dehydro-D-gluconate Chemical compound OCC(=O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O IZSRJDGCGRAUAR-MROZADKFSA-M 0.000 claims abstract description 23
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 claims abstract description 17
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- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims abstract 2
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- 238000000034 method Methods 0.000 claims description 18
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Cephalosporin Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
Description
Opis
Izum se odnosi na postupak za mikrobiološku proizvodnju 5-ketoglukonata prema zahtjevima 1 do 7, gena za glukonat: NADP+-5-oksidoreduktazu prema zahtjevu 8 do 10, strukturu gena prema zahtjevu 11, vektor prema zahtjevu 12 kao i transformirane stanice prema zahtjevima 13 do 15.
Poznato je, da bakterije, kao primjerice Gluconobacter oksidiraju glukozu u glukonat, a glukonat dalje u 5-ketoglukonat, koji se zatim dobiva iz medija bakterijske kulture.
Mikrobiološka proizvodnja i dobivanje 5-ketoglukonata interesantni su za proizvodnju askorbinske kiseline, a naročito za proizvodnju L-(+)-vinske kiseline. Sada se L-(+)-vinska kiselina dobiva iz vinskog kamena, nusprodukta koji se taloži kod proizvodnje vina. Time je proizvodnja vinske kiseline ovisna o raspoloživosti ovom sirovinom. Neprerađeni vinski kamen također je u pravilu onečišćen organskim materijalima, tako da je za dobivanje vinske , kiseline potrebna skupa prerada.
Na temelju ovih nedostataka pokušava se razviti alternativne postupke za proizvodnju vinske kiseline. Alternativni način postupka čini naročito konverzija mikrobiološki dobivenog 5-ketoglukona.ta u L-(+)-vinsku kiselinu (usporedi također DE-patentnu prijavu br. P 44 40 191.4 -44). Mikrobiološki postignuta iskorištenja 5-ketoglukonata su sada još premala za ekonomsku prođu ovog postupka:
Tako primjerice Gluconobacter oxidans tvori kod rasta na glukozi uvijek 2- i 5-ketoglukonat, pri čemu se dobivaju po koloniji specifična različita iskorištenja: za tvorbu 2-ketoglukonata opisuju se iskorištenja u području od 4% do 97%, a za tvorbu 5-ketoglukonata iskorištenja u području od 4% do 35% - uvijek u odnosu na konvertiranu glukozu (Weenk, G., Olijve, W. i Harder, W., 1984, Ketogluconate formation by Gluconobacter species. Appl. Microbiol. Biotechnol. 20, 400-405). Kroz različite uvjete kulture postiže se prvenstveno stvaranje uvijek jednog od oba ketoglukonata: tako vodi pH-vrijednost od 4, temperatura inkubacije od 25°C i titracija s CaCO3 do pojačanog stvaranja 5-ketoglukonata (Stadler-Szoeke, A., Nyeste, L- i Hollo, J., 1980, Studies on the factors effecting gluconic acid and 5-ketogluconic acid formation by Acetobacter. Acta Aliment. 9, 155-172). Više pH-vrijednosti i više temperature pospješuju naprotiv stvaranje 2-ketoglukonata (Ameyama, M. and Kondo, K., 1958, Carbohydrate metabolism by Acetobacter species. Bull. Agric. Chem. Soc. Japan 22, 373-379).
Stoga je zadaća izuma da pripravi postupak mikrobiološke proizvodnje 5-ketoglukonata, kojim će se tvoriti povećan udio na 5-ketoglukonatu. Nadalje je zadatak izuma da pripremi tvari koje se primjenjuju u takvom postupku.
Ovaj zadatak se prema izumu rješava time, da se povećava ekspresija gena za glukonat: NADP+-5-oksido reduktazu mikroorganizma koji tvori 5-ketoglukonat, naročito povećanjem broja kopija gena. To ima za posljedicu stvaranje povećanog udjela na 5-ketoglukonatu.
Za povećanje broja kopija gena ugradi se gen za glukonat-oksidoreduktazu u genski konstrukt odnosno u vektor, koji prvenstveno sadrži regulatorne redoslijede gena za glukonat-oksidoreduktazu, naročito takve-. koji pojačavaju njegovu ekspresiju. Zatim se mikroorganizam koji tvori 5-ketoglukonat, naročito Gluconobacter, odnosno Gluconobacter oxydans, transformira s genskim konstruktom koji sadrži gen za glukonat-oksidoreduktazu. Pri tom mogu kao proizvodne stanice, po sebi razumljivo, služiti također takvi organizmi, koji nemaju vlastiti gen za glukonat: NADP+-5-oksidoreduktazu.
Izoliranje gena, kloniranje i transformacija slijedi prema metodama poznatim u molekularnoj biologiji. Izolira li se primjerice gen iz Gluconobacter -a, prvenstveno iz Gluconobacter oxydans-a, uslijedi izoliranje, koje se bazira na poznavanju redoslijeda aminokiselina odgovarajućeg proteina. Za kloniranje gena primjenjuju se specifične genske sonde. Gen može nakon ligacije sa, za proizvodne stanice prikladnim sistemom vektora i ekspresije, biti unesen tekućim metodama transfera u proizvodnu stanicu i tamo doveden do ekspresije.
Primjenjuje li se kao proizvodna stanica primjerice Gluconobacter, prvenstveno Gluconobacter oxydans kao metoda transformacije nudi se naročito bi- i triparentalni konjugativni transfer.
Nakon izoliranja i sekvenciranja dobivaju se redoslijedi nukleotida gena za glukonat-oksidoreduktazu, koji kodiraju (šifriraju) za redoslijed aminokiselina naveden u tablici 1 ili njihovih varijacija alela. Varijacije alela obuhvaćaju osobito funkcionalne derivate koji se dobivaju kroz deleciju(e), inserciju(e) ili supstituciju(e) nukleotida iz odgovarajućih redoslijeda, pri čemu se međutim ne mijenja aktivnost glukonat-oksidoreduktaze. Odgovarajući redoslijed naveden je u tablici 1.
Uz gen za glukonat-oksidoreduktazu vežu se prvenstveno regulatorni genski redoslijedi, koji naročito povećavaju aktivnost gena.
Tako se može mutacijom regulatornog genskog redoslijeda tako utjecati na djelotvornost vezanja regulatornog proteina uz DNA za reguliranje gena za glukonat-oksidoreduktazu, da se time pojačava transkripcija, i time poveća ekspresija gena.
Nadalje, mogu se uz gen za glukonat-oksidoreduktazu vezati kao regulatorni redoslijedi također tzv. "enhancer", koji putem poboljšanog uzajamnog djelovanja između RNA-polimeraze i DNA također pojačavaju ekspresiju gena.
Kloniranjem gena za glukonat-oksidoreduktazu dobivaju se plazmidi, odnosno vektori, koji sadrže gen i prikladni su, kako je već gore spomenuto, za transformaciju mikroorganizma koji tvori 5-ketoglukonat. Stanice dobivene transformacijom, kod kojih se prvenstveno radi o transformiranim stanicama Gluconobacter-a, naročito Gluconobacter oxydans-a, sadrže gen u replicirajućem obliku, to znači u dodatnim kopijama na kromosomu, pri čemu se kopije gena integriraju homolognom rekombinacijom na proizvoljnim mjestima genoma, i/ili na plazmidu, odnosno vektoru.
Primjer za izvođenje:
Određivanje enzimske aktivnosti
Kod enzimatski katalizirane oksidacije glukonata u 5-ketoglukonat reduciran je NADP+ u NADPH i fotometrijski dokazan (Okamoto, K. 1963. Enzymatic Studies on the Formation of 5-KetogIuconic Acid by Acetobacter suboxidans, J. Biochem- 53, 448-452).
Određivanje produkta
Kvantitativna analiza glukonata i 5-ketoglukonata uslijedila je putem HPLC (R- Klasen et al. 1992. Incapability of Gluconobacter oxydans to Produce Tartaric Acid. Biotechnol. Bioeng. 40: 183-186).
Pročišćavanje glukonat: NADP+-5-oksidoreduktaze.
Anionski izmjenjivač: Ekstrakt staničnog homogenata (bez stanica) G. oxydans-a nanesen je i vezan na anionski izmjenjivač (DEAE-Tentakel, Merck Darmstadt; 5 15 cm). Enzim je eluiran s rastućim gradijentom NaCl (0 - 500 mM) kod 350 mM NaCl. Aktivne frakcije su očišćene i dijalizirane preko ultra filtracione jedinice (Amicon; cut off: 10 kDa).
Afinitetna kromatografija pomoću boje: dijalizirana enzimska otopina nanijeta je i vezana na obojeni afinitetni stupac (Blue Sepharose CL-6BTM, 2,6 20 cm (T. Atkinson et al., 1981. Triazine-dye affinity chromatography- Biochem. Soc. Trans. 9: 10-13). Enzim je eluiran s rastućim NaCl-gradijentom (0 - 500 mM) kod 300 mM NaCl. Aktivne frakcije su skupljene i dijalizirane preko ultrafiltracione jedinice (Amicon; cut off: 10 kDa) uz 25 mM histidin/HCl pufer pH 5,6.
Kromatofokusiranje I: Dijalizirana enzimska otopina nanesena je na mono-PTM -kromatografsku kolonu uravnoteženu s 25 mM histidin/HCl puferom pH 5,6 (Pharmacia, Freiburg; 0,5 20 cm). Podešavanje pH-gradijenta, a time eluacija proteina uslijedila je aplikacijom 50 ml poli pufera PB74 pH 4,0 (Pharmacia, Freiburg) na kolonu.
Gel-filtracija: Skupljene aktivne frakcije aplicirane su na kolonu sa gel-filtraciju (Sephacryl-S100 HR, Pharmacia, Freiburg; 2,6 100 cm). Eluacija je uslijedila pod jednakim okolnostima. Aktivne frakcije su ujedinjene i dijalizirane preko ultrafiltracione jedinice (Amicon; cut off: 10 kDa) uz pufer 2,5 mM acetat/NaOH od pH 4,8.
Kromatofokusiranje II: Proteinska otopina nanesena je na mono-PTM-kromatografsku kolonu uravnoteženu s puferom pH 4,8, 25 mM acetat/NaOH (Pharmacia, Freiburg; 05, 20 cm). Aplikacijom 50 ml polipufera PB74 (Pharmacia, Freiburg) pH 4,0, podešen je pH-gradijent na koloni i eluiran protein. Nakon provjeravanja čistoće pomoću SDS-PAGE, uzet je alikvotni dio očišćenog proteina, naglo zamrznut u tekućem dušiku i uskladišten kod –70°C.
Aminoterminalno sekvenciranje.
Za aminoterminalno sekvenciranje amino kiselina premješteno je 100 ug pročišćenog proteina na PVDF-membranu i ispitano s plinsko-faznim sekvencionatorom (Appl. Biosystems, model 470A ), (P. Edman et al., 1967. A Protein Sequenator. Eur. J- Biochem. 1: 80-91). Dokaz otcijepljenih derivata aminokiselina (feniltiohidantoin) uslijedio je metodom HPLC.
Priprema genske sonde.
Molekularnobiološki radovi provedeni su, ukoliko nisu odijeljeno izvođeni, prema T. Maniatis et al. (1989, Molecular cloning: A laboratory manual 2nd edition. Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y.). Sekvenciranjem po Edman-u određene su amino kiseline 2 do 18 glukonat: NADP+-5-oksidoreduktaze (slika 1A).
Nakon transmisije proteinske sekvence u korespondirajuću sekvencu nukleinskih kiselina, uzimajući u obzir degeneraciju genetičkog koda, pokazala se visoko degenerirana DNA-sekvenca.
Za konstrukciju gno-genske sonde sintetizirane su PCR-klice (Primjer) koje odgovaraju krajevima ustanovljene peptidne sekvence (Slika IB). Na 5’- krajevima klica konstruirana su restrikcijska mjesta za enzime EcoRI i Sacl, da bi se kod kasnijeg kloniranja postigla veća selektivnost. Nakon izoliranja klica stavljene su ove u PCR-reakciju s pročišćenom, kromosomskom DNA iz G. oxydans-a (Y. Takeda et al., 1991. Cloning and Sequencing of the Gene Encoding Cytochrome c-553 (CO) from Gluconobacter oxydans J. Ferment. Bioeng. 72: 1-6) kao matrica (S.J. Gould et al., 1989. Use of the polymerase chain reaction for homology probing: Isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate debydrogenase from several species. Proc. Natl. Acad. Sci. USA 86; 1934-1938).
Fragment od 66 pb izoliran je nakon preparativne elektroforeze na 4%-tnom agaroznom gelu i rezan restrikcijskim endonukleazama EcoRI i Sacl. Nakon ligacije pripremljenog fragmenta s odgovarajuće izrezanim plazmidom pUC19 on je ubačen u E. coli. Fragment od 66 pb dobiven PCR reakcijom je sekvenciran, da bi se odredila nedegenerirana, kodirajuća DNA-sekvenca aminoterminalnog područja proteina.
DNA-sekvenca dade se pripisati dobivenoj peptidnoj sekvenci (Slika 1C). Sekvenciranjem PCR-fragmenta pronađeno je područje, koje je kod dužine od 45 pb odgovaralo GC-sadržaju G. oxydans-a (60-65 %, J. de Ley et al., 1970. The status of the generic name Gluconobacter -Int. J. Syst. Bact. 20: 83-95). Ova sekvenca primijenjena je kao uzorak za sintezu gno-genske sonde (Slika 1D).
Uspostava restrikcijske karte gno-gena.
Na bazi rezultata PCR-pokusa konstruirana je gno-genska sonda i primijenjena nakon označavanja digoksigeninom (G.G. Schmitz et al., 1991. Non-radioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with Terminal Transferase. Anal. Biochem. 192: 222-231) sa eksperimente Southern-Blot. (E.M. Southern, 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 8: 503-517). Pored toga izrezana je DNA iz G. oxydans-a s različitim kombinacijama restrikcijskih endonukleaza i nakon odvajanja restrikcijskih fragmenata agaroznom gel-elektroforezom premještena na najlonsku membranu. Nakon hibridizacije s genskom sondom pokazao se po fragmentu uvijek samo jedan jasan signal. Izračunavanjem veličine hibridizirajućih fragmenata izrađena je restrikcijska karta (slika 1E).
Kloniranje gno-gena.
Nakon pripreme djelomične genske banke G. oxydans-a kloniran je putem hibridizacije kolonije 3,4 kb BamHI-fragment, koji je nosio cjelokupni gno-gen.
(M. Grunstein et al., 1975. Colony hybridization: A method for the isolation of cloned DNA's that contain a specific gene. Proc. Natl. Acad. Sci. USA 72: 3961-3965).
Sekvenciranje gno-gena.
Sekvenciranje kromosomskog područja oko gno-gena (F. Sanger et al-, 1977. DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467) dalo je redoslijed nukleotida od 1510 pb s otvorenim okvirom čitanja od 771 pb, koji je kodirao za polipeptid od 257 aminokiselina molekulske mase od 27.300 (Tablica 1). Redoslijed nukleotida imao je mjesto sa vješanje ribosoma 10 pb ispred start kodona (J. Shine et al., 1974. The 3"-terminal sequence of Escherichia coli 16S ribosomal RNA: Complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71: 1342-1346) i tipičnu terminatorsku palindromsku strukturu 160 pb iza stop kodona (M. Rosenberg et al., 1979. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13: 319-353). Iz izvedenog redoslijeda aminokiselina proizašla je sekvenca peptida dobivena sekvenciranjem proteina.
Ekspresija gno-gena u G.oxydans-u.
Kao vektor DNA za (G. oxydans izabrani su derivati plazmida RSF 1010, budući da je replikon na ovom plazmidu dopustio replikaciju DNA u širokom spektru gram-negativnih bakterija (U. B. Priefer et al.,1985. Extension of the host range of Escherichia coli vectors by incorporation of RSF 1010 replication and mobilization functions. J- Bacteriol. 163: 324-330). Kao baza za daljnja istraživanja za prijenos gena u G. oxydans primijenjen je plazmid pRS201PR (R. Schroeder et al., 1991. Expression of the core antigen of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors- Appl. Microbiol. Biotechnol. 35: 631-637). Prijenos plazmida postignut je konjugacijom (C. Condon et al., 1991. Conjugation and heterologous gene expression in Gluconobacter oxydans ssp. suboxidans. FEMS Microbiol- Lett. 80: 173-178).
Plazmid pRS201PR sadržavao je pored promotora također gen za temperaturno osjetljivi derivat cI-represora (cI857), koji je regulirao promotor. Radi deregulacije promotora eliminiran je dio cI857-represora plazmida pRS201PR (pRS201P). 1,25 kb BamHI/SalI-fragment, koji kodira za glukonat: NADP+-5-oksidoreduktazu, spojen je s odgovarajuće restrikcijski izrezanim pRS201PR i pRS201P. Plazmidi pRS201PR-gno i pRS201P-.gno preneseni su konjugacijom u G. oxydans. Određivanje aktivnosti pokazalo je 6,5 do 85-struko povećanje aktivnosti glukonat: NADP+-5-oksidoreduktaze u rekombinantim kolonijama G.oxydans-a. (Tablica 2).
Produktivnost 5-ketoglukonata u rekombinantnih kolonija.
Za određivanje produktivnosti 5-ketoglukonata rekombinantnih kolonija G. oxydans-a istraživana je akumulacija 5-ketoglukonata u fermentacijskom mediju (U. Kotera et al., 1972- Isolation and chemical structure of new oxidation product of 5-ketogluconic acid, and a hypothetical pathway from glucose to tartaric acid through this new compound. Agric. Biol. Chem. 36: 1315-1325). Dnevno su uzimani uzorci za određivanje metabolita, i pomoću HPLC-mjerenja kvantitativno određeni. Pokazalo se povećanje stvaranja 5-ketoglukonata kod povećane ekspresije gno-gena (G. oxydans pRS201P-gno) u usporedbi s aktivnosti glukonat: NADP+-5-oksidoreduktaze u kolonijama divljeg tipa (G.oxydans wt, G. oxydans pRS201P) za 11 % (Tablica 3).
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Claims (15)
1. Postupak za mikrobiološku proizvodnju 5-ketoglukonata, naznačen time, da se povećava ekspresija gena za glukonat: NADP+-5-oksidoreduktazu mikroorganizma koji stvara 5-ketoglukonat.
2. Postupak prema zahtjevu 1, naznačen time, da se ekspresija gena glukonat-oksidoreduktaze povećava, povećanjem broja kopija gena za glukonat oksidoreduktazu.
3. Postupak prema zahtjevu 2, naznačen time, da se za povećanje broja kopija gena, gen za glukonat oksidoreduktazu ugradi u genski konstrukt.
4. Postupak prema zahtjevu 3, naznačen time, da se gen za glukonat-oksidoreduktazu ugradi u genski konstrukt, koji sadrži regulatorne redoslijede gena za gen za glukonat-oksidoreduktazu.
5. Postupak prema zahtjevu 3 ili 4, naznačen time, da se mikroorganizam koji stvara 5-ketoglukonat transformira s genskim konstruktom koji sadrži gen za glukonat-oksidoreduktazu.
6. Postupak prema zahtjevu 5, naznačen time, da se Gluconobacter, prvenstveno Gluconobacter oxydans, transformira s genskim konstruktom koji sadrži gen za glukonat-oksidoreduktazu.
7. Postupak prema jednom od prethodnih zahtjeva, naznačen time, da se gen za glukonat-oksidoreduktazu izokira iz Gluconobacter-a, prvenstveno Gluconobacter oxydans-a.
8. Gen za glukonat: NAPD+-5-oksidoreduktazu, naznačen time, da ima jedan kodirajući redoslijed nukleotida za redoslijed aminokiselina naveden u tablici 1 i njihovim varijacijama alela.
9. Gen za glukonat-oksidoreduktazu prema zahtjevu 8, naznačen time, da ima redoslijed nukleotida prema tablici 1 ili jedan DNA-redoslijedom, koji u biti djeluje na isti način.
10. Gen za glukonat-oksidoreduktazu prema zahtjevu 8 ili 9, naznačen time, da sadrži regulatorne genske redoslijede.
11. Struktura gena, naznačena time, da sadrži gen za glukonat-oksidoreduktazu prema jednom od zahtjeva 8 do 10.
12. Vektor, naznačen time, da sadrži gen za glukonat-oksidoreduktazu prema jednom od zahtjeva 8 do 10 ili strukturu gena prema zahtjevu 11.
13. Transformirana stanica, naznačena time, da sadrži u replicirajućem obliku gen za glukonat-oksidoreduktazu prema jednom od zahtjeva 8 do 10, ili strukturu gena prema zahtjevu 11.
14. Transformirana stanica prema zahtjevu 13, naznačena time, da sadrži vektor prema zahtjevu 12.
15. Transformirana stanica prema zahtjevu 13 ili 14, naznačena time, da je ona Gluconobacter, prvenstveno Gluconobacter oxydans
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