HUE026630T2 - Eljárás L-triptofán elõállítására javított, Enterobacteriaceae családba tartozó baktériumtörzsek alkalmazásával - Google Patents

Description

PjárásiL-íeiptefá» etbalHfâsâra javított, EfitemhüeterÍiteesg csatádba tartósé bifktèriasnfhrxsek alkalmazásával

The present invention relaies K? fetoiback-resissans variions of ;i-d<ua>xy-l>-3rid>im>· hepalosonaie-l-phdsphate synthase (ÖABP sypthase), reenthhmanî híieftmfiPúsms öl she Etsíerobaeieríaeeae fatally soittsmisg these essayistes and as« ihesoef for she fesomnisstsve tpasssifeetssire of organic ehesmeal eoshpounds.

Prior Art

Organic chemical compounds, in paniculssr aromatic Lwsmlno adds, are used In human medicine and la !:ho phamsaeeuifeal industry, in she food isuitssiry and in aniotal nutrition.

If dtfanie chemical compbuds are soentlpned feeiom « of snore epmphhds selected from she grosip consisting of tho ecmspounds of Use shiklofefe tstefehujk pathway, soeh ay she atmnatfe 1.-amiue seid% L-tyrosioe, L>phenyk1auine end Ldrypiophan, ehorismsste, sOierofeaeiis, msus^dnntm, tetrahydrofokue ssnd ubiquinone are meant thereby. lia aromas ie i..-isstsisn> acids sp preferred, nod L-tryptophan and P-pessyMasssne are particularly preferred, II Is knosrn ffest organic chemical compounds are rsmnnlheiared hy ferrueniaiion of strains of the Easerpheeseriaeeae, in partleolar Escherichia colt (i, eolfr and; lersaila mereescess. gceaoas of its great ll|ortua«% there is eoÄuous work: on itnprovmg lie asaasddetarhsg process. Proeesa improvebtents can relate so fermesWailoP: technology sdeasnres, such as lor Mantpfe stirring ansl sppply of uxygso, or the .composition of the nutrient media such as for example the choice of the soger issed or the sstgar eoneetifeafidn during the fermentation, the workup to: she prodoet form;, e.g, by ton exchange ehromtstpgrsphy, or the intrinsic pcrtprmtmee characteristics of the mfrfocrpnism itself lit wild type strain«, stries: regulation mechanisms proyenffhe production of metabolic products soehi at ammo: acids beyond their own reqatretsmufe, and: their telea.se into the medium. the eotistrtseiioh of strains: which: frosts the poducerk viewpoint overproduce organic chesnieaj eisrapoonds therefore requires this mesabolk: regulation to he overcome.

For elimination of the epskrol meehatbsms and snsptovepsonl of the performance ehifegetaristies of: these .microorganisms, nsetisods: of stitsfegatsesss, selection ;pd. mutant selection are used:- In this manner,; strains which ate resistant to ssattmelaholifes sued as let saample the threonine anafops o~ sun inn- h-hydroxyvaleric acid {AHV} or ammtruphic for meiislolites significant for tpgpfefc: pd produce i..-ssmfeo acids such as for exemple L-threonnse ate obtained. Resistance to the tryptophan analogue 5-o\ethy1-DL4ryptophan td-MT) for example characterises a strain which produces L* tryptophan,

For some years. methods: of reeombioaht: DBA technology haveidso been used tor the targeted strain improvement of L-atstmo a®ids:prodsicip|:WSrains of Use Fotcrobaetemeeae family, for example by amplifying individual atnlno sell biosynthesis genes or ehanghsg the properties of specific gestes ssnti studying the effect ost predsiCtson. A summary of Information on the sell soul molecular biology of Escherichia: ebltabd iinldnalk Is to he fosstsd iss Bcidhardf fed): isehericeia cols apd SalmsneilSi frellular and Molecular Bhsiogy, 2nd edition, Ai|M Press, Washington, D.C., USA, (1996), A sntmrtaty os! She metabolism and the podaciion of I.-threonine las been published hy :Dufeá|ov (Advances Isi Biochemie«! Engmemog Yo&amp;?9, 1 13-136 (2603)).

The shikimate metabolic pathway for the synthesis of inter aim ehoftsmate, anterohaeim;: mcpgufnone, tetrabydrofoiat«, ufeiqumo# smd the atotaaik amino eci# Is strongly reguláiig» temmlm·. btstsymhem is tabulated in B. fco.il both, at the genetic level by situation and reprssslost and also by end product inhibition of «nxymes (Frost and Draths, Annual .spvkw of microbiology* 49:557»?$ (1995); Pinard, Genes to Colls 1(8):717-25 0996)). Via this pathway* the prodnciiou of ' aeamatlemetaheiit&amp;s is snatched precisely to the requirements of the ceil. 7 he literie acid pathway 1$: mainly regulated by control of the irmial reaction, which is catalysed by three different isoonaymei of DAMP synthase iencoded by the genes nroF. aroG and aroB). In E. cob, the aroö gene product snakes up 80% of the overall activity of all DAMP synthases. (Tribe et si.t Journal of Bacteriology 127(3):: IÖS5-I097 (1971); Pittard, 4Éánes to Ceils 1(8):717-25 0996)}: aroG » 95% inhibited by the aromatic amino acid B-ph«oykkame:> AroP is likewise 955» inhibited by L-tyrosine, while AroH can be s:sp to 697¾ Inhibited by L-trypiophart (Gamakaris and Pittard, Journal of Bacteriology 120(2):599-7 < 1974>>, Since Aroli is only partly inhibited by ^»tryptophan, the shikimic acid pathway is not completely iahíbitedj evet! at high concentrations of «11 three asnlno acids, so that some chommk acid can always slid he homed fey the remaining residual activity, and la then available for other biosynthetic pathways. As the genetic kvel, the fognlsfot Tyft lit presetted of phenylalanine and tryptophan represses the transcription of aroG (Baseggio at ak, Journal of Bacteriology 172(5):2547-57 (1990); Davses et ah. Gene 35(3}: 323-31 (1905)).

The nucleotide sequence of the wild form of the gene aroG coding for the 3-dcsoxv-D-arablno-hephifosonato-?-phosphate synthase of .Escherichia coli was determined by Davies and .Davidson (Maejsk Acids Beseareh MM 131: 4045-4058 (1952)) and including the opsiream and dowoske&amp;m lying regions is publicly available ln ihn database of the National Genter for Biotechnology Information (NCBI) of the ^tionaicÜiraary ;W Meâtee (lethesda, MD, USA) under the accession number HC90O9ÍS (region; 704.656 ~> 705.998 and npstrwmr/downsiream).

Is the patent application BE 1 276 721, the beneficial: effect of the use of an aroG allele on the production and preparation of the pontatie annno aetds L-phenyi-alaame and B-tryptophaa with strains of the gemts Escherinhia is dsserifeed, where to this ease it is as alkie wherein the praline at position ISO Is replaced by a kocise. In the patent application WG2005/05677Í, alkies are described wherein the valine at position 57 is replaced by so alanine or the cysteine at position 61 by arginine. Kikuehi or el. (Applied and Bnvitonmenta! Microbiology 03(2):761-762 (1996) in addition describe alleles with matat Ions at the positions 146, 14? and 202.

Since the biosynthesis of organic chemicals of the shikimate metabolic pathway is very éómpiea* Is replated in many ways and moreover is Interlinked with vannas other metabolic pathways to the eels, it is desirable to have available toany variants of 3-desoxy·D-arabino-heptalosonate·-?-phosphate synthase which differ in susceptibility to inhibition by L-phenylaknine. Owing to the complexity of the hlosyntkeik path way, If sarmot be pedktod with which variant improved production of the organic ehetpka! compounds, preferably L -ttypiophao and i - phenylalaoiae,: can he achieved.

Purpose of the invention

The inventors set themselves the task of providing oskel variants of S-desoxy-fr-Unfruno'· hepiuIosonats~7~gbosphate syufhpo, which I» eosuparixoB to the wild type (Wf| enaytpe possess mctMsed feedback resistance towards t-phenylmianme and |h«s provide new bases for improved presesses fir the fermeBmilve asapufacm*« of organic chemical compounds, preferably EfreyptoghaB ^ir#e^efe»tek«Äipi^*pfp»isn^j:Pfth« Entershncieriaceac family. iDescnptso» of the invention. A suMeci of the invention ate alleles of the aroö gene, namely nucleotide sequences (Db«A|, which code for feedback oesisboti variants of frw emsyœe S-desoxy-D-arahlnodtepiulosoBaie^E phosphide synthase (DAMP synthase droG^ oharaeterked in that their amino acid sequence has the ami«« acid sequence of SEQ IB Mo.¾ Ihclndihg deletion and/or insertion of írom Ί to21 amino acids, and essentially comprises a length of did amino acids and contains within the corresponding amino acid sequences the amino acid sahstii étions in the amino seid sequence of SfrQ 113 Mol2 selected from the group a I substitution of L-alaaine at position 33 by E-valine, b) subsiitutioa of I-alanine at pasikon 33 by L-vallne and substitution of L-serine at positloa 211 by L-ph«nylaiaoine, and the ammo acid sequence of SB(| 11> No.2, where appropriate, additionally contains one or taorp df the «nhstilations selected front the group consisting of c) substitution of Ldystne at position 14 by an amino acid selected from the group consisting of L'-asparcgine and E-glutamine. preferably 1,-asparagine, d) sahstitntion of fr-valine at position 47 by an amino acid selected from the group cottsisting of L-sianine, E^ysine, L-argMna and E-histlÄe, preferably L-aiaÄs, e) substitution of E-threonine at position 74 by an amino acid selected front the group consisting of E-alanine. Edysine, L-arginine and L-histidipe, preferably E-alanine. 0 substitution of L^glutamk adid at position 11 by ap amino acid selected from the group consisting of L-aspariic acid.. !..-serine, E-alansne and §..---hreoBise, preferably E-aspartic acid. g) substitution of E-dysine at position 84 by an amino fpf#::s*4eemd from tbs group consisting of E~glutamlna, L-serine, E-threonine, E-ghttamie acid. L-aspartlc acid and L-asparagine, preferably JU glutamine, h) subsithtioh of L-aspamc acid at position §5 by an atnino acid sclecled from tbc group conslstmg of L-alanine and glycine, preferably glycine, i) substitution of E~giutamte acid at position 231 by m. amino acid selected from the group consisting of L-nspariie acid, E-serine, L-alanine and 1..-:ineonioe, preferably E-miaume, j) substitution of l.-lysme at position 244 by SB atsmo acid selected front: the group consisiag ofL-giulamme, E-glutamic acid, L-asparlie acid, L-scriae, L-threonine asui f^asparagme. prefrrably: fr-glutamine, k) substitution of E-aspsrngine at positloo 254 by an atnino acid selectel froia: the grpUb consisting of E-ibreonlse, E-serine, E-glntamlc: acid and ikaspartic aenh preferably Efrhreooine, 0 substitution of l-proiine m position 259 fey m amino acid selected from the group edasrstlag of L-serine, fr-feremfene, preferably fr-fereomnO;, m) sabsttfetion of fr-alanine at position 26® by L-proline, n> substitution vfL~mtim at position 2?2 fry m mim mé mimmé from: tbs poop cousistmg of fr-oyskUue sud fr-oietbidifeie,: ip^ferafeli^systéiab, 0) suhstitatioR oÉMysin® at pPltfetó76 fey an ammo seid selected from the grrmp consisting öf ^'gluÄis«, L"g|»isS*®felÄ fr-aspáfefo aelfe t-sértőé, L-ferénu.me and L'-ssiíárbgluós ptb^i'áfely L* glutamine» p3 subsifrutíorí: of fr-aaparoo add at position 280 by ait sutom seid selected from ifea group consisting mi L-sertue» fr-alanlne, L-glutantfe seid ttod L-threoolne, preferably L-gMamfe astfe q) substiOÄsy Of |,>fIfeismk solfr ot position 313 fey « sutom seid selected: from its gspp constating ofL-seríne* l-aiaaine, L-asgartfe: add aad L-tfrreoofeei preferably L-aspartfe aejd, # sohslttotloq of fr-gfrnamk: soll ut position 3 Iá fey m ρφο acid sefeeted front tfee group consisting Of E-glutamine-, ;L-seriae, L-fereottlne,, L-asgartlc acid: Sad L-asparagine, preferably L-gltsiautfee, s) sofrsiitOilpR Of fr-alanine at position 319 fey su amino add seleefed from the group coasistiag Of L-thrcrtniOe, L-vaiine and L-scnne., preferably L-threonine, 1) substitUtienof L-alaniue a? position 342 fey an anttPo add selected from the group consisting of L-serine, 1--9000¾ and t-ibrenaine* prefetafely L-sedne or fr-valine, u) substitution of L-asparagnu·: ut position 343 bÿ m mimé acid selected front ifeö grööp consisting of fr-aspartic nclfe fr-gklaofre seid, E-serine, t-alapios má L-threonlne, preferably fr-alanine.

Preferably., tb« ROböäbd pulyppidus have a Isaph fetsfrsspoudiog io :35b uríIoo solda, ikcfersbÍR ssi’O Isblsfrd polynucleotides according to SEQ ID No, I, efraraeteneed tu that ifeslf sequence contains the foHowing substitutions: * sfpdolOouttS tbymiRs or « at positions 9<3 aad 632 thymine. .diso a subject of tbc invention arf faoOmblRsnt otfanle Chemical compounds selected from tbs group consisting of the compound*! of the sblkkoate metabolic pathway, preferably the aromatic fr-amloo acid-prodaclng ruicroorgaolsutU of fee EsicfP|sbtOfla«aaa family» which contain a feedfraefe:-réSiaí&amp;ni aroO alfele according té fei Äeatifefr- -WhÄ: codes for the d-desoxy-P-afafeiuo-heptnibsonsue-T-plospbaie synthase arnfe, aud winch prodRes sail sompouRds Ir increased atnount ahd accumulate ifeem feu the call or the otediors). A subject of the invention are profersfeiy rocombluant L-lrypfophaR-prodaemg mfefoorgsuisms of fee Euterolssotonaceso fenrOy, wbfei eoRtaiu a feedback-resistant aroö allele Póordíag to fee feyeotiop, wltiofe codes Ip the 3-despy-D-afabÍROfeept«losortPe-7-pfeöspbaío syotfease arofr, and whip produoa frsfryptopfeah in increased amount md aecamulate it in the ooil or tbc medium.

Also a stsfejeçî of the invention are L-amtno scid-prodnctug tnieroorganisms of the fruferofeacterl&amp;ecac feplly- AhiOb p psispstArt L-fbenylttfebbfe and febieb produce i,-0'ypö|shsm in 5nereased aamuni acid accumulate n in the cell or Ose medians L-pheoylaiaoine is genera Oy used it; concentrations jfesm > (greater fona/eguni to) 2 to < (less citas/ equal ce) 15 nslfo As the starting poibt fór the comparison, la each esse the rniereorganisms less resistant to L~phe?iyialanine, on which the m4Mv>H aesbrÉuglo the mvemidn ws$ dut jpÉsmnd, were used.

Also a subject of the invention is a method for the fermentative tnsnaiaeture of organic chstnlcal oothpoonds selected from the group crofoistlhg of foe compounds of the shikhuate metabolic : pathway, preferably the aromatic L-amino acids, is; particular l-tryptophan, with use of recombinant microorganisms of the Cofc'TvdxK'wrUeea« family, whleh to :par(ic«hK already pfodpe ss|| organic chemical compounds before the loss of feedback inhibition aecobfingto che ùmhifohabü&amp;dhiWhiih at least: the are G allele or nucleotide sequences coding for the igene product thereof is or are exjsresseif, preferably enhanced or overexpressed,

However, Cite wild type strains and commonly used laboratory strains such as inter afin I>H5u, O.H5nmcr, WAItO, .MG 1655, MÇ4100, YK?k9, H56Ô and MM 152 are not foehsdeb Osnoag the microorganisms producing L»aal$tf&amp; acids before she loss of feedback hfoihitiun according to the icivefaion.

Preferably the microorganisms described sere used.

When L-amino acids or amino acids are mentioned below, thereby: are tue&amp;et one or more gotlsp acids including salts thereof, selected from ihr group consist ing ni L~asp&amp;rag|pg* igi|ç^onp«,;:L* serihe, L-giuiamate. L-giyeice, L-alanine. tL-cysteine, L-vaiio«, Lmmthiotdne. L-proline, L-lsdlsaeme, 'ti4ö»ci.ne, L-cyro&amp;ine, L-phenyl-alanine, L-histidine, L-iysine, hArÿpôphah, L-arginine and Ifo homoseftne, Particularly preferable are t-feypophao and L-phenylaiaafo«. in this context, the term "expression” describes the impiecóeSíntfon of the intraceliubtr activity of on« or more «nsymes or proteins in a cnicroorganism, which afo encoded by the correspondingly expressed DM A,

In this context, foe terns “eohaacemenr describes increasing of the intracellular activity or eoneentratieo of one or more esxynKs or proteins in a micreorganisifo which arc encoded by the corresponding DMA, for example by ioeruaalug the copy number of she gene or the genes, of foe GifF or the ORPs or of the allele or the alleles by at least one (1) copy, fonctionally linking a strong promoter with the gene, imitating the endogenous promoter and regulatory region or foe ribosome binding she which is located upstream of the structural gene, or using ή gene or allele of 0RF which codes for a correspöndlttg eoxpK or protein with high activity and where appropriate combining tbq§e measures, “Endogenous genes“ or “endogenous uaeiUdtide sequences* tire Understood to be foe genes Of open reading frames or alleles or nucleotide suspenses present in the population of one species.

Where approprias«, il can he of advantage to effect only a moderat« iscretsse in the intracellular activity or eoneentrution of one or ttrore cneynves or proteins in a microorganism, sin«® an excessively strong increase can for example lead to defective ceil division or altered cell tnorphnlogy, enhmnating in toxicity (Outhtie and Wickoer, Journal of Bacteriology 1.72(10):5555-5.162 (1090): Genevaox P, ct ai.; EMSO Reports 5(2); 105-200 (2004)).

The term "moderate increase" describes Sh« increasing of tfes intracellular activity or concentration of the rekvsnr grotcm by at moat i0-foidf Mold* 6-fold, 4~foíd, 34feld, 2-fold or Lf* fold fessed m that of the wild type protêt» or on the activity or cooceniratkm of the protein I». the microorgtmism or parent: strain that is imn-reeomhinaht for the relevant enayme or protein. The rhiotosrgSsilstO or paraoï straîîj timt is non-reeomfeimmt is understood to be the microorganism on which th® enhancement or oversagefesstos according to the invention is perföÉÉbd.

The ropy mmbm 0 the m&amp;G atkk aeeotdtog to the tmmniim is preferably increased fey at most eight (S$ copies, paHieplMly preferably fey at most ffeur (4¾ copies. Saefe moderate expression is for emmtple achievable through the use of plasmids with iacT'5 allelefoopf miied gases (Glascock and Wetcksrt, Ogee 223, 221-231 (1998)} without induction fey 1ETG addition, which leaps to very weak expression of the aroG gen« cloned without its own promoter. The transcription start of the aroG gene was located 4d base pairs Before the ÂTG start codott (Gaseggio et al., Journal of Bacteriology 2592-253? (1990)) and oot trspslerred onto tbe plasmid togetber with the nucleotide ssiptenee coding for the DÁBP synthase.

For the creation of mwG mtnoîfonm which are used I® the microorganisms used for the method according to the invention, methods for directed motagestesis described in the prior art can inter alia be used.

Methods of site-directed matapnesis wtth ose of mutapnlc oligopocleotides ft, A, Brownt Gesiechnofogte % Einsteiger [Geoeric «agtoeerittg f|f fesgtmtefagSpektrum J:kadethtscher Verlag, Heidelberg, 2943? or the polymerase chain reaction hs described to tli® manual fey Gait;

Dhgrmodeot tde Syttfhesls;: Â Practical Approach GET Erase:, Oxford, OK, 19:84? or fey felewtoo and Graham (PCR, Spekitton Akademischer Verlag, Hetdelfeerg» 1:9991,: caa ha: used. Por thá: conStfftetioo of mutations in the otdG :geo«, főt example; the Quiek Change sitmdlrecied mut age nests feit (font Stratageoe (Amsterdam, Gefheriands): can fee ttsed, %|th use of these methdds, the aroG gene described in the prior art 1$ amplified by moans: Of the polymerase: chain reaction (PCRt starting fromisolated total DMA of A wild typo sirmtn cloned iotp sdtfabktptastnid voetetw and the DNA fiten subjected to the matagonoata process, By mpaos of "GeneSGEing" (Gene Splicing' by Overlap Extension, Horton, Molecular Biotechnology 3 ; 93-99 (199333, she point mutations ce« already fee obtained by PGR, The mutations crested can fee determined and cheeked fey DMA sequencing for example by the method of Sanger ct .d. (Proceedings bitbe fefetlonai Academy of Science HSA 29 (12$ 3453-596? (19??)),

Tbe alleles created cars for exstpple be sscprpprtdetHstfo sntiabla strttlns fey transfortnattoe and tbs method of geos or a He Hr snbsHtatioe. A common method ;is tbc method of gene sdfestfonioo: described by ilamdfoo ct ah (lemrsai of Bacteriology 129, 96(7 - 9622 (19993 by means ofa 'conditionally replicating pSCIOi deHyattve pMAKTOS or with pH03 (Link ct ah, Journal of Bacteriology 179 : 6228-623733· Gther tnfeihdds described in the prior art such M for example that of Martioea-Motaks et al. pbnrtfäl of Baciörtöldgy 1999, ? 143-7198 0999?) that of Boyd et at (Journal of Bacteriology 182, 892-897 (2996)) can equally be used.

It is also to paasferr&amp;e alleles «remed lato varions stratus bÿ «oojipfie» :fe ttifeSbOCtSOU,

More detailed explaftáfibuáhéh îhb ïenÂdibty of genetics au# sUoiseular biology cats be found hi kfiews fextjtfepks of geoetícs aad suoleesd&amp;r biology such us for example the semboob: by Birge fBacîerial and Bacteriophage Oeaetics, 4* Bd,< Springer Verlag» New York (USA). 29()1) m tie textbook by Berg, Tyroocako and Stryer (Biochemistry, 5!h Ed., Fieetnan and Company, bfety York (USA), 2002} or the snamtal by Sam brook cl at (Molecular Cloning, A Laboratory Manual, (3-Vohane Seu. Cold Spring Harbor Fres^.?Ç»Mi|pidg; Harbor (PS A ), 2001). 1¾ achieve oyereRpfeMlim:, fer example llaícopy somber of the relevant genes or open reading Barnes ebb bo ioereasedy or tiroipfobsoser- and; regtiatney region or the ri&amp;sisome binding site which is located: apstrsmn of the strbslaral: geos sas be :ina:ts:tedo Expression:; cassettes which are isscorporated apsiteasts of ihe structural gene operate to ;fee oaotoyyey< L· addition, by means of inducibk presoetors it Is possible to increase expression in the dSiife« of fermentative L-îryptophao snanufeeter«, iirfeetmore fc use of prötooters fer gene axpresstfet, wisich enables diffetessi chronological:: gene eygrespofo can also be abyafeageous. At the level of the trapiaiiootsl regulation of gene expression it: ib possible to increase the frequency ofdstliatldh fa»&amp;ehment of the ribosome io the m-RNA) or the rata Pi elongation (prolongation phase). I&amp;rongis measures for prolonging the sifetone of the m-RNA, expression: is also improved:, Furthermore, the etrcytne activity m also enhanced by preventing defrpdpltos of the esayme protein. The genes, ORFs or gene coostrovts cats either be gresesst is plasmlh; with differe«t copy number or be integrated and amplified io the chromosome. Alternations moreover, overerpresslon of this relevant genes cars be achieved by alteration of fee médiats cosnposstsoo end culferhsg procedure.

Methods for ovorespression have bees adequately described to the prior art, for example Ip Mskridas eh ah (MicroMOfegiPpi Reviews 60 (3), 512-333 ( ippdjy By fee use of vectors, the copy .number k increased by at feast one (!) copy. As vectors, plasmids, as for example described ín ÖÍ can be used, Phages, ibr exsmspte fee phage mu, as deseribed to BP 0332443, or fee phage lambda (a) can also be used as vcclors. An increase In the copy smother cats also be achieved by ismorporafmg a forther copy irsfo a farther site of the chromosome, for example Into the air site of fee phage X (Ya and Court, Gusse 223, 2?-SI (1003)).. Ids US 5,030,307, it is stated feat tbrotsgb incorporation of expression cassettes dr promoters such as for example sac promoter, irp pronsoter, Ipp promoter et Pt, promoter and Fg promoter of the phage fe fef example epstreass of the chromosomal ihreonhse opérés, ass socrease to expression could be achieved, it could be shown that the tac promoter is 3 times stronger than fee (fp promoser aspi 50 times stronger than fee lac promotes: (de Boer et al, Proceedings of tise National Aeadestty sFBeleoees of fee 0SA 30:21-25 (1033)}. life prosrsoters of the phago 17, the gear~hox promoters, the tsar promoter or fee promoters of feu genes rtsO, rstpR, csrA, esrB, ostspA, fesA, pepd rpll or rpsG can be used in a similar essence, Such expression cassettes or prosstoters can also be used, as described in BP 0 :503 792.. in order to overexpress plasmid-bound gestes. By use of the laeiF allele, the expression of fáé promoter gene fusions can in sum be control fed (Glascock ami Wesekefg Gesse 223, 221-231 (1993)), tbc nsa of these plasmids w oboát mdaesioss by ÍPTíl aMitsas leads to. very weak expression of genes, pfpaod wÄtt» theliddwn promoter. Furthermore,, it is passible that the activity of the proovMers is increased fey oiodification of then setptence b> mesas of one or more nucleotide subs#«troas, : %y: ««d/sr á#tioo(s). Issspfepitutlos of promoters by a promoter for example determined to genome-wide comparative expression analyses with uniformly high expression to the course of a fermentation process produces uniform enhancement. HifMep chronological g eon expression cart for example fee achieved : through: use; of fie: growth phase» dependent is promoter^ as described by Walker et ah (idomai of Bacteriology IS? f1269-80 (3699)). The Me 6( elongation is influenced by the eodoo usage, and through the use of codons M t»K,NAs eonmtonly ocettrting to the pareot :sit»ins the gene expression can bo amplified. Moreover, sdhtitutiori of a start codes to the codés AT% at 77% that most eommottif occurring is Escherichia cob. cas emisiderahiy improve expression, $mm the codon AUG ts two to three times more effective than lor example the codöps GUG ásd ÜUP (Hhudyakov et ai, FEES Letters 2320 ):369-71 (1988); Eeddy et al.:, Eroceedittgx of the Hatiboafi Aosidesty of Sciences of the USA 82( i ?): 5656--60 (1986)), The sequence envifosmgirt ol the átért codoo can also be pplmired, since ioíéraétisg eOects Imtweeo the start codon sod the Hooking regions have been described (Steostrom et ah. Gene 273(2):259-65 (291)i t; Hoi ét Mté EMID J ómmal 3(3): 623 -9 (1984 t). 'fitosé skilled in the art will bad general directions for this inter alls to Chaog and Cohen (IpOrsai of Bacteriology 134: 1141-1156 (1678)), m Hartley and Gregott (Hehe 13: 347-353 (1683)). ip Amaan and Brosins (Gene 40: 183-160 (1985)), in de Broer et ah (Ffdoeedtogs of the Natiosui Academy of Sciesees of the tinned States of Amefiett Üt 21-25 (1983)p is LaYaiiie et ah (BIO/TFCMNOLOGY Π: 187-163 (1993it, is 60370897/13356, in Lies« or at. (Plasmid 2o 222-224 (1661)), 1rs Quandt and Klipp (Germ gy- -61-106 (1689)), in Hamilton el a), (Journal Of Bacteriology 171: 4617-4632 (1989)), in .lessen and Hammer f Biotechnology and BioengioMriog 58: 191465 (1998)) and wkm*m textbooks of genetics and stolMhiar biology,

Blasmid vectors replicable in InMobacmHafUsc soeh as tor example cloning vectors derived Stoss pA€¥€184 (Bartoioste et at; Gene 102: 75-78 (1661)), pT re-99 A (Amann et ahpöene 66: 301-315 (1638)) or pSGMi derivatives (¥oeke and iastmt; Proceedings of the National Academy of Sciences USA 89(21): 6537-6561 (1633)) can he esed. Is one method according to the invention, a strain transformed with a plasmid vector can bo Used; whoreis the plssntid vector carries at least the aroG allele or nucleotide sequences coding for the gene product thereof.

The term translbrmmioo w understood to mean (he uptake of as isolated nucleic acid by a host (tnicroorganisrs).

Through the enhancement measures, in partieuiar overex pressiοn, the activity or eonceoiMkm of the relevant protêts is geserelly increased by at least 10%¾ 338% S8%> 75%, 160%, 130%, 289%, 306%, 490% or 500%, at most to 1000% or 20006¾ based on that of the wild type protein or on the activity or cosceniraitoo of the protein to the ndetPorganisbt or parent strain ihm is son-recombinant for the relevant enayspe Or protein. The microorganism or parent strain ihat is noo-fecosthlnant is understood tő ibe the thicroorgartisst, on which loss of the feeubatk inhibit tors or overexpressios according tstihe invention is effected. for the dyicrmuiíiííon of the extent of tbc feedback resistance of a vada»* according to the invention of 3-des«» y -ö- :«whi»o-h«|>«áO§ööat«*í?*jphósph8to ayifthsse* for example the thioharhlîurate <may after Schoner end Herrmann {The Journal ofTBiolcgic-al Chemistry 251(18>: 544Η5447 (397% modified after War&amp;vdesar sod Saslaw, Biorhimicá et Biophysica A«á 14 {i9521 iis used. This test is based on the periodate oxidation of PAHP to tornsylpyruvafe, whom reacts wfth tftiobarfeätesfö to give a coloured comptes. A 100 pi mkt»psphpsphifO buffer paM, p!4 f| and 75 pmol of phosphoenoh pyruvate <PEP) and 03 pmof of etf(hföse-4-phosplmíe (149 ! is inetsbated far y mins ai 3?*C in the water-bath. The reaction is stdrMd !% addftioo of 50 pi of crude extern, obtained hy nitrasonieasioo (Simpson et si.. Journal of Bacteriology 107(3): 798-905 (1971), and stopped after 10 odos by addition of 300 pi of 0.6 M trichloroacetic aoid solution. Precipitated protein is eentrtegsd down, 100 pi of solution i (0.2 M sodium pflodaie in 9 M are added to dip pi of the reaction mixture, and incubated idr :*i nrins at 2M93 is the mier^hatb. To this are; added 490 pi of solution If (Ô.75 M isodlfon arsenate in 0.5 Μ Ma2SO< and 0J5 M SjSOl, Incubated at room temperature (Or I anus, and then 3 ml of solution ill (0.04 M ihiobarhitursts in Ô.S M NaJTL) are added and the mixture heated for 15 mim I« the boiling water-bath. As a blank sample, wafer Is used. The bls§k sample remains colourless and the crude extracts tested become more or less intensely pink; the contest of DAHP is measured via the change in the extinction at 549 nm after cooling of the solution (extinction eoefftciedt: f# 334 χ t #M*mf% for the determination of dte extent df ibe leedback resistance, the catalytic activity of the 1>AHF synthase variants is roeastnnd in the presence and absence of 2 mM 1.-plmnylalapine.

The concentration of the protein can be determined in site get via 1- and 2-dimensionaJ protein gel separation and subaegttetd optical: ideSttlieation of the protein concentration with appropriate evaluation software. A eoshtwon method fût preparation of protein gels; and for tdenilÄonw iff proteins is the procedure described by Hermann et ai. (Electrophoresis, 22:1712-23 (2091)). The protein eoucentratiott can also: be deteriUtned: by Western blot hybridisation with an antibody specific Tor dtp protein to be estimated JSambçdôlç et ah, Molecular cioning: a laboratory manugk 2!!<< lib Çplp Spring llarbor Laboratory Press* Cold Spring Harbor, N,¥,, IMS! h»á sttbseptteuf optical evaluation with: appropriate software for mmmimtim. determination (l..obans and Meyer 3998} Biospektrum 532*39; Lottspeich, Angewandte ChetmoAS; 200-2647 (19991.

Chemically, a gene pf Allaio is a polynucleotide. Another lectn iftff this is nucleic cold, in particuiat deoxyribonucleic acid.

The terms polypeptide and protein are mutually interchsugeable· A section of a nucleotide aepenee which codes or can code for a protein or polypeptide of rlbommiets acid, to which according to the prior art no Emotion can: be assigned Is described as en open reading frame (OBF). After assipmertt of a function to tltC: relevant section of the nucleotide sequence, it is generally referred to ae e gene, Allci.es ate generally understood toibc aftornaftve forms of a glvepigcne, Tim Kuans are eharpterped by differences in: fhe ttueledfldc sepnence,:

The protein encoded by a tmeleptide asp norms, lie, mMT> a gene or an allele or the esteddel ribonnelete add is generali y referred to as a gene product. A subject: of the invention fs a method for manufacturing organic chemical compounds selected from the group Consisting: of the compounds of the shskimate metabolic pathway, preferably the aromatic L-äfninö: acids, in particular :I,-trypfephan, 'by fennetriaiioU of the recombinant microorganisms: of the Eaterohaetariaeeae family according to the invention, characterized in that :s) the mtc'f&amp;organiiSîns producing the organic chemical compounds, in which the aroG gene or the nucleotide: sequences dr alleles coding for the gene product theu-of are expressed, in particular overexpressed, whereby the loss of the feedback inhibition is achieved by a mutagenesis measure within the: aroG gene, are cultured in a medium under conditions under which the organic chemical compounds is accumulated in the medium or in the ceils, and, b) the Organic chemical compounds are isolated, wherein, where appropriate, further components of the fermentation broth and/or the biomass in whole or In tractions (> 0 to 100 remain: in the isolated product or are completely removed.

The, ip particular recombinant, microorganisms with an expressed or overexpressed aroG alléié, which are: also a subject of the present invention, can produce the organic Chemical compounds írom glucose, saccharose, lactose, fructose, maltose, toolasses, where appropriate starch, where appropriate ceihdose: or from glycerol and ethanol and where appropriate afro from mixtures. They are members of the FnteeofraCterlaceae family, selected from the genera Escherichia, Erwlnla, Providencia and Serratia. The genera Escherichia and Serratia are preferable, in the genus Escherichia, especially the species Escherichia coll is to be mentioned, and In the genus Serratia especially the species Serratia marcesceuS,

Recombinant microorganisms are generally created by transformation, transduction or conjugation, or a combination of these methods, with a vector which contains the desired gene, the desired ÖRT, an allele of this gene or ORE or parts thereof and/or a promoter enhancing the expression of the gene, allele or QRF. This promoter is the promoter derived by enhancing mutation: from the endogenous regulatory sequence located upstream offre gene, allele orORT,

For the production: of the strains of the family EnterobacteriaeettS: accumulating the organic chemical compounds, which contain an aroG alléid,, strains (starting strains or parent strains) which already possess the ability to concentrate the desired organic: ebem leal compound in the cell and/or to excrete it into the nutrient: medium surrounding it or to accumulate it in the fermentation broth are .preferably' used. The expression “produce” can also be used for this. In particular, the strains used for the measures of the invention have the ability to concentrate or to accumulate > (at least) 0.25 g/1, >0.5 g/1, » 1.0 g/i, > 1.5 g/1, > 2:.0 g/1, > 4 g/1 or > 10 g/1 of L-amino acids, preferably L-tryptophan and L-phenylalanine, in < (at most ) 120 hours, < M hours, <48 honrSi < 36 hours, < 24 hours or < 12 hours in the cell and/or In nutrient medium or fermentation broth respectively. These can be strains which wore produced by mutagenesis and selection, by recombinant DMA techniques or by a combination of both methods.

Said L-amino acid-excreting «trams produce one or more, preferably one or essentially one amino acid selected from the group consisting of L-asparagine, L-threonine, L-serlne, L-giutamate, L-giyente, L-aianine, L-cysleine, L-valine, L-methmoine, L-proiine, LAsolepcine, L· leucine, L-tyrosine, L~ghenyklapifis, L-lyssne, L-trypíophan, l^rgi8Íoe.«í p*$|erah|? £g|ec&amp;á from the poap consisting edf L-drreoame, t-bomosetine,, E-leucine* L-valine, E-alánűk, Msoieauine and t-hisiiditse, ihéi&amp;Sft L-ssnitm acid of atóaó àdd âsb''éemprfê«s:ll« -sahs testi,

Th® term >!ope w essentially ose amioo adl8 takes acsonnt of frm ihet ihat p addition te the desired L-amlro seid one or pore other said L-amino acids (side ammo soils) can he prod seed. The content of those side amino acids is » t) id at post 40%, preferably > 0 to ai post 20%, particularly preferably > 0 fn at: most 10% and qoito espeelsliy preikshiy g ft ίο at most 3% based oft He quantity of the debited L-amino acid.

As LArypipgkao-prodtfring or excreting stratfts of the genus Escherichia*; to pameulaî m the species Escherichia coli, suitable as the peront strain, the following eats tor example he mentioned: » Escherichia eoh JP473$/pMU3028 (U$5,750,34% * Eschenchia coll IFftOil 5/pMU9l i'ü$5,?3d,34% « Escherichia coh W i b4ípGH5} í WCj94/08ö31 i * E. cob AGX(7ipGX44) (hiHRL 0-13343) (ÜS4J71,614) * E colt AGX6ipGX50)aroF ]£NRRL 8-12264) (US437 i ,6 i 4) * Escherichia cob AGX!7fpGX50,pACiS(34pps Ç WO07/0S233) * Escherichia coli ATCC 31743 {CAlt 82409} * E cob CS34/pi>?3 HEpDM 136 CAW 30795) (WOri/öi ! 30} « Esehenctu« col i iB 10;2/|>5f..R PS;·: <U$5..939..295). i..-ir\ ptoph&amp;n-produeiog φ excreting shrams from the Enterobaetenacese family preferably possess, inter aha, one nr more of tbc genetic or phenotypic characteristics selected front the gfdttg consisting of: resistance to 5-peihvbDi.-tryptophan, resistance to 5-Euoro-tryptophan, resistahce to 4-meihybDL'trypto-phftd* resistance id h-PCfityi-fll.-ftypiophan, 'resistance to 4-ΠΡο*οΑίΤρίορΡρη,: resistance to tufftmto-dryptophan.ifesistäftee To anthramiate, resistance to tryptazad* psistpsce : id rudoie, resistance to indole-aery Ik: acid, reiidh’Cpeni for phenylalanine, rcqniremcnt Pr ifroape, phtïre appropriate ability to mii ko saccharose, enhancement of the tryptophan opérons* preferably of the anthranilate synthase preferably of the ièedbaek reGUant form, a partially defective tryptophan y i«tRN A synthase, an attenuated tryptophan uptake, is defective trvpiopbanase, inactivated: repressor proteins,, enhancement of serine biosynthesis, enhancement «EpbespbbetKdpyruvata synthesis, enhaueemepf: of P-eryihrose-d-phosphme synthesis, enhancement of :l-deoxy'D-arabino-hep-tuio$omrte'7-phosphat« (DAHPs synthesis and enhancement of cbottsioal« hiosyfttbcsia.

Daring the sktdies on which the invention is based, it was found that, after expression nr okepspression of the aroGipileies, toteroor|aoistns of :the Baferobaefenaedae: lanrily produeesEamind: acids, iis pamcaktr t-trypto|Í8b, ift gréarer pahtify attd: accomftlath them in tin del or in the tnedifttn.

The nucleotide sequences of tie genes or open reading: frames IÄF) of Escherichia colt belong to the prior art and can he obtained fron· the genome sepettce of Escherichia cob published by iiaftnet et al. i Science 377: 1453Ί462:|1993)), ft Is kftbwn that the N-tormitmi amhm acid aMlftpftipe catrlo cleaved: off by enjptses intrinsic: ip the host {maihioams .pstfeppepitdase)r.

Pro;« Salmonella typ.im»;»imo and Shigella Sexneri, which also belong to the g«ters>hseter«eae family, the nucleotide sequence for ;he aro<) gene is also known (Access;»;; No : N€ 01)3197 (REGION: 823772-824324) and Accession Ko.: NC0Ö4337 {REGION: complementary (T? 1488-572541)}}. Födhet nucleotide sequences for foe aroG gene are found fro;« the following EnterobaGema-eae: Shigella boydll (Asiesssfoo No.: CPihHXKfo): Shigella dyssofotiae (Accession No,: 00()00034): Shigella sonne; (Accession No.: CP0b(s038;; Salmonella enterte;; (Accession bk;,: &amp;§8il2MY> and Serratia proteamacuíans (Accession hi».: €9000826).

The aroG gen« of Escherichia coli K12 «s »her als» ílescribed by (he follÄsgttatemahts: h4iîït;«s: 3<-:desoxy->i3-ar8htno>t;e|ô«i<sson3îe-7-'pbos(;hai«: $fnth.ase (ÖAHP synthase:} ercsG

Phnciio»: Iron-depéndent tnetáUnnnayme which is regaktei or inhibited: bosh by |en«ttc regulation via an«»aa;ion and repression.:. g«d also «iHoaforkaUy fey L~ phenylalanine. The esuynie condenses pbosphoeBolpyTnvafo (PEP} No;» giycoipis: and erythrose-A-phosphate ; H4P) kfoirthe peiifose phosphaté feÄway in tfee ifoltta} rernkfon of the aromatics biosynthesis pathway to give DAMP,

Reference: Ray et ai. Jobrhsii bi Bacteriology ί30(3:2):550Ο-·4: (1088}(

Davies et ab, N seietc Acids Research 10Π3) :4045-8 (19823|

Prost and Drstbs, Atpfoai review oP mierofetbfogy, 49:552-79 {1995};

Pit-ard. Genes to Ce|y 1(8) :717-25 (199$)

Access«»; No.:; KC0Ö0913 (Region: 7|4J5ő'->f85.9ö»>

The polypeptide encoded has a lengd; of 350 amino seeds.

Ai tehnfeiivfe: gone 141734 name:

The nnciete aokl seqoenoes cap be obtained kom the dstofeases of the Pstfooed Genter for Bloteehnofogy information i'NCBl) of (be National Library of Medicine (Bethesdo, M13( LSÀ), the nucleotide sequence database of the ffoygpesh lÉ&amp;focalar Biologies Laboratories (EMEL, Pfoidefoerg, Germany and Cambridge, UK.) or the DNA düÜÉiiüe of Japan (13DBÍ, Mishima, Japan).

For better clarity, the known sbgaehoe for the aroG igene of Escherichia edit is show» folder SEQ ID No. 1 and the known sequences for the aroG genes of Salmonella typhimuriimr arid Shigella. Oexneri under SEQ ID No. 3 and SEQ 113 No, 5. fhe pmiefos encoded by these reading fra;:nes are shown as SEQ ID No, 2, SEQ Hi No. 4 arid SEQ ID No. 6. The known sequence few the aroG geifo of Escherichia col; including the upstream at;d downstream located ancleotsde sequences is shown under-the SEQ ID No 9, and the protein encoded by ibis reading ffofoe is show® under SEQ 113 No. id. lis genes pr ppp jeadtng frames àewÜk^! m She st&amp;feéife^.pssages cart be used aeeordiag: k» lile invention. : FhrthemäSre, alhdex of the genes or n pan reading tamss which result from the 4e$m-mtcy of Ét genetic: code or by iyoettan-neuirai setiSp ifeÄllöhs can be used* "£be ose of endogenous genes or endogenous open reading frames is. preferable,

The alleles of she aroG gene which contain fuaction-aeafca! sense; mutations tncluáS inter alia those which lead so at meal 30 m to as most 20, proferahiy so at' most Id or to at.·most 5, pits especiidiy fefeferahly to m most 1 of to at most 2 or to al least one conservative: amino acid substitution Is the protein encoded by then·*.

In the case of the aromatic amino adds, whso phenylalanine, tryptophan and tyrosine am substituted pf one afe deferred to os eenservailve; .substitutions. In the ease of the hydrophobic amino acids, when feuern«, isofeoetne and valine are substituted for one another, these are referred to as conservative substitutions. In the case of ihe polar amino adds, when gfetsmme soli asparagine see substituted tor one another, these are referred to as conservât ive stsfestiubious. In the esse of the baste amino adds, when arginine, lysine and histidine are substituted for one aooiher, these are referred so as conservative substitutions. Its the case of the acidic stmhso acids, when aspartic acid and glutamic acid ere substituted Ibr one another, thesefare referred to as conservative substitutions. in the ease of the hydroxyl group-containing amino acids, whea serioe and threonine are substituted for ope another, these are rbferred to its bohservative substitutions.

Similarly, those nucleotide sequences which code for variants of said proteins, which additionally confetti at the: bo or iDdermsoes a lengthening or shortening hy at least one 11) amino acid can also he used* This lengthening or shortening antoitnts to not more thus 40, M, 21), )0, 5, 3 or 2 amino acids or amino add residues.

The sdtable aiieies also include those which eods for proteins in which at least one 11 ) amino add is inserted or deleted. The maximum somber ofsuch alterations described as sndels can relate to 2, 3, :5 or 10 hat in no case snore Shan 20 amino acids.

Those skilled 1st the art will find directions for the identification of DMA sequences fey hybridization inter alia In the rhannal ’The DIO System Users ön ide for Filter Hybridization“ from Boehringer Mannheim OmfeM (Mannheim, Oarmany, 1993} and in Ltehl or afe (tnieraailonal foornal of Systematic Baeterlolagy 41: 255-260 (1991)). The hybridisation lakes place under stringent conditions, fa other words hybrids are oniy femed in Which probe and target sequence, fee. the polynucleotides treated with the probe, are at least 70¾ identical. If is known that the stringency of the hybridization including the washing steps is influenced or determined fey variation of the buffer composition, the temperature and the salt eoueeatrailoo. The hybridigetion reaction is generally performed at relatively Sow stringency compared to the washing steps (Eyhsid Mybrldlsation Guide. Hybaid Limited, Teddmgton. UK, 1996).

For the hybridisation reaction, for example a buffer corresponding to Sx 3SC buffer at a femperature front ca. :50*€-68°€ can he used. Here, probes can ttlso hybridize with polynucleotides Which have less: than 2¾% identity to the sequence of the probe, bitch hybrids are less stable and are removed by washing under stringent conditions. This catt for example »*; achieved hy lowering the salt ebneealrstiots to 2x SSC and where appropriate fisén to 6.Sx |S€ (Ths $10 Jlpfesn. :$||er’s: <3uids for Piks* Hybndwattotg Boeht inger Mannheim, Mtm-tlseim, Germany, 1993} during winch a temperature from ca. 50%'-ore, ca. 52^0-68^0, ca. 34%;-68°0, ca, 56Λ€-68Ν2, ca. 58*C-68eC, ea. nrC-ferC, eg, 629068%', ca. 6-^0-68%-. es 66°C-68*C is set. Temp6fat»m ranges frofe*·9#* 64°C-68®0>r ca, 66“D-6i"0 kró preferable. Where appropriate» it is possible to lower the salt coàeèatraiîon.to a e«mceatratioo: sskres|kîsdk;g to 0.2x SSC or O.ix SSC. By Increasing the hybridixatlon temperature stepwise Bom |PSC fd 68*0 in steps of ca, i-2. -(.:, poiyöödeotide fe«gon'»fs> whnkt lot example possess at least 70% tkkt least 80% or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, át least 95%, M least 963¾. at least 97%, at least 98% or at: least 993» identity k? the stetppa^P::^hite|^^e used or to tie nucleotide sequences shown io SHQ ID No. t, SEQ ID No. 3 or SBf| IP No. S css be isolated. .iprtber direction* for the hybridiaatiem are obtainable os the market is the fewin of so-called kits (tug. Did busy Hyb- frost Roche Diagnostics öatfeÉh Mannheim, Germsny, Catalogne No, 1613368). furthermore, for the manufacture of L-atsitto acids, is particular L-tryptophas, with strains of the Enterobacteriacese family, in addition to the expreisstott of We sroG allele, it earn fee advantageous to enhance one or .more engymefs) of the known biosynthetic pthwfeyf m enzymes fel the anapkrotio metabolism or enzymes lor the production of reduced nleotisamkle-sdemae átnueíeofkie or glyeoiykts enzymes m PTS euaymes or sulphur metabolism eppnes. The use; of esdogesoas geaes is generally preferable.

Thus for example for the production of Ldryglophan, one of more of ifee genes selected froid the group » at least one gene of the tryptophan operos coding lor atuhramiale synthase, anthraaf-ate phosphoribosyl transferase, phospktsrtbosyl anisrShil&amp;to isomerase* tfee intlole-O'-giyseitstfepfeoapfeate synthase end the kyptopkan systhpc (Applied ρ$· itpdpnmstsfü- ÄrÄfspt 18(¾. 181-191) 0979)), * fité SerA gptte coding;isf:3xpfensphogiytterate dehydrogenase (WD87W I t 3d) * the seri /gene epdktg fee hitosphoseriae phosphatase 19. < pi unt * the setG gsue coding for phosphoserhe aminotransferase (W0S7 01130) * the atpF gene txsdtitg Rtf !.. -tyrosine-sensitive DHAF synthase <WO87f0i 130; EPÍ27072I) * the aroll gene ifef fe'-ttyptophaw-sensklve DHAF synthase (WO97t0nS(>; EP1270721) * she pps gette coding ffer phospfioenolpymy&amp;te synthase ( WO96/08567} * the pæk gene coding for phosphoenolpyruvato carfeoxyklnese f W02004/1896125), * the tktA gette coding for transkctolase A t USS,168^136):- * ffee too gebe coding for transkeioiase B (i./S3,ife%0:5l|, * the gene product of the yddO Bscheciebis coli open reading frame {OR.F) (Accession Number NCÖ0Ö9U (Uegion 154431.7-:345193) of the National Oesker for Biotechnology Information (ΝΓΒΙ, Berhesda, MIX USA), 1Π449918), cast fee stmttiieneonsly ekbaheed, in particular oyefexpfessad.

Furthermore, for the manuifecture of L«atmno acids, in particular L-tryptophatu it can bo advantageous, m addition to t|® loss of the feedback inhibition of the aroQ gene, to eliminate undeshed skie tondrons (Nakayama: “Breeding of Amino Acid Producrag Microorganisms“, in: Overproduction!, of Microbial Products, Krnmplanssl, Sikyta, V&amp;nek (e<k.), Academic Frost,, London, UK, 1982)

Hus for esmtupe for the production of L'tryptophsn otto or more cd the genes selected fron) the group * the írná pec coding for tt^|»^ha«a$e (UP,371,614), * tip trp&amp;ígííris cpdmg: fur tlm repressor of the ftp gpdfoo (US4 J'f (,§ \ 4) * site tyrA gens coding for ehorisomte mumse T end prepbenats dehydrogenase (ijS4.;S/!,6id}( * the pbeA gene coding for ehorismam pfepfemmte dehydiopnsse (ÖI4Í7L6I4), * the rntr gene coding for the ttyptophamspeifle transport proiein (USSS§,345}, * the tnaB gene ending |bf tryptophan permease (l;S5,756,345), * the aro.P gene epihng for the transporter ipr ttrotpstfe antinn poids (US5.?5bJ4d|,; * the sdaA geoe coding far i..-seriue deaminase (EP#l4d$394, * the pgi gene coifing for ginixese·6 phosphate isomerasc (WÜ87/Ô1 BO), * the tyrB gene coding for the tyrosine aminotransferase (WO87/0È Mb) can simultaneously be attenuated, where appropriate eliminated or expression reduced. Where appropriate, these measures ate performed additionally to or in suitable coordination with the stated measures for iheenhmmement of genes for increasing the tryptophan production. in this context, the tertn '‘attenuation” describes the reduct tots or eEmmauon of the intracellular activity or conesmtutloo of one or more enzymes or proteins in a microorganism w§feh arc encoded fey the corresponding PNA, tor example by »stag a weaker promoter than in the microorganism or parent strain that is nomreeosnhinant fm the corresponding enzyme or protein or a gene or aileie which codes fof a corresponding enzyme or protein with low activity or inactivates the corresponding enzyme or protein or the open reading frame or the gene and schere appropriate combining these measures.

Through thé atténuation measures, the activity of concentration of the relevant protein in general decreases to Ô to ?S%, 0 to 50%, 0 to 25%, ti to Ul% or Û to S% of the activity or concentration of the wild type protein, or of the activity of cencestration of the protein in the microorganism or parent strain that is nomrecomfelnaot &amp;r the corresponding enzyme or protein, Microorganism or parent strain that is oomraeombintmt is understood to mean tlw mieroorpmism car which the attenuation so elimination according to the invention is performed.

To achieve attenuation, for example the expession of Pc prtea of open reading brames or the catalpie properties of im enzyme proteins are decreased or glmluated. Where appropriate, both measures can be combined.

The reduction of the gene expression can fee effected fey suitable culturing, by poetic modiication f mutation I of the gene expression signal structures or also fey aatisertseiKMA leclmolugy.

Gene expression signa) structures are ter example repressor gehgsy activator genes, operators, promoters, attenuators, ribdsdte« femdmg sites, the start codon and terminators, Those skilled lit the art will find information ο» this later alia lor exemple in lensen and Hammer (Biotechnology and Btoeoglueermg $&amp;r i#:h49S (1998», in Carrier and Keaslhtg (Bioteelmulogy Progress 16; 18-64 ((999)), French and Gerdes (Carrent Opinion In Micfobitddgy 3; 159-164 (2000)), KawattO et al, (Goclete Acids Research 33(19), 6368-6270 (2005)) and in known textbook* of genetics arid molecular biology such as fnt example the textbook by Kntppers pMolekulsre Oeneiik” j'kteleeniar Gen®&amp;s|, 6!fe Mlitida,: Georg Ihletne Verlag, Stuttgart, Germany, 1995) or that by Wahttaeker ("Gene and Klone" |(leseS :8hil Clones], VCH VeriagsgesellsehaC, Weinhenn, Germany, 19901

Mot&amp;tions· which lead to alteration or .lowering of Hie cal&amp;ly tic properties of eaxytxte proteins are: fenowh: iront the prior art. As examples, the papers by Clin and Goodman (Journal of Biological Chemistry 272; 8611-8617 :((992):), ¥ano «1 al. (Prnccedrpgs of the National Academy of Sciences of the United States of America 93: 5311-5515 (1998)), Wente api Sehaehmattn (Journal of Bioiogledl Ghemistfy 266: 20833-20839 p99l}> stay be rnemfottM. Summary accounts can be found in known textbooks of genetics and molecular biology such m for example that by Hagetsann (‘’Allgemeine Genetik" (General Genetics], Gustav Fischer Verlag, Stuttgart, 1986).

As mbtsliobS, transitions, transversions, insertions and deletions hf at least one (1) base pair or ttecleotide are possible. Depending on the effect of the amino acid sttbstifotion on ensyme activity due to tbs mutation, these are referred to as inlsseoce mutations or nousertse mutations, The toissense mdfntidn bads to substitution df a given amino aeld lb: a protein by abothef, where id partienlar this Is a non-conservative amino: seid substitution, As a resalt, the fanctior.nl: capability or activity of the protein is impaired and reduced to a value from 0 to 7556, Ö to 50%, Ö to 2536,Al to 10% or 0 to Sis, Fbe nonsense mutation leadS itb a stop codon in the coding region of the poe anti thus to premature interruption of translation. insertions or deletions of least one base pair m ope gene lead to iramé shift: mutations, which have the result that wrong amino acids are incorporated or translation stops •'prematurely.· If it stop eodpo arises itt the coding:: region as a result of the mutation, .this also leads to premttlure Interruption of translation. Deletions of at least one Ü? or severs! codons also typically lead to omuplete failure «film enr.yme activity. The redoction of gesse expression b^' suppression of te stop codpn «relation in the coding region by sttilable t-E.NA suppressors is described: tp ψΟ Ö3/0747I9.

Directions for generating: such mutations belong to the prior tsrt ant! can be obtained from known textbooks ei genetics má moleetdaf biology snob as for example; tie: textbook: by Shippers ("Molektdarc Genetik’· 6tSi Edition, Georg Thieme Verlag, Stuttgart, Gcrhlany, 1995), that by Winnecker ("Gone and KJone", VCH VetlagsgeselNehafi, Wclnbeitn, Gerthany, 1990) <sr thai by Hggesrtan« (“AHgenteiae Genetik", Gustav Fischer Verlag, Stuttgart, 1986).

Suitable mutations in thd genes can be incorporated into suitable StMns fey gene or aile!® substitution. A common method is fha : method of geno substitution by means of a eomVttottallv repbeating pbClbl derivative pMAE.703 deserlfeeh by Hamllfos et ab (Jourtts! of Bacteriology 171: 4617-4622 (1989)). Other methods described : hr the prior áft stich as for example that of:Mmtlttaa-MOfélés et al.

Cffeurmi of Bacteriology \Ζ\· 143-7US p^9$por thai pfeioÿd: et si, (l«Í difcaefeffofogy 1:82; §42-84? (glfofe)) can likewise be utilised

Il m sis© possible to transfer mmatfofcs tahe partfenlar genas ér mdtatrhhs wfelfefc rdatö: fo tfee exptessioa of the particular gases őr open reeling frames seta varfotss strums fee conjugation or tfeusdneiiom

Where appropriate, foe measures for enhancement sík! tor attenuation cars be combined in any way.

The performance of the isolated bacteria or of the fermentation process with mas of the pare with regard sa one ar more parameters selected from the group consisting of the product eotteenfratfou (product per unir volume). product yield (product formal per carbon sonree consumed} and product formation (grodud: formed per «all *Äme and time) ar other process parameters and combinations thereof is; ipereasod by at least: %$%, at fousi 1%, M least 1.5¾ ar si least 2% fessed on the osscrostrgsssisss: or paresst straisr that is noSprecootfeiohuS: ar ifee: forraeotaftoo process wisfe use of the sara«.

Accordihg to life Invention. the sniemotgaoisms produced sfe caltureiilh tie feafeh poeess, la the fod-batch proposs* in the repeated fed-hatch process: ar In &amp; continuous process (IMfdSifeddSdSStlJ -M:4^:5,763,2.30). fomtmanes ooneommg such: pt'Ocesses are available In the testboole by flhmiel (Btopuuexdcchmk I- Einführung m d;e Bloverhihrcastsefeuik |Eibpedcess tocfeoology I. introduction to bfoprocesS teclmotogy] fönsiav Fischer Verlag, Stuttgart, 1991)) or la the textbook fey Storhas (Biere aktoren and periphere Einrichtungen (Bioreädots and peripheral snstalSatlans] (Viewsg; Verlag, Brarmschwslg/Wiesbaden, 1994»,

Ip a batch ppsesis, With few exceptlpp» such as for exatnpie oxygen anti pH: eoffeetigg agents, all ingredients are ossemfeied In the fern t# a batch ami the nikroorganistn is cultured in the -medium obtained.

In a fed-batch process, the microorganism Is firstly cultured by a hatch process (batch phase), hollowing this, an Ingredient essential for tha puductfon of :te product, olsa wfeere: appropriate several ingredients, is led Into the culture continuously or diseonttmtoosly (toed phase). In tire case of the production of E-amino acids, this is or carbon source, A: repeated fed-batch process is a fed-hatch process in which, after terrhhkdion of the fenoeositioh, &amp; part of the ferinenfation broth 1¾ used as inoculum for the start of a further repeated fed-bstch process. Where apptopnate* this cycle can be repeated several tlöMI ifopafoâ -fod-haiili: processes arc for cxatnplc described in WO 02·' 18543 and WO 000)14843, in a coatsooous: process, following a batch or fed-hsUch process, one or more, ansi Where appropriate all, ingredients arc commuons!)· fed onto the culture and fermentation 'broth removed at the same time. Continuous processes are for example described in the patent texts US $,763,230, WÖ 03/014840* WÖ 05/014841 and WO 05/014842. »be clfitttfo: medium to hO: used must appropriately satisfy the retpirarnentS: of the particular strains, Descriptions of culture media of various midoosganlsprs! arc contained In she manuaí ffoanna! of Methods fobpehertd Bacteriology” of the American Bocleiy for Badetfology (Washington D.CL, 0¾½ 111¾ The tsms ««Hure tedha»* lepaemation mediae*, .ptá nutrient soedhxm or· rnodinm are omfetdly fiPexchangcnhle,

Try general, a g&amp;lturn tedium ter A nohlams on« : or mote c&amp;rfeön sooreofl), nitrogen sottelsf te phosphorus scntreefsh

As the c&amp;rho» source;, sugars and. earhnkpbáths noch as for: example glucose* saccharose, iaetos«, frtsefose, maltose, molasses, starch axtd?wfmite appropriate caillée, oils and Ä Such as for example soya o U, sunflower oll, peanut ois aatl coédmn Sat, fatty act# .such as tor example palmitic seid, stearic add asul linóiéin a:;isi alcohols such its tor example glycerol sod ethanol andMfganie adds soteps: fey example acetic add can fee used. These substances can be used singly or as a mixture.

As id mtrogen source, orgitidd nitrogen-contammg compototde suth *» peptones, yeast extract. Soest eixtred!,: iptall extract, convstedp Itipor, soya bean msafte urea* or Inorganic compoonds such as ammonium sulphate, a»mtôtùat» chloride, a;nmonhun phosphate, ammonium carbonate and arnmonmm nitrate nan.fee used. The mitogen sources can he esed singly or as a mixture. ás; the phosphorus source, phosphorto acid, potassium dihydrogen phosphate or dlpbitmsium: hydrngen phtssphate or the Torres ponding södlum-contaming salts can fee used. ,. Hk Idulture medium mast further estein sells of metals, suefe as for example: roagnesruio sulphate ox fro« sulphate, which axe accessary for growth. Finally, essential growth sabstanees such as amino acids end vitamins :#re used in addition to the aforesaid substances. Apart from this, sulfstfeie precursors can fee added to the cuttere medium. Said ingredients can fee added to the culture In the form, •#ffe slpsle batch or fed try us appropriate during't'hb mbtuflug,

The fertttestation ts generally performed at; a pH from 3.5 to 9.8, In particular 6.(1 to 8.0. To edistrti Ihe pH of the caitare, basic compounds such as sodtdm hydroxide, potassium hydroxide, ammonia m apucoos ammonia or acidic compounds speh as ptephorle apid or sulpimric acid are used as appropriée. To control foam evolution, not Team agents such as for example fatty acid poiygiyeol esters can be used. To maintain the stability of plasmids, subside selectively acting substances, e.g. antibiotica, can be added to the medte. in order to maipiam aerobic conditions, oxygen or oxygen-eoniammg gas mixtures such as for example air are passed ; info the codera; The temperatm« of the eedtore normally lies at about 3S0(T*o 4§SÇ; and preferaidy about 38<fC to 40*0. Through the activity of the microorganisms, concertfratids or accumulation of the Ipammo acid An the fermentation or culture broth takes place. The culturing is continued until a maximunt of the desired i..-am.ino add bas formed. This target is normally reached whhin from if) hoars to Mi hours. With confípnous processes, longer co harsog ti más are possible. A fermentation hrofh or cukoré broth Is understood to be a fermentation medium in which a tplproetgaxsism has been cultured for a certain time and at a certain tafapefaSerev

Ott termination of the fertneniatkm. the fermsoyatiem. hrofh produced thereinto contains a} the biomass (soil mmé of the nncroorgsirtsor fhrmpd ss a result of the pnlifcratton of the cells of the microorgbufshr, ;h| the L-amtrm acid: formed In the course: of the fermentation, e) the orgusie : aide products foroiod itt the eoorse of ihe fertomdailou má d): the «smponents of ihe fetmeiitaflon m#ipm/formenla1 bn media used ásd Oie ingredients such as for esÄplo vitamins seed its thsasalOa or salis sech as magnesium sulphate »|t cuOsiumed by the fermentas smb

Tim culture or fermentation broth produced can then be ce-Íeét«d and tbs desired Lräsblao seid Of Φ t,-a:mito>:Scidseö;ataiss:sg prqdsset obtained or isolat#

Ip orté process Vartans, where approprias* the fermentation broth is eoneeturaíéíl add the L-afosOo apid lises purifiai or à-olaled is pme or almost ptÄfhÄ Typical methods tor Sise puialmatloss of L-suntrso seida ssp loss exchange chromatography -extraction processes aol tif&amp;imeai

Wils activated ehareoisl. lo this manner, largely pare 1..-amino acids ere obtained wish a cobteiti of> 9b tyt>%:b;95 wt.% > 96 wt % J: ¢7 wt.39 > 98 xvs.3« or > 99 ws,%.

It is also possible· lo another process variam to produce a product from the i'ersnesitation broth produced:, by ctShpiatdv t I0d%> :or aiotost completely, M, -ämm (ban or greater than p| 96¾ >95%, >97%, >99%, removmg she biomass of the bacterium contained in the fermentation broth sod leaving the remaining components of the fermentas ion broth largely, ie, up so 3Ö%-100%, 4939-1ÖÖ39, 50%“ 1003s, 603«·-100%, 7039-1(10%. 8039-1003¾. or 9039-1003¾. preforabtf gpafof S81&amp;* >b05a, >7039, ;>80%. >90% or >9S% os' even entirely {10039) in Our products

For She removal or separation of the biomass, separation methods such as tor example eerbrifegasioss, fibrasism, decantation, floccolatÄbr a eombfeaiiorruf tiess arc used, 1 he brush obtained Is sheo thickened or esmeerstrated by known methods sach as for esample by sneasss of a rotatory evaporator, ism: il su evaporsfop* falling filar evaporator, bym verse pssnesis, by bassotltsation or a coorbirsatis>i) bf these.

This esmeenirsted broth 5s sheet processed into a preferably possr&amp;ble, Une particle powder by methods of tfeebe drying, spray dtyin$j, spray granulation or by other ftmmms. This pourable, tine psffielc powder: ctm in torts be convsri#|p# % pearse-gralned, ^Élpwabie. storable and largely dost-ied prodtsef by suitable cosnpaaing or gfesmtaAors processes, Dmang: this, the «Jper ds overall tip to more than 90%: removed, so that the wafer obesesst in the producf is: lese ihass 19 wiTAfess thae wt, 5%, less ihass: m, 43« or less thssn > m.%.

Tie isnalysis of feasoioo acids lot the dasermfeasloo of Ilse conceosrassoo at ttrsa or shore lime poios(s) To the coarse of The fermentation can be effected by séparas tors of the t-ssmtao aetds by ice exchange chromatography, preferably cation exchange chromatography with subsequent poss-eotussm derivatfeatfeo using: nhdjydnrs, as described in Spackmsssr et at, {Analytical Chcssbsfry 30; 1190-1300 (1958)), festesd of nStshydtin, ortbiá-phtíkiialáehydc cats also be uses! for the poss-cofemo derivssiksstiom A feylew article ott ism, ekehsitsge ebromatograplsy is so be found %pekerlssg (Magarlne: of Cbixitsmlograpisie S deuce) 7(6), 484-487 (1989).),

It: Is: Mm possible to perform a pre-columtt dmivatmation for ex-ample usmg ortho-plitaólsldebydé: Or phenyl feo-hfeeyanase and to separate the ammo add derivatives formed by reversed phase elfbshategrsSpby (RP> preferably the forth of high pèrfofmànce liquid chromatography' (HPLCf, Such s method is for eyansple sleaersbed m Lindmfh et at (Analytical Che-rosrry 51; 1167-1174 (1979)).

Detect}«» is effected by photometry (absmptfen, ffeorescenee). A staronary description of hntlup acid asraiysis east inter »lis fee found in site rextWöí "Rio&amp;n&amp;îytsk" lBsoa«alplS|by Lotispeissh sM Zosissss (Spektrum Akademischer Veriag. Heidelberg, Oermassv 1098.;.

The process according to the invention is used for the fermentative umauiacture of orgsmie ehesasostl Compounds selected írom the group eopsisdug of the cemppsmds of the shtkimate tnetaholie patbcmp the amsaaspo: Lmmlsio acids: L-tyrosme,: L-phsnylaksrtnë and iHs-yptpphM, cfeocisipai.«* eaterob&amp;cflrp menapnippne,: tetrahydrofokse and ubiquinone are prsderuble. L-tryptophan and L-pfeeny hissisue are esspeesai Jy prefesable. sEWJzmii listing

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Civ .Asfi 01'/ Asp Cys His tip rig .¾¾ Asp illy Oily lips Git- 8ra Asb 2/5 235 ' 255 ' 2s0

t«a age gag asp cae gtt get. -gits gtg a&amp;a ga« ggg ctg asc a sa ges ?6S ‘7'7'.7 O77 7 Alii l.yt iil s '.77:7 7:0 -:7 7:7.0 V.7.7 :.777: 0:.7 77 St y 1.777: ÄÖ.33 7.7::7 7: : 0 245 250 255 gee 77tg et* syaa í7ífg gtg «77g ata gat: ttot as/a cat get a&amp;c tag ta.'; 83 6 <3îy 52®'» Óra Als Mlrs VgJ. Sígs .1.:.« .S-Sp .80s 5®r HiS Als 5.74:3 .Sas 0:3,7' 200 2SS 230 aga caa tte a»:S sag sag a tg gat gtt tgt get 7isc gtt ege esg esg 864 :77::7 7:3 77 7·7ΐ7 :77:7 Av-33 Sitii 0-777 ASip Val 7.' 7'77 A.Uí ASj? V':St CV77 Sil« 00.73 275 ' 255 285 ste gec. gge gee: g*a sag get a et alt gge geg atg gtg gsa age esi; :H2 lia Alá S3 y Oly fit« lys Als i ts: Ha Oly Vai Siet Val St« Ser Mis 250 255 500 7ttg gtg gsa gge tat esg ágé ctg gag aga ggg gag aeg at g gge est $68 l.ay Vöt Site Sly As« Site Set 1,·«« Site Set Oiy Sty S'tc· 1st. Ala Syr 355 ' 510 .0.5 220 gge sag age ate age gst gee >.:ge 73*777 gge egg ass gat «ee gst age 5.0:08 07 7; lys S-s;·. lie Ti3.t «BO Sis Cyss 23« SUy 37:p isi'« Asp ’·57.3' AS';ÿ « : a 325 ’ ' 830 350 etg .tts çgt gsa 37eg gag ass ges gta Asa gag- egt ege ggg tas U2S3 3.33:,7 /.-S'.S Atg Stfi i..>?37 Ä.3S As« iUs V«5- Sy« Ala Arg Arg 81 y 3 S Ói 5-55 338 <2\Ô> 2 <2H> 3SÖ · 212> FRT < 213> Escheric-hk coli <4m> 2 ’Mat As« ïy'37 8i>7 Ass Asg Asp isik? Arg île lys St« lie lys <2:337 /.«ts t ' 3 10 Si «au ?eo /773: Vai Aia Lee Ia3« ollxs lys î!h» Àte· Als The St« As« ils 20 25 30

Ate Ast Thr V«3 Ata 81a Als Atg lys Ala IXe His lys île lay lys SS 40 55 öl y As« Ásp Asp Ary Ls« Ias l?ei V&amp;l SU Qiy Pro Cye 2er lis Säle 50 55 20

Aap Pr« SQQ. Ms Ala »ys Qi* iyr Als ihr Arg *:« LOS Als ;..is« Ass 55 30 75 50

Sis Qi« SSsyj >3ya Asp >51« Aa« Qi« 11® Val îfcsi·. Aztf Val lyr S*h« >51» 55 SO ' ' 55 'ays ix» Ar« ihr ihr VAX Qiy 2«p Ays Qiy Aea 11« Ase Asp Pih Hiss U5Ö 5 05 1115 hat Ä5p As* S*r 5;ha Ql* :;>a A&amp;P Aap ölv 1.*« Ar* 555 a Mä Ar;; I.y* 115 120 ' ' ' 515

Ahr. i.a>3 hau Asp lis As.« Asp Ser Oly l.s« Os o Ale Ala Qiy 05 s Ohe 1515 155 ' 140 ï.ess Asp Mer 15.* ihr Oxa Qis Tyx Ase Ais Asp iss« Hai Sax irp Oly

•40 1015 1 SS· IW

Ale lia illy Ala Ary Ihr ihr Qi« Shr öle 551:. 5Us Ar«· Ql« Ae« Ala 1Ö5 1755 Π0 5sr Qiy im Ser Cys Pro Val ciy 5?ha Ays As« Uly ihr Asp Gly ihr ISO 105 1515 11* Oys Val. Als lie Asp Ai* lie Ass AS.*. Ai* Sly Ai*. Pro Oie Cyss 155 050 200 sOse 1.«« 3ar '0*5. ihr Ay* tip Oily Sit* ösr Aia lie VsS. As« is·:! «er 2115 015 120 QS.y A;$e Qly Asp Cye Ois ! 1* I;6 As« Arg öl.y Qiy Ly;; Ol« pro As« 220 2515 235 210

Ayr Oer Al* Aye Ais Vai Als Qis Vel 5>ys Qis illy Sas» As« Ays Ale 240 200 250

Qiy Lea Pro .Ai« «1;; Pal P*t 11« Asp Phe Oer Síi* Aie A»;·. Ser Oar ΐ 00 2Ó0 270

Ays is iß 5-he Ays Ly* tu» iMac Asp Vsi Cys Ai* &amp;sp· V*.5. Cya Sie Qi« 275 280 255 ils 1.::> Qi« SI* Oie Los Ai* 11* 11a Sie Vai Pier. Val öl« Sar Lis

2015 ' ' 255 ' OSO

Les Val Qia Qly Ass Ql« Ser Les öl« Ser Qiy öl« Pro Aas Ala ivr 305 010 315 320

Qiy Ays S«r lia ihr Asp Ais Qya 11* Giy Trp Qis Aap ihr Asp Ai* 320 030 305 les Ae« Ara Qi« Aas; Ala A;;« Ai« Val 5,-ys Ai« Arg Ara Qiy

340 .315 ' ’ " ISO

<2 H» 3 <2Π> 1053 <2 i 2> DN A < 213> SsIhíöííóIísí typtrimttrit»» <22 Ö>

< 22 ! > CDS <: 222» (1)..0053) < 223» smG máim régim <4ûÙ> 3 »feg e»fe t&amp;t c»g me §&amp;c sat its css act ma gaa alt aac gag feta «8

Mai: As« ?yr Oie As« ä«j> Asp As« Arg 11« &amp;ya Sis ïl« As.« Siti Are;

i S ' .Î.0 iS tfe» cct teg gtc geg teg ctg gsa tag ttt cet «ce «tg «sa «et gc» 33

Aea Fre Asc Val Als I.«« Ae« Si« Lys Ks« Sic Al« SAï Si« Aas Al« >v as st

gea aat «ce ait gel cac «co ege s&amp;a gcc sic est asa sfefc etc «sa 1;M

Alit Asa ?fe:v Va.;. Alá Sis Al« &amp;r« Ays Al« Ala Kis Ays Al« As« Ays 35 4Û it «ge »st gsc gal egt ceg ctg gfcg gig aie ggt «et tat fe« «it cä'fc 10?.

Si y As« Ase Asp Atg Lee As« Val Val lie Sly Vre· Cya t&amp;r il» Oi.a SS SS S3 gal ec« «es ge«; gijg a«s gag tat «es gas egt. t.feg etg geg eia ege 330

As« SA.;·.; A.la Ala Al« Ay« Ai « Tyr Alá Ai.·:;. Ar g As« Aö« Al.a A«0 As« S«' ?S 35' ss<f gat gag ett aas gge gág ett gast alt gl« stg egt gte ist tat gag «tfe ASS Síi; Ls« il;« Si'/ C-Ii« lant SiV; lis Val list Arg Val 'Ayr 555,*. Sí.·..:

SS SÖ ' ' OS

sas ecg cgt. see «ce gtc ggc tgg «sa ggry ecg· alt sa-s gat teg cac SOS

Ays Ar :.; A .re 'Ihr ΤΑ.« Val S i y rr.A Lys C:iy Aas lia .A AA. Asp Av* Fia IVÜ ' 105 ' lib «ta gai «ae age tec cag ait aac gae ggt etg egt ait geg ege «a« 3Si

Mai Ase Ass .ter 30.« t:i.A ils As« Asp ·*': ; y As« Asg ils A.ta .Arg Ays US 130 135 et.g et.g eig gai apt ase gae «ge ggc etg cet gee gee ggc gaa tte 333

Aise Les le:: A s y Ils ÄS« Asp Le.r Sly As« e Al« Ala Sly fils APS Ï ïô i 35 ' 130

ett eat Atg alt ssg ctg tea feat ctg get gat its aig agt igg ggt 4HO :i«e Asp wet lis the Are sis Tyr Ae« Ale Asp l·»« fiat Ser tr-p Sly is« :l«0 ISO ; «0 gee este gye a&amp;-¾ egg act «et Si«» fcc* ceg yt3;. esst ege g«;s ístg g?;y 528 A3.« sie es y- Xi.» Ài'û 'Sisr COS; Si« Ser 03 ft V» 3. fii.e's fi rg Si« XS.»

255 170 ÜS tes ggc ccet tyt eey ytc ggt tôt »«» «At «gt »ce gsit «y« sscy Si» S«f GX» Se» Sssr. <2y» Cto vÿssï. <3iv C«« :..yxs Ast« Xi.y TKr As'ig Xi» sCiss.

isc * .m ' ISO »fct s«a yte ge« satt gae ge« ate »es.· ge« «ce ggc geg ec« ce» toge 525 U« Lys Val Ala IS« Asp- Ala lie .«se Alis S1« GXy Alá i?ro iüs Cya i»5 200 ' 205

Ist« etc ts.ses ysss.; »ek *»« v.gg g«t csit tcy gog akt gta ssat ess.as age S'?2 Cég i.ge S«r 0»). tfcir Ly» fctp Gíy »iss Ser; Ais» 2 its lX's.1 Asus Ihr See 210 .as ' 220 gae as« gee gs»c tg« stet <v.c att ctg ege gsye ggt s.a» geg cesi »ae »20

Ost» As» 02y Asp Oys fi.èss riss Ile »»« Arg sls.y Gig Ly» AI« Sie·« Asjs 225 ' ' 220 235 ' 220 t«t .»ge gog egg eat get get gag gtg ««&amp; ga« gg« etc see a»a gog 358 '3'yr Gsss' Ais» Si:»·; fii si SVi: AG« Giss Val A y:':· G.Ui Si y Less TA» Lys Alii 225 250 255 ggg ctg .acg eest ts»g gts; gtg atc gat »te age cat goe a«c es"« ;:gt Si» öl» :0β» siót fi.es; Al» 0»1 Kct ils As* sfiiss S«.v K.ia Ai» As"»;. 3«r C»a 200 255 220 «ssg ca» ott cas say «ag a «g gag get Cyc g ce gst ge« tge eag csg 553

Lys 02a Gisss 05.« 3.>ys giss fiiét Oie Oa.s Oye» A.la Asie '».ai CysS 0:0". Ois

275 ' 280 ' ’ 2SS sitii syeg ggc ggt gaa aa» gets sitt «te gge gtg «cg gt» gsg «gt cat SÍ2 ïi.e Ais» 02.y giv Xi .s «ys: Ai.a 21» ïi.a Ci» '»as Ost V»2 Sie Ses. fi.is 250 ' ' 205 ' 300

stt.g gt:» ga» gga oao eag agt. ctg g»a age ggt. s:«g ccg: efc>g aese tse.: MO I.sssi «sis. Ois.s C ty Asses tils', ses: s: Oses sis.:,: 5-ajs Gi v Slss Ose'; Osss T si s T;2.t 305 ' 310 lit. 520 ggt ;»«» »«c ssi.t sice sg.ac gee î.ss·:.· »tt gge tgg gsia gát. ace sg.at as;« 2002 02» 2sys See lie Tis-t Ase Ai» Cys il» Oly txp Sis; Asp t&amp;s Aso A3.« 325 130 335 s;t.g ctt. «gt. e.«g tis.g teg ,yss« tjs.sg gtss. ss.a* gess «gt. e«c s;s.;c ta» 1055 ïasis Lee Arg Gis Leu 3ec Alss 3U« V»3. i.ys A3.« fi.tg Atg sïi.y 350 555 350 < '2Í .Ö > 4 <2 ri >3 50

< 2?.2> F8T < 2D> SslmoaeUa tvpkionsriss?! <4Ô0> 4 îfiit. Ae« '*tyx Oi s; Ass.a A»p A«c s.eu Arg 2 le l.v» G.sa iiss Ass» 03s; Osu • 5 ' 20 ' 35 îas» OrsS' Or« VsS.3 Ai* Les> 3;»« 531« Lye 00« s-'ro Al» ilsr ölsí AsSSS Al» 20 25 30

Ai«. As» tbx v»i si.» His Ai a A*« Ays AXa lia His »ys Ua X»«« Ays 4S 40 4 s

Gl y As» Asp Asp S.sy is;« i<«p V;U 0«3. .Π.» Gl.y p.a» Cy» Sy;;.· XI.« Hiss SG SS SO .Asy Hast Xl.a Ai* AXa I.;,.'a Gl s Tyr G.Tg AG s Ar ·:.; s I.ga Ais Aas T.r a δ 5 TO " ’'s 00

Tags· Gla .Aas.. Säst Öi;y Gis; .Gas ßU Xi» Val 3i»i. A.sr; Vaa Tyr »ha Qi»

HO 30 ' OS Lÿss P.a» Aaa; '.£.»* ï>vr. Val SG.y 'f.Ap Oy» Si y t«vj Xi« A»h &amp;§£ P.a» Bia •5.00 i OS 113'

Vf A A Asp A. AA AAA SASA GAa A i A Afi.» &amp;S}S G ; y AA S: Â:AJ Si A: Si» .A :S:S ÎA/iS

US X20 ' ' ' US :;<&amp;« tóií iss« Asp ïî,a iss» Mj> S«r Si y iss« Hi fi A3.« isi* Si y Sis Sh« : SO U&amp; 3 40 X>«ss Afsp 34« P XI« Ï3sï Ha» S3.« Oys Os« A3.» 3Up ·.«» 34« P Se.s T.Ap Qi y 3.40 ISO 3. SS 2 50

Ma XX« Giy Ais Asvç ®»·* 'Sirs'. Úts Sas Sia \>«i 8i«· Si'«· CSia Ghaa Ma 3.05 UO :i 7 5 fis: s Giy r.A r< G- A s G sa G .so 'va À fTiG. s Paa Lys As» Giy T;': s Asp G fy SSiss

3.00 ' ' ISO ' XSO

Xi» «y» V»i Ai.» XX« Aap A3.&amp; Xi» Aah AXa Ai» SX·/ AXs Px» âii» Cys XSS 200 005 P5« Les.; S«sr Sis 3. Th.A X.ys S.asa Oiy Hifi fiefr Ma lia V»X San ïif.A 8»*· 220 2 XX 220 Kï.y .San Gly Asp Cas Sis Us U« ?.·»» Au SXy Giy l.-ys Ai.» Pa.p âsn 225 ' 200 245 240 ïyx Oax Axa G3.a Hia S'ai AI» GU Vs3. i.ys GU (aiy i.«h 'ihr X>y» Aia

24S 2SO 2SS

Giy lAv» l'HfC P.ao Gi» Si»2 35*P Xi* A»p .Has S«ï Ois Ai» .Ass G«« Oys 2 SO 25S 220

Gy s Gif; !:··»* öl.» l.ys GXrs sAaa GU SiU G v A Sia Asjp irai Ov» Gin VT is 3:2 S " 200 ' 205 XX« AXs Giy Giy St» Xys »2« XX« Us Giy :%s.l 34« » vaX Gis Osa sis sgo îss los I.ea V«3 Sis Slÿ Asa Sis. Ser Levs Gie Ser 0.2 γ G2a ÍArö I.Cs 23hr Tvr 305 310 BIS 320

Sic Tá'S sár île tas·. Asp Ala Cvs> lie Slv ire Ois Asp Tar sse Ala 32 S ' 330 335

Les Oes Arp 02s Les Oer Ala A3.s Val Lys Ais (Lrq Arc Si y 340 325 ' 350 <2105

< 2 ! ! > IÔ53 <2I2>DMA < 213> Shigella íteserí <230

< 22 î> CDS < 222> (i).,(1052) < 223> aroO ooiliïsg regiôa <400 5 acg asc tat e«g ass qac pat tta ogc etc ass cas ate aas gag Lia 35

MsÇ s;.« Ty.ï Ois .Sas Asp Aep Las Ary lia ,LyS Sla lis .Lys' Sla Les 1 5 .3.0 ' 15 ai.!; ccî. cet g te ses tfcg ctg gsa aas etc ;:ce ges set gaa «st get §6

Las Osa Aris Ksi ALa Lesi L-sa Sis Las Poe r'tc Aie Sic- Gis «es Ale. 30 25 30 gcq a«o .agg gát er;o pas. sec ega aaa gcg a te scat a,eg afce ctg saa 143

Ai.« Las ter Vei Lia Ors A3.« Arq 2.y;i Alii 23e Sis Lys 23e Lac Lys 33 40 45 qgt «ai gai·, pat ege aeg ttg gtg gcq atc Ope ccg tgc Lcà act cet ,1233 (33.y Asr. Asc Asp· Aeg Les :.a s Val Val 21e Si y Lr c. Cys Sar lis Lis 50 ' 53 00 pet, ccc pte gcq get aaa gag tat gcc sot ege ctg ccq gcg ctg cgt 240

Asp Stro. Va 3. Als Sis Lys Sia Oyt Aia T5r ?i.rg .Las Leu .Lié .La-c Arg 53 30 ' 75 00 gas pegs ctg sas gat gsg ctg gáa acc gts stg ege gtc tat tet gas .205

Sis Glu Les Lya Asp Sla '«as Sic lia Vai Mac Arg Vai tyx í:ha Gis 35 30 35 aag ecg cgt esc agg gcq ggc egg aaa ggg ctg act sec gat ecg esc 334·

LyS Pro Arg ÎLr 'Ihr Vei Oly Trp Lv&amp; Si y Las lia Asc Asp Lacs Ois 100 ' 103 110 stg gst sac âge tcc cag atc sac gac ggt svp ogc ata geo cgt as« SS4 tet Aap «sa Sac Os«· Sla île As« Asp Oîy i.eu Srp lie Ala Arg '«ys U5 lia i V5 ttg ctg c:.? g-At set saa: ose: ago ggí: ctg ces gcg. gag p:gt gag ttt 432 îa;c Les 3..AS Aap ils Las Asp Ser Giy :Lac Ars Aia Ai« Giy Gic è'Ls

130 230 ' ÜO etc gat oiV.g Aie set oet City tat etc get, geo etc aty apc tgg pgc 400 33.es Asp Met lie ïî's.f Arc Ois l'y.r 33.es Lis Ass> 32.es .tet Sa.r 'tirs (32 ÿ i-ίδ 3 SO 103 tso

ges ate -¾¾¾. gos egt ate «eg g«a t.cg eag 9'fc§ ose «9« gv« «>,%· 9*9 SSO

Ma ils Ci y ALa Sr:g Tir Ttr ··:·· « Sat Ci λ Vai Sis Arg Sis.; Lés Rsa ISS Î78 ' its

Set ggt ctt tet tgt ccg gts ggt ttt «.sa sac ggc ses gac ggt aeg SIS

Ser Civ tes Ser Cya Ceo Vai Civ CSe Lys .Rsa S3.y Vár Asp Civ VAr USö iSS 3 50

sit «sa gig get ate gat gee att sat get gee ggt geg oeg esc tgo SOS

Xie Lye Vai Ms Sit Asp Ms Xis Asa Ria Ais Sty Ria Pro Sea Cys i30 208 080 ate ctg tee ga.a set aaa agg ggg «sc Ltg geg att. gtg aat see agt 072 PRe Lev Car Val ihr Rys Tag Giy Kis Ser Ria lie Val Ass The Ser 2)0 2iS 828 agt a«e gye gat. ige eat etc att et.g ege ggs ggt sa« gag cet aac 028

Si y Asa Sty Rap Cys Sts lie ïie I.ea Arg Si y Sty R ys Sit Pré As« 220 230 235 280 tse age geg asg erre gtt get gas gtg aa« g»a gag etg aat; aaa ge« 000

Tyr Sas Ais Lys 8ta Vat Ais iS is t'ai Rys Sis; Cly As« Aas Lys Ais 240 208 2S0 ggt ctg esta get. eag gtg atg ate svat tie age e.at get aat teg tee 82 0 •iiy i.vs'a Are Ata Cie Vai fïet Lie Asp Sise Car Sis Ai« is is Ser Ser

V 5 ’0 2 00 V 8 V sas css tte aaa ahg esg aig gat gtfc tgt get gat »gtt tge esg cag 004 l>va Si a Pia Lvs Lys St a Set Asp Vai Cva Ala Asjs Val Cys Sis Sis

à" S " ' 280 ' 80S ait gee ggt age gas aag gee ati att g-ge gtg sty gtg gsa age eat Si-2 a lii A.ia slv t: y ..:i a rys «: « i.;e : ;e v>-iy vai Mat Va; t;s aar κi.e 280 ' ' - 2S5 ggg ctg gtg «as ggs aat tag âge etc gat ago gag gag ceg etg gce tat ΟΌ0

Lev Vai Cts Oiy Ass SA.« Set Asc Asp Set Ci y Sie Pro Re» A.ia Tyr 380 3i0 3 t 5 320 ggt sag ege ate «ec gat gce tgc ett ggt tgg gaa get «ee gat got 3808

Si y Lvs Rar tie ihr Rep Aie Cvs iie Si y ïrp Cio Asp Ter Rsp Ris 320 338 330 ctg its egt c«a ctg gtg soi·, gea gfco aaa gtg ego tge ggg iaa 1003

Lev I.ac Ar g 81a i.sv Vai Rsa A.ia Vai Lys Ai.« Ar g ârg Ci y 34« 345 358 <2!8> 6 < 211> 350

< 212> FRT < 2 0> Sh»ÿd ta flescen <4ÔÔ> 6

Met A.e« Tyr Civ Sa« Asp Asp Lee Art Lie Lys Civ ïie i.-ys Sis; Lev i 5 lis 15 R.;:« Ore Art Val Ais Le« Lsv« Civt .Rya S!«s Pro Ria Tot Rio Ast Ria 20 20 38

Ms Ass i'ïi.r. Vei Aï» Oi.s Aie Arg Lys Ai» I Ls Gis Lys T.iss Lep Lys 55 4S>' ' 45

Gîv Ass Asp Asp A«? Les Ms Vei VM ïîe Sïy Are Cys Ser iis Gis 50 55 SD

Asp ï?r» Veï AÏS Si:« lys Gis ïyr. Aïs Ter Are Ms X<e» Aïe Gsss Ary 55 70 75 50 8λ Où; 5-ett lys Asp Gi« lse fii.U Lie M«ï Met Arg Ο»:. Tyr Phé 8.5» öS 05 55

Lys Pre Arp X*hr *ihï. Val Sïy Trp lys SU* Ass lis Am Asρ Pro Mis 5.00 ' Ï05 ' UO

Gtet Asp Ass Ssr 25» GÜe Ass ASp Gïy Le» Arg Xié AÏS Arq Lys US MO 125

Lp« Lee Le« Asp ï.ï.a ASS Asp S&amp;t Gry Le» Ors Ai.« AÏS Gïy Gis .Pie 530 5.35 530

Les Asp Mît lie 70*:· :* Mo Gis 'Ϊν.ϊ !e JU.» Asp X.OP Met Ss.î 7'rp Giy •45 550 Ï55 ISO

Ai» Xts* 8iy Ai» kzq 'the fhs Sia Ses ÖI» Vei iïia &amp;r§ Sis Ses Ai* 555 570 5?S

Ses G3.y lse Sas Cys Pré Vsï Gï.y -Aie Jys Aso Giy 7’hr Asp 8.1 y Vhr 7 S0 505 55s

Us lys AM Ms 5'5» Asp Sis ïie As» Aï.» Aie 5ïy Sia Pro Sis Gy» 55S 300 305

Pis ÏXég Ser v»5 Ihr lys trp Sï.y 55 s Ser Aïe 5ÏA V»i ASP LAï. Ser 5 i O 155 200 G.ïy As» Giy Asp Cys Ars Ï5e Lie Le» Are Si y Sïy Lys 85s Crs Ass 025 23s 355 340 yyr ser a 5 s x<ys Gis veï Aie aïs Veï Lys G·.« Si y Ls« Am lys Aie 245 250 255

Sis 5-7« Aïs Aie Gis MM Met Aie Asp es* s** Aïs Aie Ass Sas Ser 200 205 270

AyS Gis 25s Lys .lys Sir Get Asp Vsi Cys Aie Asp Vsi Cys SX» Gis 275 200 2S5 ï 5« Aie Oh y G iy Sis Lys Ai» Us Me Gly Val Get Vei Gis Ses Ois 230 205 330

Ass Vei Gïïi Giy Ass Sis Ser AOs Asp Ser Giy Sis Arc Ley Ais Tyr 305 ' 3X0 355 320

Ci y Ays Ser X5e Ihr Asp A3.a Cys Ue Si y Try Gi« Asp Ihr Asp Aie 325 330 335

Ls« Ae» Aïs 8ia Ae» V«i Ass »is V&amp;5 Lys Ais Arg Arg ßiy 340 545 350 <2 H» 7 <2Π> 1053

< 2 Í 2> DMA < 2 \ 3 > Êseàsiichïa:sols <22 0>

< 22; > CDS < 222> (1),.( 1053} < 223> amû coding reglos <220> < 221 > m»58»«8 < 222> mum <223> c -> 1 irsnsHion <22Ü> < 221 > mutation < 222> (032),.(032} < 223> c -> t tr&amp;nsHiôa <4W> 7 sty «ai f.iis cay sec; ««c y:at us cyc «te sss gsa «te «sa «ay et* 43

Met iœ ï'yr Sla As b Asp Asp tea Ar« lie üys si« Xie Lys SI« -hm : ' 5 :H? ï.5 ett «et est «te ops ttg et«· «s* ses tes «ce «et set ysa est «et Ot tse Mo Pro Yai Aï* Less .tea S1« Lys the hto Als The 01« Ma Ais 30 35 30 gty est se« glt «ce est «ce eg» ses ysy et·;; «at «Sy .>te Ma «;>.« L44

Vei .Me ïhr 'Vei Aïe Ht» Aia Ary lys Ala île Kis Lys Ile Le« Lys 35 ih 4.5 ygt asi yst «ai c«c et« fct« «et yty att ««e ces Lye tes a U. est iÿt Ôiy Ass .Μρ Aap fit« M'a .tes; Val Val .Vie SV y Me Cys îLs.î; iis Sis «0 ’ “ 55 50 «st est «te yey yca asa gay tat «et set ege fey et« he« ctg e«t 340

Asp Mo VT A Ata A I a Lys Otu Tyr Ai« VA'.v A : ··.: té',: At a Aie Le« Arg §5' 50 ' 5 5' SÖ g«« «a« et« saa «*i gag et« «sa «te gts stg ege «te tat ttt «as 2S3

Sis Gîa Lev Lys Aöp SSv l® vLU.s Vis; Vei Stet Ar« Vet Tyr Piss; 0.5« 8S SO ' 35 a«« a-o« e«t sea a?eg gty ggs tyg aas «g« ctg atu «so gat ec« est 3 ;t

. ν’ Ae?;; t;;a Th ;; ils;; Vat T i v 'hue· Lys TI y Lev Vie Aha Ahe Pro 0 i A

VOL ’ V55 ' ViO

atg gat sat age tto sag ata aac gae ggt ctg cgt aea etc cgí: aaa 3M fise Asp As ft Ser Llie 02« ils As« tea SO.y LAa .Aj.g 2 le A 2 a -AAA Lys

IIS !.:>0 ' ' 5.2S ttg ctg eut «et até «ac. gac «ge ggt ctg scs gcg gea ggt gag ttt S3i :àac Le« :;<?:« Ass lie Asc As« Ses Oly 22e« 8ro Als &amp;1« Si y G2« S'«« .130 ' 135 X50 «te gat eeg «te ace «rca ««&amp; tat »t« «ca gac ctg atg *§« Agg ggc 380

Leg Asp Hat Xi« T:>r Lsg Gift iXee Iss» Si a Asp Leg Üst Ser Cigi <5îy 2 33 2 50 XÂS Χδδ ges aft ggc gea cgt ace ace gaa teg c&amp;g gtg eac ege g«a ctg ges 533 A2» ils Giy AS.» Aeg XCr. Thr 02.« Snr. Gift VaX 222s Atg 02» Ses« Aia 25¾ 2X0 2X5 tes gag çtt tea àg-t eag gee ggc tte asa sat ggc aec çae ggt aeg 5?t

Ser 02 y î*3 Ses Cys 2'rg Va2 02 y 2Ά c Lys .Ses SX y '0':;. .A AA· SX y Cftï

230 ' 250 2 SO até aaa gtg gai. ace gat. gae act sac ge;e g ce ggc g'cg es« esc sge 5S4 22 s Aye V'ai A2a 1.2a Asp· A2a 21 Osa .A2a Alii Oly Aia Saas His Cys ISS 300 255 tCe etg etc ata af;g «as tgg ggg eat teg: gag stt gtg aas ä«« age 633

She Lee Otse Oai ysiï. 2:ya 25;g Giy Aie Set AX« IX» lui Asc tiix Ser. 525 ' 32 a ' 520 ggt aae ggc g«t aga cet s te »tt .«ég· aga gge ggt aa.a gag e>at asc 7220 G2.y Asc 02.y Asp Cys Hi.fi X2a IXe La« Ara Olt' 02y .ays öla ä?sc Aon 255 250 505 720 cas «gc gcg sag eae «ta get aas gtg sac g»a gag et* aae aas gaa '208

Tas Ser Sia î.ys 222.« V»2 Aïa SX« VaX 2.-ys 02.« 02 y Lee San i.-ga Aia '235 255 255 gge etg cas gc.a a«g gtg aég ata gat tfcc age eat get sae tag tea 02 0

Sla 7· a a Ose a.Aa O.la 5!a2 Met. 2 2 a Aaa She Ses Ois 2>2.a sasa Äa.a Ser 550 HAS >'?·) ana «An tea aaâ sag aeg aty gat get· fege get g»c yte tyc cag eng Oi53

Lye Gift She Lys Ays 02 η Oct Asp Vax Cys ΑΧ» Osa- Vol Cys iS!.« Oic 57S " 7gg ' egg stf 5.5« vïgfc ggc gaa gag gee «at· att. gge ety stg gte ana age pat :32 22

Xi« AX« 02 y 22Xy >52.c Lya Aia 2.2.« Xia SXy VaX S-Set. VaX iïi« Ses His 550 ' ' ' 555 50s> ceg gtg gaa gga aast· oag aga ate gag aga ggg gag «eg etg gae tec 3>50 X.«« 5-02. SXO 02.y Aaft SXii S«« 2a'i« SX« Ses Giy 01« Pro Asa Aia Lyr 300 320 525 720 gga sag age ate ace g*t gee tgc ate gge tgg ges gat aea g*a gat 20f>8

Oiy .’ays Ser Xie 'Ihr Sag Aia Cys 2Xe 02y i'sp 02.« Asp 'far Lag Aia 305 ' 520 335 ctg eta cgt ear etg gerî sat gea Aits »«» gegs ege ege ggAï t«s 2 053

Ltift 2<sa .Lag Gift 2.«a fi:a As.« Ala vat ey:$ A Is Arg Arg i32.y L30 305 350

<2H)> § e 2 \ ; > 350 f 2\2> PRT

< 2 Î 3-> EsolsorsdO'ii «oil <40D> S Ηκϊ. As« '2 Sift Ai:ft Up Up ïy: T A::i Lys G.i >i Xi ft': Ly.ft: Sia Sa« I § ' Ï8 5.5

L«a S«o Val Sia L«« Las Si« Lys DA« S«« Sis SA·« Si« As* Sis SD 25 DD

Val Ass Sai- Val Sia Sis Sia Ascç Lys Sia ils Sis Sys iis Us Sya 35 5v' ' 55

Si y Sa« .Asp .Up Ä«p «sa Lse Vai Val ils Siy Dr* C'y» Sav il* ilia SD 55 m

As« Pro Vai. sia Sia i.ys Si.« iy·* sia i’Ar Sas las« Va« Sia Sa« Αΐ§ SS ?S ' ?»' Dô

Si« Di« 1.*« Lys Assp Si« 1·«« Dia üs Vai Ms« Arg Val 'î'yr Via Di«

85 SD DS 1.YS Fro to T,iu' i'iif vai. Si y up tps Siy uv n« äs» Asp Pro Dia iPü 185 lis

Stet Aap As« Sa« Phs Sia Xi.« äs« Asp illy X.·«« Sag il.« .Sia A«y i.y« US ISO us ta« 5«« Las .As* il* As« Asp Sas Siy ï·*« Pro Ai« Als Sly Si« Miss

US iS* ÍSD

Le« Asp HeA Π* tlir Ft* 05« tyr La» Sia Asp Lav Hst §w ?«ρ Sty

US iSÖ iSS ISO

Ais 3!« Qiy Ai« Arg thr. tör Si« Da* Dia Vai Dia A«p Dl« La« Aia iS* iiD i'i» 5«« Diy La» Oes Cys Xu·; Vsi cliy 8h« X-v« As« Siy Sir« Äsp Siy Da«

ISS ’ ' 185 ' i PS lia Aya Val Äia ila Ssp A.la Us Aas Ai* Ais Siy Ai« Dip Ais Cys IDS 2D8 £05

Das Da« PP« Vai IPn Lys Tap Siy Dis S«r Ala ïi* Vai. As« Sör Dar

US 215 SAS

Siy Aaa Siy Asp Cva isis il« lia U;a Asg Giy Siy Lys S.1« D«o &amp;«» 223 2.50 235 250 lya öss« Als Lys Dis Vai Alá Ô» Val Ly* öi« Siy Dev As« Lys Ai* 245 2S8 255'

*lv ï,«s Pro Ais öls Val SSst Ile As'g Vs« Ses- Ms Ms As« Set Sat 2 SD 2*S

Dys öl« Ahe Dys Dys öl« Mes Asp Val öys «.la Asp Val Cys Sla Öls 27« lag am

11« Dia S.lv öiy SD« Dva Dia 11« 11« öiy Val ltot Vei öl« Das Kis 250 235 DSD 1-«« Val öl« ö.sy As« SI.« Ses De« öl« Dar öiy öl« Pro Da« Ala Dys DOS 31Ö llS 120 ölt Dvs üs.r :ö.«s Shï Asj» Ma Cys 2.5 « öl y ?ί.ρ Gitt ösp Ί5ΐ Asp Ais .12 S 230 325

Do« De« Arg öiít i.e« «la Da« Ala Vei Dys Ala Arg Aíö öiy 210 335 33* <2 i(i> § < 21 !> 2Ö54 <2K>.> ON Λ < 2 ! 3> Esdoîikliis cols <22Q>

- 22 5 > CDS <: 2221- (501).,(1 S$3) · 223 ·'· &amp;roC- coding region <40(11' 0 ecgtlat tyi cgacyteest tgyogegaoa ttttcaoggg egtoaggyg« tseotageoe 50 gestoagotg eggpgtctgc ctg« «cotta itagtctgeg ci «οροί ala goeegcoaot 1:20

gccgcaocgc tg$cmG%ct tagsglygge gteeyeaata atgtggcoag tttiylcett IDS tiealsgyst getoctgtsa fcggtegttat gteggafcaac cifc&amp;î:te«s.«e agtgcattt.g 230

caçpiiyeata tsegyoateg gittaegatc ieayocegyt tatgaaaegc ageayagaet ISO c tt g seat aa tt as táóácá «agyag 11at ag t tagaae t tgiagyag ag a to tag tt tt 3S* icgxxäecaat ctggogtttt tel to ?. teat teceggatta ateegateal agtytaseac 320 ooogtttaos eattetgseg gaagataisg atiggasyta ttgoactoeo taagalaagt 480 atygoseoae tygaaoagao atg ast tat tag aac gas gat tta ago a .to asa 532

Mel Asa Tyr Glss Asa Asp Asp Das Arg 11« Dys 1 ' *' 10

gas sto ass gag tta eti cot «at yte gos tig ctg gas ssa tto a«a SSI *1« 11« DyS Silt Dg« De« Pro Pro Vsl Ala Da« De« Si« Dys Pits Pro 15 2* 25 got set gas aai geo gey aat «cg git go« oat got ega ass gag ate «25

Me Ihr öl« As« Ala Ala Ass TAr Vsl Ma Ki« Ale Ary Ays Ala lie Ο 35· 46 erst, sag sfcc ecg sas ggt aat çat gat «ge ctg ctg act gtg stc ggc 67;

Sis X>ys 3:X« Leu l.vs oly Ass Mp kap Sts Le« Ls« Val Vax île Sly 65 56 55 eca cgc tea att. cat aas cct gtc geg oca aaa g«g tat gcc set ege '155 S v·;'.· Cÿs Sár S ; v: Süss Av:p Avo· VAX Als Als T.-y a A:i u Tyr S3 a 'vitt· .Avy 50 * 5-5 70 ' 7 5' tta ctg geg ctg egt gss gag cto asa gac gag ctg gas see gta atg Tlí ·.?;« Sa;; AXa X.v;» Steg OXe Sia X,«·.; Aye Asp Sie S&amp;w OX« Sic VaA Siót 88* S5 S8

ege gte cat tet gas sag ccg egt see acg geg ggc egg «sa ggg ctg SSX

Asg Val Tyr LAs Sts tvs Pie Sag Xisr ·(».;» vsi Qiy Trp Lys gxy p«« 55 " 300 163

ate sac gat ccg cat: at.«· gat sat age tec oag stc a.ao gsc gat ct.g OSS

Xla .As« step Oxo Sis Nat -isp äs« Sax tea 0.1« ÏXe Aas .Vg·.. Oly X.a«

Xi b ' U 5 126 eg?. ata gec cgt. asa ttg ctg ctt gat stt asc gac age get ctg «ca 6X7

Arg Xis Als Arg Ly« i-se Lev Le« Äsp Üs Ast Äsp Sex Siy La« Αχ» i2S X 30 3.35 geg «es get gsg t.fct etc; gst «eg ste sec ca» cas tat etc get gas 555

Als ,AXa tilg ßi« Pb.« X.-e« Aas Set île TAr ·ΐχί.· Qi» Tyr Le« XU« Asp 5.66 ' ÜS ' 156 .3 35 ctg stg age egg ggo gca stt ggc ge» egt ses ace gss teg sag geg 3.0X3

Ass« Met Ses Trp Si y Ais XX.« QXy Ais Arg 7302 ttx gi« Ose als Val

iSO 356 XV’O

esc ege gas ctg gca tes ggg «et tet egt ec» »Ce ggc ttc ssa aal. U3-5X

Ais Arg 01« I-PS Ai«. Ost OXy iss Ost Cys Ire V.ai OXy 16s Aya Äs« X 7 3 .18t! 185 gge sec gac egt aeg set «sa gtg get st« «st gee stt «st gee gec .1108 SO.« Ώ;ϊ. Asp SOy 7':'>v Xis Lys Val Ais lia Ay.p Ai« 3' VC .Aas Ala Ais X50 ' 335 ' 366 ggt geg ecg cae tgc tte ctg tes gts seg sas tgg ggg eat teg geg 3.X57 OXy Ais £6:s Pia Cys Jive Les Ser VaX ii'.x Aya Trp ßly 3ä.ta Our Ais 365 " 33.6 ' 3X5

set gtg s«t see age ggt sa« ggc trat tgc cSt sie stt ctg ege ggc X20S

Xle VaX Äse X'Av .Ae« vXi.y A:VV: Qiy Aap Oy» Xäia X' A:V XX.« Arg SX y 268 5 25 ' ' ' 3ÏO ' 635 çgt sas ga» ect sac tac age gçg sag cat get get gaa gtg ass gas 1233

Sly Lys Qi« ívre. As« ïyx Sex .Ala Oy a Ri·» Vsi Al» 01» V'ai Lya QX« 2i6 ' 3X5 256 ggg ctg asc aas ges g>gc ctg cos gca esg gtg st.g ate gat ttc age 1361

Sly Oe« Aas Ly a AX a SX.y Leu yro .Ais Sirs Vs 3. Set Xis; Asp i?he Sax 655 260 665

eat. get as* teg tec asa ess tec as.a ssg cag atg gsc gtc tgt get X3-XS

;3Xs Ais ÄS« la';’.' .tax 35yg OX a Six* Sl.y» Lys Qi.» tg>>; .Asp XL's;. 6:y;S &amp;3.S 676 ' 225 ' ' 6S6 gsc get ege esg esg stt gee; ggt ggc gss asg gee stt act gge gtg 2 337

Aap Vai Cy S Ai.ii (XX V: XXa A5.S OXy ci y 01« X.ys AX a XXc XXO OX.y %sX. 605 ' 260 ' ' 655

gig gas »ge sst ctg gty gs« gye s«t tag «ge et« ysg «ge gye :134S

Ort Vai 0.1 e Sfc.r Sis Ae« Val Sie Siv äs» Sis S«x ﻫ« Sie Sex Sty '.•SO 305 3,10 335 gag ceg cty gee tae ygt sag «ye «to see gat gee tge «te gye tgg isS.'i

Sie Sx« fee Ai« Syx Oly Ays Sex Sie ϊΑχ Ast .A1« Cys : le Sie i'xe 3x0 .505 230 g*s gat ace yafc ycé eeg te« egt es« eég yeg «at gea gts sa« geg 0S41

Oly h&amp;p ΊΆχ tev Als &amp;«» Xev Axg <3t« A«« Ai« Aga Ai« v»l tya Ais

;35 340 siS egt ege ggg tsa ggetsaatfcy1 'teggatgege eytesgsgtg gegtateega 1003

Arg &amp;xg Giy 350 tgaafccacc-s esggsefcgst aagtegeges geytegeste sggeastgtg eteesétytt .5.SS3 ageaaeesa« «ageegactc aettgeayte ggettéetea tttta&amp;aaga stgaegs.tts i 'ils sttogettfca cectygtttg eaaeogeggo tgctttcget gegatotegt eayesttsee iTi.j eagat«atsg ogtttesgsy gtttgsastt etegtegaac tcxstaeacca ycygeseyce is33 ageeyggats é ta&amp;getesa gaatetette ttegeteaty tt«fcca«gat a tt teseeag .1.053 ege&amp;sgtaaa gagttaceyt gtyesgegst gate«cgegc teaeegetet teataegege 1353 eagaasagti testiccayt a«ygyate«e ycggtesatg gteagegeea ggeattcsgt 301.3 éágéggeayt tett te étaye éasgittege gfcaacgegga t 2055 <>I0> U3 <2U> 350

< 2S2> PST < 213^ Hschoilchis ooii <400> 10

Set Ass Syr ilia Asa Asp Asp A«« Axg 31« Ays S3« lis Ays 01« Ae»

i " 5 10 IS i<as Px* l;ro gal Ala As« Lea Ole Ays LSe Oxo Ala 'tax S.lw Asa Aia 2e 13 30

Ola Age Ohr V&amp;X Aia Sis Ai« lira Ays Ala its His Ays lie Aee Avs 33 40 45

Sly Asa Asp Asp As» Ae« Aeu y at Vax its O.t y Ore Ays oar Al« ills 30 ' ' »3 40

Asp Pro Va.I Ala S3« Ays G1« 3’yx 01« AA.r Arg i«xs Ae« Sla .tea Arg

SS 00 '15 SO

Ole Oie Age Ays Asp Sie A&amp;s 01« lie Va,i Oat Arg Val tyx Hsa Sla SS SO 35

Lys Pro Arg Thr Thr VsX Gig ?rp Ly a Gig Leu Xis As« Aep Pro äüs iöö IDS no

Stet Ae« As« Ser Sirs CUn Xi« Ass Asg Gig Lee Arg Xi« Ais Arg Lys

UL ·30 ISS

Le« Le« r.e« .Asg X.te Aas Asg Las Gig X>e« Hro Ai« Ala GXy gl« Pt« ϊ.-.ö .US 14Ö

Ura Aap üet Xi« Th« Pro Gis Tyr L>«a Als Aap 5.·«« Hst Ser Txp ö.ly

H5 X SA ILL ISS

Ai« IXe Gig .AXs Arg 'i'Lr Ter öl« Ger HX« VAX eis Arg Öl« Ls« AXe ISS X'XÖ I?5

Ser Gig Le« Gar Cg» Pro VsX Giy Sire Lys Ae« üly GHx Asp SXy ïivs

XtC XAL iso IX« Lys v«i. AXS iie Asy Ala iie &amp;s« Ai.a .Als CU y Ai&amp; Asc His Cÿs iSS SOÖ X03 PLe Sri« Ser VAX ‘XLr X<ye \?rg Gig Hie Ser .Aie Xi« Vai .Ae« GAr Ser XIO ' XXL ' X28

Gig As« öiy Asp Oys Siis Sie île Le« Arg Gig Giy Lys ·':·Ar Aris Ae.«

XXL >30 XXS MS

Tyr Ser Aie Lva Hia Vai Als öl« VsX Lvs Gi.« Gig i.e« As« Lys Ai«

XXL XL« XSL

Gig Ls« Ars Aie Aie Vei Her lie As» HAe Ser Sia Aie Ass Ser Ser Xöö XtL X7ö

Lge Gis Ata Lgs Lys Gie Paî: Asg Vei Gye Sia Asp liai Cyss Gis Gis

XIS 230 XtS iie Aie Gly Giv Gi« Lys Aia Xie lie Gly Gai Hei «si Gi« Ser Hie SLÖ XSL 38Ö

Le« Vsi Sie Gig Ass Sin Lar 1er« Gi« Ser SXy Gi« tre Le« Sie tyr 305 " SIC Xi 5 3X8

Gig Lgs Ger iie TLr Asg Aie Cys iie Gig Tr« Gi« Asp THr Asg Ai«

XXL .33.0 XXL

Le« Le« Arg öle Le« Aie As« Sie Vel Lys Ais Arg Arg öiy 340 345 Ï50

Claims (3)

1. :Jg£ayj«m*ok I, iNokíeörtd^sáökvesda (DNiSJS: amely &amp; 3'Uez<i3ciiwÎ>-iîf«/émi>he}>{«lo2oaàt>'7''lôS2ÂsÂiè&amp;' iplÄB-ssintaz) enziro visszacsatolást» gátlásra reziszt«»» változatát kódolja, azzal jellemezve, h®g$: otAoaavászekveneiája a SBQ ID NO: 2 szerinti aminösáv-szekvcneia.: amely tsrtaímazhat 1-2Ô eminosav deléeiót és/vsgv iaszerelót, ás lóppgébetí 3S0: anlinosav hosszúság«» ás a 8EC| i!> NO:2 szerinti SíníKösag''SZélveö£Íá»ak megfelelő amtnösíp-'Szekvanela a kővetkezőkből glló csopflbíP választott a»őnosav-sz«bsztitáoók;:s· tartalmaz: s) a 23. pozícióban k-aiasún hel>«tt«58»tdsú::PValto»ai, b) « 33. pozícióba« L-alania heiyeilesrtóse L-vallonsI és a 211. pozícióban lévő L-szerin helyettsattóse k*.fen Uttlantanal é» a SEQ ÏD NO:2 szermtí mmmm-mMmsk Älssfa egy vágy több» &amp; következőkből álló csoportból választott szubsztitúciót is tartalmaz: c) a 14. pozícióba» L-lizi« helyettesítése: l,>aszparág|n: és: :L*glötäMie választott aml®0SSk'k8.ls ólőnyősen L-aszparagmítái, .11 s 6Ί po^loióbett L'-vaHn helyettesítése a kövctkezékból. á||ö csoportból választott ammössvvíU: L-alatsis, L-Hztn., 1>argioin és L-hiszUdta;· előnyösen L-alansanal e) a 24. pozícióba« t**íreonm helyettesítés« a következőkből álló ösöporthől választött altitioSSkval; i-sianisk L-iízhp l-etgsm:» és L-hisztidm; előnyösen L-ai&amp;ninnaí, I) a 31. pozícióban L^ghttamlasâtf'-'iëlyÂsftése a következőkből álló csoportból válásatört a««808ay*al: L-assptrsagirtsav.. L-sæcrin, L-aknin és L-íreoitsn; előnyösen L-aszparagi«sayv»L g) a 84. pozícióba« Ldlzs» helyettesítése s kővetkezőkből· siló csoportból választott ímsloosavval; L-ginrattmt, L-szcrio, Lrtreomtt, L-glutamissav, L-aazparagíasav és L-aszperagsn; előnyösen L-gínt:t trónnak b) s |S, pozielöba« i>aszparagi»sav helyettesítése a következőkből álló csoportból, választort aminosttv vaí : L-alasln és glicht; előnyösen glicht»«% i) a 23b. pozícióba« L-glutasrtinsav helyettesítése a következőkből álló csoportból választott arfdftosavvaj;: ;Laszpín-aginssv, L-s*sris, L-aiaión és L-íreonht, előnyőse« L>aiaró»aal j) g 244 pozícióba« MÄ1 helyettesítése a tőeetlezőkM álló csoportba! választóié ammosawal k-gl«tttr»ln, L-gkusnbstxav, L-asaparagínsav, l-szeríe, L-treonítt és L-aszparagis; előnyösen L'gltstarninnak k) a 254. pozícióba«' L-aszparagia helyettesítése a következőkből álló csoportból választott aßrtsosavval: L-trcooia, L-szarin» Lvgiptansipksv és L-aszparaginsav; előnyösen l· treonisrnak 1} a 259. pozícióba» L-pmltn helyettesítése a kővetkezőkből álló csoportból választott amittosavval: l-szens és L-treottin: előnyösen L-treoamnal, ni) a 2ŐŐ. pozícióban L-alanm helyettesítése Lapról innal, tt) a 272, pozícióban L-szerin helyettesítése a kővetkezőkből álló csoportból választott atninosavval: L·cisztáin és 1, -.«κΡίοηirt; előnyösen L-dszteianel, 0) · s 21h. poá&amp;lóban L-ttztp hsjyetíeskése a ktvolkazókbii éllé csoportból választott asrktösavval: l.-glutaatift,· L-gtmatitoav, .^as^síagíasisvj. k-szerlo, Löreonhr és: L-aszparagio; előnyösen t-glaíamitm&amp;i, p) a 2kö. pozícióban L~axzparaginsav hetyePeshése a követk szókból |H6 csoportból választott amfrmsavvak L-szerlo, l.*ála:nia, L-gl «tara insav és L-treonin; előnyösen i,-glm.soooxsvvss, Φ a II pozícióban h-glutaminsav tselyettcstíése a követkazÖkhÖI: álló :«pÉI választott arnmosavyal: L-szcrm, L-alanixt, L-sszp&amp;raglosav és L-treotiin; előnyösen L-asápáragiassvvst, r) a 3lő. pozícióban t-gíutasninsav helyettesítés« a kővetkezőkből álló csoportból választott ansíoosavvak L-glotatnin., L~szerln, L-treoom, t-aszparsglnsav ás t-zszpsragxn; előnyösen L-ghiümmml. s) a 319. poziciobart L-alamo helyettesítése a kővetkezőkből álló csoportból választóit aminosavvai: L-treoma, l-vaim és l-szerin; előnyösen l..-íre.on innal. 1) a 342. pozícióban L-alaots helyettesítése a kővetkezőkből álló csoportból választott LzFtfe és L-treonia; előnyösen L~szerinnel vagy L-valinnal, v§ a 343. pozícióban L-aazpasragte helyettesítése &amp; kővetkezőkből álló csoportból választott stmioosavvak L-asz.psragtnsav. L-glntam&amp;sav.iL-szerin, L-slanht és L-ímonhi; előnyösen L-alanlonal. It M h igénypont szerinti hnkíéótld-szekvehéía (DNS), azzal jellemezve, hagy sz Emrn&amp;hmm-imms családba tartozó oxikroorgaslzoiushöl származik. 3. fektot:, azzal jellemezve, begy 1. vagy 2. igénypont szerinti aókleotici-szckvcnciát tüSlüi*i: ás adóit eSétbón éépllkllódik EhmmMèimiàèmè családba tartozó ahkroorganiiönösbán. 4 Az hnterobmfäriäm#* csalóéba tartozó mikroorgasignnssv azzal jellemezve, hogy # tartalma* 1- vagy 2, igénypont szerinti ookleotld-székvescsát, és ahol az expressMlt D&amp;iéF-szinlázt kódoló rmkieotid-xzekvencus jelen van. 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3. 44. Eljárás a sikimisav métabolisas ötvonal vegyi! letelnek csoportjából válpziéít: . szerves|émtáí vegyidet. előnyösen aromás: L-aminosav, vagy ezen vegyületekét tzréatnmzö ételmtszér-adalék: előállítására Enterobáctmacm#· családba tartozó tniktoorgapizmóS MmlM történő foramntáeíf vat, ózza 1 jelleta ez ve, hogy é) e szezveskémia! vegyöiétet termelik 4-13. igénypontok bármelyike szerinti mlkroorpnizmnst olyan körülmények között tenyészti tik tápközegben, amelyek között a vegyilet a tápkézegbeo, vagy a sejtekben lelbalmözódlk, ós b) a vegyöletet izoláljnk a íeromrdáctés léből, ahol adott esetben a fermentációs lébeő tévő egyéb ve,gyűlések és/vagy a biomstssza egésze vagy része (> ö-KH)%) az izoiált termékben marad vagy teljés mértékben eltávolításra kerül. I. 5:, Eljárás L-trlpíofon vagy L-trlptotetst tatialmaxé élelmiszer-adalék elöállitásátá: a 4, Igénypont szerint!, Etearobocterteceoc családba tartozó bakteriam alkalmazásával történő levmentóelöval, azzal jellemezve, hogy aj 4-13. igénypontok bártnélyike szérmti, MrípteSans termelő tn ikröorganizmost olyat; körülmények között tenyészünk íáppzegbea, árnelyék között a vegyüld a tápközegöen vágy a sejtekben telbalmoződik, és b| az foippíofánt vagy L-trlptoflní tartalmazó élelmiszer-adalékot Izoláljak. a fotzüéníáeios léből, adete: esettben a fermentációs lében lévő egyéb vegyöletek és/vagy é biomassza egésze vagy része % folÖÖfor az izolált termékben ssarási vagy teljés mértékben eltávolításra kerül, lé, A 14, vagy 1$. igénypont szerinti eljárás, ászai jellemezve., bogy az L-trlpteőfo ibrmmbn#eí'tecepc családba tartozó bakteriam alkalmazásával történé formentacioval van előállítva, ahol adott esetben egyidejűleg további egy vagy több. a következőkből álló csoportból választott gén expressziója egyidejűleg fokozva ven, speciálisan, a géntermék tél van termeltetve: * a« aotmmióksmíázó íbs^:íkílbö^ü*&amp;o?:raskál->lKkaíefást mêôk3'§lie^r»)~fbs;xS&amp;--s^imâ^ es. triptofán'SítuHátf kMoló MflAií«gír«a legalább ag$ gv«je, * a kdbsafögHcerát-dehídrogenár.í ΜΉΐοίό ser A gér,, * a i< t»;í les «er i s - lósa faíík ; kódoló »esR gén, « a fosafoszom-atsinotransz&amp;rázt kódoló sert? gén. * as Miroznira êrÂeny PHAP-szjíR&amp;d Mdeló aye# f#»» * m LArRdoíaom érzékeny DlMP-sziostät kódoló aroH gdg '*^a::tbs2ÂfeBolpi?u«àî^laiâÂt ködeid pps gén, * &amp; fesK&amp;esölpsrevákkaílösIkloáKí kódólé· pefcA gás, * a tfasssdtsiolóz A-t kódoló sktA gén, * á :líOííSzk:0kO:Íkk :B"S: kódoló SRIB gén és * m fáú<J Msghmchm leolvasási keres; (ÛRF) góáterméke. Hr A 14*1% jglitypoídök bármelyike szerinti eljárás^, őzzel jellemezve, hegy m M#tÄb EtíMitMétérmmm esaládba tartozó; teaktéHsm álk8lmazás|y4ÉM:ónési§ ilbwggÉfetóval van el$MlÉy% öböl adott esetben egyMpiieg ttsv^ll egy vagy több, a kővetkezőkből álló esopdrtMl választott gén taékéálád;csillapítva vám adott esetbediélimtöálva vas vagy express«!ó|a esdkkeotV(t vmi » s triptolaoazt kódoló tnaA gdrg * a rq:;· opston te presszóm kódoló trpR gén, * &amp; konzmái-taolá/ T-t és pt«fe^t-d|feí#diób&amp;»t ÂMddÿrA gén, * a konzmát-ntuíáK: F-l és preíettátHlehidrogsmázt kóds>ló pite A geo, * o trlptoíaara specifikus trassszporliehéspóksldoiO satr geo, * a íjipicfáJí-períneázt kódold íttaB gén, *:M stromas atrdaosavsk transzportétól kódoló ar# gá% * az l.-szerio-doardoazi kódoló sdaA gets, « e gltikoz-ö-íosziát-isoiííefázt kódok* pgj gets, V 8 íiro»t8>aminotraTtszferézt kódoló tyrö gén és * is gip reguloa represszorát kódoló gipR gén. IS, A :14-17, igónypvntok· bármelyike szerkói eljárás, tuz<td Jziíémzzw, hogy a mÉíoorgagszoiusk szakaszos eljárással .rálápílÉsos szakaszos aljárássaii ismótóR mépláMsos szakaszos eljárással vagy iolysOr&amp;tos eljárással tenyeázdpk, és/vagy a sikirmsxv rastaboliktss ótvo:aai vegyStetstoek csoportjából választott szerveskémlal yegyüler koneen trâeléjàt a fersnootlgld aoráts ogy vagy tops poototr meghatározzuk, A meghatshnKzotí: DANÜBIA