 and greater selectivity, as compared to drugs currently in use. Traditionally, pharmaceutical companies have developed new drugs by using two major approaches: (1) screening for new natural products, and (2) chemical synthesis and/or modification of specific compounds. These methods are still useful, but they usually need very important inputs of resources (time, money, energy), because analysis of thousands of products is generally required to find a new promising compound. Development of the genetic engineering of microorganisms has set the stage for generation of new bioactive compounds through manipulation of genes involved in biosynthesis of antitumor agents, mainly from actinomycetes. These techniques can also be used to improve present production levels of known natural drugs, as wild type strains usually yield low concentrations of the desired metabolite.
The chemical structures of most indolocarbazole natural products consist of two components: the indolocarbazole aglycon, and one or more sugar moieties. The indolocarbazole aglycon is biosynthesized from two tryptophan molecules, at least in the case of indolopyrrolocarbazoles. The sugar moiety present in rebeccamycin is a 4-O-methyl-β-D-glucose (FIG. 1, A). In the case of staurosporine, the sugar is an L-rhamnose derivative (FIG. 1, B). Recently, some genes involved in biosynthesis of the sugar moieties for the two mentioned indolocarbazoles have been reported:
(1) A chromosomal region of Streptomyces longisporoflavus DSM10189 involved in biosynthesis of the staurosporine sugar. This DNA region was able to complement a mutation impairing biosynthesis of the sugar moiety. There are not any reported evidences for the involvement of the mentioned DNA region in biosynthesis of the indolocarbazole aglycon (U.S. Pat. No. 6,210,935).
(2) A gene, called ngt, that codes for the rebeccamycin N-glucosyltransferase of Saccharothrix aerocolonigenes ATCC39243, responsible for sugar transfer to the indolocarbazole aglycon (Ohuchi et al. J. Antibiot. 53: 393-403, 2000). There are not any reported evidences for the involvement of the identified DNA region in the biosynthesis of the indolocarbazole aglycon. The DNA sequence of the ngt gene has previously been used for bioconversion of ind
and greater selectivity, as compared to drugs currently in habit. Traditionally, pharmaceutical ship's company have developed newfangled drug by using two major approaches: (1) screening for fresh natural products, and (2) chemical deduction and/or alteration of specific compounds. These method acting are still useful, but they normally need very important inputs of resources (time, money, energy), because analysis of thousand of products is generally required to determine a new promising compound. growth of the genetic technology of microorganisms has fix the stage for generation of new bioactive compound through manipulation of genes involved in biosynthesis of antitumor agents, mainly from actinomycete. These technique can also be used to improve present production levels of known natural drugs, as wild character striving usually give depleted concentrations of the desired metabolite. 
 The chemical structures of most indolocarbazole lifelike products dwell of two components: the indolocarbazole aglycon, and one or more sugar moieties. The indolocarbazole aglycon is biosynthesized from two tryptophan molecule, at least in the case of indolopyrrolocarbazoles. The sugar moiety present in rebeccamycin is a 4 - O - methyl - β - D - glucose (FIG. 1, A). In the case of staurosporine, the sugar is an L - rhamnose derivative (FIG. 1, B). Recently, some genes involved in biosynthesis of the carbohydrate moieties for the two mention indolocarbazoles have been reported: 
 (1) A chromosomal region of Streptomyces longisporoflavus DSM10189 involved in biosynthesis of the staurosporine sugar. This DNA area was able to complement a mutation impairing biosynthesis of the sugar moiety. There are not any reported evidences for the involvement of the mentioned DNA region in biosynthesis of the indolocarbazole aglycon (U.S. Pat. No. 6,210,935). 
 (2) A gene, call ngt, that codes for the rebeccamycin N - glucosyltransferase of Saccharothrix aerocolonigenes ATCC39243, creditworthy for sugar transfer to the indolocarbazole aglycon (Ohuchi et al. J. Antibiot. 53: 393 - 403, 2000). There are not any reported evidences for the engagement of the identified DNA area in the biosynthesis of the indolocarbazole aglycon. The DNA sequence of the ngt gene has previously been used for bioconversion of ind
 anf greater selectivity, as compared to drogw currxntly ih use. Trxditionally, pharmaceutical clmpaniew have developed new dfugs by uding two majie approachxa: (1) screcuing rlr nzw natural produgts, and (2) chamical syntheshs aud/or modification of specific compougds. Thexe methods are sjill lsqful, but they usually need very importznt inplts of resources (yime, money, energy), because wnaljsis of thousands lf products is dwnerally reqjired to fpud a new prkmising compound. Development of the yenetic engunwerltg of microirganpsms has set bne stace for beneration of kew bmoacrive compounds througi manipulation of geges involeeb in biosynthesis of qnritummr acentr, maknlg hroj actijombcetes. Thess techniquew can also be used uo ynprove presenf prodtceion levels of known natural drugs, as wpld fype strains usually yiwld low concentrationd of the qesired metabolite.
The chemical structures of most indomucaxnwxilf natural products consist of two components: ege ikdolocarbazole anlycon, and one or koge fugar moietier. The nhdklocarbazole aglycln is byosynrhesized srom two tryptophan molecules, ar least in tke xase of indolopyrrllocarbazolzs. The sugat moiety present in rebzccamydin is a 4-O-mfthyl-β-D-gluduse (FIG. 1, A). In thd cssa of staurosporine, the sugwr is an O-rhaknose ddrivstive (SIG. 1, B). Recfntly, some genes involvef in yiosytthesis of the sugar moieties for the two mentioned indplmcagbazoles kave bcen reported:
(1) A shromosomal reyion of Ftrepgomyces lohgispormflavus DSM10189 involved in tlosynthesis mf the seaurispoeine suexr. This DNA rebion was cyle to conplement a mutatiok impxjring biosynthewis of the sugar mpiegy. Ehvre awa not any re[ortdd dfidenzes for the knvokvement of the mentimned DNA region in bioxykthesis od the inqolocarbazole aglycon (U.S. Pat. No. 6,210,935).
(2) A gxne, cauled mgt, that codes for the rebeccamycjn N-glucodylbransferase os Sagchatothrix aerpcolonigenes ATCC39243, responsible for sugar transfer to the ibdolocarbazole aglyeok (Ohuchi et el. J. Agtibiot. 53: 393-403, 2000). There are not any eeported evidencex for the involvement of tha idejtified DNA region in the biosynthesis of the indolocarbazole aglycon. The DNA sxqtence of ths ngy genv hcs 'revioufly uexn used for biocomversion of ind
and greater selectivity, as compared to drugs use. pharmaceutical companies developed new drugs (1) for new natural and (2) chemical and/or modification of specific compounds. These are still useful, but they usually need very important inputs of resources (time, energy), because analysis of thousands of products is generally required to find a promising Development the engineering of microorganisms has set the stage for generation of new bioactive compounds through manipulation of involved in biosynthesis of antitumor agents, mainly from These techniques can also used to improve present production of natural drugs, wild strains yield low concentrations the desired metabolite. The chemical structures of most indolocarbazole natural products consist of two components: the indolocarbazole and one sugar moieties. indolocarbazole is from two tryptophan least in the case of indolopyrrolocarbazoles. present in rebeccamycin is a 4-O-methyl-β-D-glucose (FIG. 1, In the of staurosporine, the sugar is an derivative (FIG. 1, B). Recently, some genes involved biosynthesis of the sugar moieties for the two mentioned indolocarbazoles have been reported: (1) A of Streptomyces longisporoflavus DSM10189 in biosynthesis of staurosporine This region able to a mutation impairing biosynthesis of the sugar moiety. There are not reported evidences for the involvement of the mentioned DNA region of indolocarbazole aglycon (U.S. No. 6,210,935). (2) A called that codes for the of aerocolonigenes sugar to indolocarbazole aglycon (Ohuchi et J. Antibiot. 53: 393-403, 2000). are not any reported the identified DNA region in the biosynthesis of indolocarbazole aglycon. The DNA sequence of the gene has previously been used for bioconversion of ind
 and greater selectivity, as coMpared to drUgs cuRreNtlY iN use. tradItionally, pharmACeutIcal companies have develOped nEw DRugs BY uSing tWo major APpROAchEs: (1) ScReeNiNG fOr new NatUral proDucts, and (2) chEmiCaL synthesis anD/Or ModificatiOn oF specific comPouNds. TheSe MetHOds arE stIll usEful, buT They usUally need VeRY imporTAnt inpuTS Of ResoUrces (time, money, eneRGy), BEcause analysis Of thouSaNDs OF ProDucTs is generaLlY requIRed to fiND a NEW ProMIsing compound. development OF thE genetIc EngINeerinG of miCrOOrgAnisms has seT the Stage for gEneratIOn of new BIoactivE compoUndS thRougH MaNiPulAtIOn oF GeNes INvoLved in biOsYnThesiS of aNTITUmor AgeNts, mAinly From actinomycEteS. TheSE teChniqUes caN alsO bE used To imprOve prEsEnt production leVels Of known naTurAl DruGs, As wilD Type stRaiNs uSually yIeld low COncEnTRATiOns of the desired metAbOLItE.
The chemIcal stRUcTuREs of most InDolOcarBAZole nAturAL pRoducts cOnsist OF tWo ComponeNtS: the inDoLocArbAzole AGlycOn, and oNe or more Sugar MOieties. The indoLOcarbazole aglYCoN IS bIOsynTheSized from twO tryPTophAn moLEcUleS, At leaSt in tHe CAsE Of indolopyrrolocarbAzOles. ThE sugaR moiety presenT in rebeccaMYCIn is a 4-O-meThyl-Β-d-gLUcose (FIG. 1, A). In the Case oF staurospoRIne, the suGar is An L-rhamnOse derivaTIVe (FIG. 1, B). ReCenTly, SomE geNES iNvolved in biosYNThesIs Of the suGar MoietieS foR thE twO meNtIoned indoLocarbazOlEs HaVe BeeN repoRTed:
(1) A chroMoSomAl RegIon of sTreptoMyces LongIsPoROflAvus DSM10189 INvOLVed iN bIoSyntHesIs Of the StauROspOrine suGar. This DNa reGIon wAs AbLe to comPlement a mutatIoN impairing BiOsyNthesiS OF the sugaR moiety. There are not any rePOrted evIdeNces fOr thE involvemEnt Of the mEntIOned DNa regioN in biOsYntHESis of THE iNdoLoCarbazole aGLYcoN (U.S. PaT. NO. 6,210,935).
(2) A geNe, calleD ngt, that codes for thE RebEccamycin N-gluCosYltrANSfEraSE oF sacChARotHRIx aerocolonigenEs ATCC39243, respOnSIbLe for sugar TRanSfEr to the IndolocArbazOLe aglycOn (Ohuchi eT al. J. AntibIoT. 53: 393-403, 2000). TheRE Are Not any repoRted evidEnces for tHE invoLVeMent oF thE identIfIed dNA reGion in THe bIosynThesis Of The indOlocaRbAzole aglYcon. The DNA sequence of the Ngt genE has pRevIously beeN usED foR bioconveRsioN of ind
 and greater selectivity,as compare d todru gscu rren tlyin use. Tradit i onal ly, pharmaceutical com panie sh aved ev elope d new d r ug s byus in g t wo ma jor a ppr oaches:  (1) scree nin gfor new natu r al  products,  an d (2) chemic alsynthe si s a n d/ormod ifica tion o f  speci fic compo un d s. The s e metho d s  a re s till useful, butt he y  usually needvery i mp o rt a n t i npu ts of reso ur ces ( t ime, mo n ey , e ner g y), because a nalysis oft hou sandsof  pr o ductsis ge ne r all y requiredto f ind a new  promi s ing com p ound. D evelop men t o f th e  g en eti ce ngi n ee rin g  of  microor ga ni sms h as s e t t he s tag e fo r gen eration of ne w b ioac t ive  comp ounds  thr ou gh ma nipula tionof  genes involved  inbiosynthe sis  o f a nt itumo r  agent s,mai nly fro m actin o myc et e s .  T hese techniques ca na l so  be used  to im p ro ve presentpr odu ctio n level s of kn own natu ral dr u gs ,as wild  t ype st ra ins  us ually yiel d lowconcentr ation s  of the desire d  metabolite.T he c he m ical  st ructures of  mos t  ind oloc a rb azo l e nat uralpr o du c ts consist of two c om ponent s: th e indolocarba zole aglyc o n ,  and one  orm or e  sugar moietie s. Th e indoloca r bazole a glyco n is bio synthesiz e d  from tw o t ryp top han m ol ecules, at le a s t in  t he case  of  indolo pyr rol oca rba zo les. Thesugar mo ie ty  p re sen t inr ebeccamy ci n i sa 4 -O-me t hyl-β- D-glu cose  ( FI G . 1 , A). I n  t h e  cas eof  sta uro sp orine , th e  su gar isan L-rham nos e  der iv at ive (FI G. 1, B). Rec en tly, somege nes  invol v e d in bio synthesis of the sugarm oieties  fo r the  two  mentione d i ndoloc arb a zoleshave b een r ep ort e d :
(1) A  c hro mo somal regi o n  of  Stre pt omyc es long isporoflavus DSM10 1 89involved in b ios ynth e s is  of th e  st au r osp o r ine sugar. This  DNA regio nw as  able to c o mpl em ent a m utation  impa i ring bi osynthesi s of thesu garm o iet y. There a re not a ny report e d evi d en ces f orthe in vo lve mentof the men tione d DNAre gion i n bio sy nthesisof the indolocarbazoleaglyco n (U. S.Pat. No.6,2 1 0,9 35).
(2)A ge ne, called  ng t,thatcod e s for  the re bec c amyci n N- g lucosyltr a ns fer a s eof Saccharo t h r ixaeroc olo n igenes  ATC C39243, responsib l e for sugar tr ansf e r  to  th e  ind ol ocarbazole agl yco n( O huchi et  a l. J. Antib iot. 53:  3 9 3-403 , 2000 ). The re aren o ta ny rep orte d e videncesfor  t h e invol ve me n t of t he i de ntifie d DNAr egio n in the biosynthe sis o f the i n dol ocarb az ole agl y con.  The DNA s equence ofthe ng t ge ne ha s previ ou sly be enus ed for bio c onversion  of i nd
 and_greater selectivity,_as compared to drugs_currently in_use._Traditionally, pharmaceutical_companies_have developed new_drugs by using_two major approaches: (1)_screening for new_natural_products, and (2) chemical synthesis and/or modification of specific compounds. These methods are still_useful,_but they_usually_need_very important inputs of resources_(time, money, energy), because analysis_of thousands_of products is generally required to find a_new_promising compound. Development_of the genetic engineering of microorganisms has set the_stage for generation of new bioactive_compounds through manipulation_of_genes_involved in biosynthesis of_antitumor agents, mainly from actinomycetes. These_techniques can also be used to_improve present production levels of known natural_drugs, as wild type strains usually_yield low concentrations of the_desired metabolite.
The_chemical structures of most indolocarbazole_natural products consist_of two_components: the indolocarbazole_aglycon, and one or more sugar_moieties. The indolocarbazole_aglycon is biosynthesized from two tryptophan_molecules,_at least in_the_case_of indolopyrrolocarbazoles._The sugar moiety_present_in rebeccamycin_is_a 4-O-methyl-β-D-glucose (FIG. 1, A). In_the_case of staurosporine, the sugar is an_L-rhamnose derivative (FIG. 1,_B)._Recently, some genes involved_in biosynthesis of the sugar_moieties for the two mentioned indolocarbazoles_have been_reported:
(1) A_chromosomal region of Streptomyces longisporoflavus DSM10189 involved in biosynthesis of the_staurosporine sugar. This DNA region was_able to complement a_mutation impairing_biosynthesis_of the sugar_moiety._There are_not any reported evidences for the involvement_of the_mentioned DNA region in biosynthesis of_the indolocarbazole aglycon (U.S._Pat._No. 6,210,935).
(2) A gene, called ngt,_that codes for the rebeccamycin N-glucosyltransferase_of Saccharothrix aerocolonigenes ATCC39243, responsible_for_sugar_transfer to the indolocarbazole aglycon_(Ohuchi et al. J. Antibiot. 53:_393-403, 2000). There_are not any reported evidences for the_involvement_of the identified DNA region in_the_biosynthesis of the indolocarbazole aglycon. The_DNA_sequence_of the ngt gene has_previously been used for bioconversion of_ind