Overproduction of threonine by Saccharomyces cerevisiae mutants resistant to hydroxynorvaline

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1992, p. 1677-1682 0099-2240/92/051677-06$02.00/0 Copy igh ©) 1992, Ame ican Socie y o Mic obiology O e p oduc ion o Th eonine by Saccha omyces ce e isiae Mu an s Resis an o Hyd oxyno aline CAYO RAMOS AND ISABEL L. CALDERON* Depa amen o de Gene ica, Facul ad de Biologia, Uni e sidad de Se illa, Apa ado 1095, E-41080 Se ille, Spain Recei ed 20 Decembe 1991/Accep ed 22 Feb ua y 1992 In his wo k, we isola ed and cha ac e ized mu an s ha o e p oduce h eonine om Saccha omyces ce e isiae. The mu an s we e selec ed o esis ance o he h eonine analog a-amino-13-hyd oxyno ale a e (hyd oxyno aline), and, o hese, he ones able o exc e e h eonine o he medium we e chosen. The mu an s ains p oduce be ween 15 and 30 imes mo e h eonine han he wild ype does, and, o a lesse deg ee, hey also accumula e isoleucine. Gene ic and biochemical s udies ha e e ealed ha he h eonine o e p oduc ion is, in all cases s udied, associa ed wi h he p esence in he s ain o a HOM3 allele coding o a mu an aspa a e kinase ha is o ally o pa ially insensi i e o eedback inhibi ion by h eonine. This enzyme seems, he e o e, o be c ucial in he egula ion o h eonine biosyn hesis in S. ce e isiae. The esul s ob ained sugges ha his s a egy could be e icien ly applied o he isola ion o h eonine-o e p oducing s ains o yeas s o he han S. ce e isiae, e en hose used indus ially. In Saccha omyces ce e isiae, h eonine and me hionine a e syn hesized om aspa a e h ough a common me abolic sequence ha leads o homose ine in h ee s eps. Homo- se ine is he b anching poin om which he pa hways o me hionine and h eonine di e ge. The egula ion o his ou e akes place a a numbe o s eps, bo h a he le el o enzyme syn hesis and a he le el o ac i i y ( o a e iew, see e e ence 8). Th eonine seems o be he p ima y com- pound ha egula es he ca bon low in o and h ough he common pa hway, speci ically a he le el o aspa a e kinase and homose ine dehyd ogenase, he i s and hi d enzymes o his ou e, espec i ely. Mo eo e , he low in o each speci ic a m is con olled by end p oduc inhibi ion o he co esponding i s enzymes: h eonine inhibi s he ho- mose ine kinase, while me hionine inhibi s he homose ine O-ace yl ans e ase. The egula ion o an amino acid biosyn he ic ou e can be in es iga ed by he use o amino acid analogs. Mu an s ha a e esis an o a oxic analog a e good candida es o be al e ed in hei abili y o egula e he syn hesis o he na u al amino acid. In ac , many analogs ha e been used o isola e amino-acid-o e p oducing s ains, bo h in p oka yo es (1) and in euka yo es (15). o -Amino-,-hyd oxy ale ic acid (hyd oxyno aline) is a h eonine analog known o inhibi bac e ial g ow h mainly because i mimics h eonine and hinde s he ac i i y o he h eonine eedback-inhibi ed enzymes (4). As a conse- quence, cells g owing in he p esence o hyd oxyno aline p oduce smalle quan i ies o he amino acids syn hesized h ough his pa hway, which can e en become limi ing o g ow h. Hyd oxyno aline- esis an mu an s ha e been iso- la ed om bac e ia such as Eschenchia coli and B e ibac e- num and Co ynebac enum spp. (13). In hese mu an s, one o he h eonine-sensi i e enzymes has become insensi i e o he eedback con ol by his amino acid and, as a * Co esponding au ho . P esen add ess: Depa men o Yeas Gene ics, Ca lsbe g Labo a o y, Copenhagen, Denma k. consequence, he mu an s o e p oduce o a ce ain deg ee and e en exc e e h eonine in o he medium. To ou knowledge, hyd oxyno aline has ne e been es ed be o e in any yeas species. I he si ua ion in his o ganism was simila o ha in bac e ia, one would expec ha , in a ce ain hyd oxyno aline- esis an yeas s ain, one o mo e s eps o he pa hway would ha e been eleased om h eonine inhibi ion. Some o hese mu an s could also o e p oduce and exc e e h eonine. The equency a which each kind o mu an a ises and he deg ee o o e p oduc ion o he amino acid would be indica i e o he ela i e impo - ance o ha s ep in he con ol o he pa hway. Addi ionally, hyd oxyno aline esis ance could also a ise by a de ec in he pe mease ha anspo s i in o he cell. In his wo k, we ha e isola ed hyd oxyno aline- esis an mu an s om S. ce e isiae and ha e selec ed om among hem hose able o exc e e h eonine in o medium, which indica es h eonine o e p oduc ion. We ha e s udied he accumula ion o h eonine and o he amino acids in he mu an cells and cha ac e ized hei de ec bo h biochemi- cally and gene ically. MATERIALS AND METHODS S ains. The S. ce e isiae s ains used a e lis ed in Table 1. Enzymes and chemicals. Hyd oxyno aline was a gi om Degussa A.B.M. (F ank u , Ge many); bo elidin was a kind gi om K. Po alla; lac a e dehyd ogenase (EC 1.1.1.27), py u a e kinase (EC 2.7.1.40) om abbi muscle, phosphoenol py u a e, o-ph halaldehyde, and P-me cap o- e hanol we e om Sigma Chemical Co. (S . Louis, Mo.); N-me hyl-N'-ni o-N-ni osoguanidine (NTG) was om Al- d ich Co. (Do se , Uni ed Kingdom); all o he biochemicals we e pu chased om Sigma. Ino ganic compounds we e om Me ck AG (Da ms ad , Ge many). Media. Comple e (YPD) medium, minimal (SD) medium, and p espo ula ion and spo ula ion media we e p epa ed by he me hods o She man e al. (20). Minimal p oline (SDP) medium is iden ical o SD medium, excep ha L-p oline (0.1%) is used ins ead o ammonium sul a e (0.5%) as he sole ni ogen sou ce (6). Hyd oxyno aline oxici y was 1677 Vol. 58, No. 5 1678 RAMOS AND CALDERON TABLE 1. S. ce e isiae s ains used in his wo k S ain(s) Geno ype o pheno ype Sou cea D273-11A MATox adel hisl CSH F4 MATa h 4 LCC MMY1 MA To u a3D52 CyhR SERI XCR13-5A MATa pl-l lysl-l u a4 leu2 DGS XCR28-4A MATa u a3 a g6 hom3 hisl DGS AHV1 o AHV6 MATa pl-l lysl-l u a4 Ieu2 This wo k Ah R Th E YAHV1 MATa u a4 HOM3-RI This wo k YAHV2 MATa HOM3-R2 This wo k YAHV3 MATa HOM3-R3 This wo k YAHV5 MATa u a3 HOM3-R5 This wo k YAHV6 MATa u a3 HOM3-R6 This wo k a Abb e ia ions: CSH, Cold Sp ing Ha bo Labo a o y (Cold Sp ing Ha - bo , N.Y.); LCC, La C uz del Campo S.A. (Se ille, Spain); SERI, Sola Ene gy Resea ch Ins i u e (Golden, Colo.); DGS, Depa amen o de Gene ica y Bio ecnia, Uni e sidad de Se illa (Se ille, Spain). es ed in SDP pla es con aining 10 mM hyd oxyno aline. Unde hese g ow h condi ions, he MIC o he wild ype was ound o be 2.5 mM. When necessa y, minimal media we e supplemen ed wi h he app op ia e equi emen s (20). Mu agenesis. Ea ly-s a iona y-phase cells o s ain XCR13- 5A g own in YPD medium a 30°C we e ea ed wi h NTG as ecommended by Calde on and Ce da-Olmedo (3). Unde hese s anda d condi ions, he su i al a e o his s ain was app oxima ely 40%. C oss- eeding es o h eonine exc e ion. Cells o he Th - s ain F4 we e g own o he s a iona y phase in YPD medium, cen i uged, washed wice, and suspended in wa- e . A sample o his cell suspension, con aining abou 106 cells, was sp ead on o SD pla es. The s ains o be es ed we e s eaked on o his cell lawn wi h oo hpicks, and he pla es we e incuba ed a he empe a u es s a ed o di e en pe iods o ime. The p esence o a halo o F4 colonies g owing a ound a s eak was conside ed a posi i e esul . Gene ic analysis. Con en ional p ocedu es we e used o s ain ma ing, diploid selec ion, and e ad analysis (20). Pa ial pu i ica ion o he aspa a e kinase. Aspa a e ki- nase was pa ially pu i ied by he me hod o Ramos e al. (16). The me hod is basically ha desc ibed as ollows. La e-exponen ial-phase cells, g own a 30°C in SD medium, we e ha es ed and esuspended in AT bu e (40 mM phospha e [pH 7.2], 0.1 M KCI, 5 mM MgCl2, 2 mM EDTA, 1 mM di hioe y h i ol 1 mM L- h eonine [19]) and dis up ed in a B aun homogenize wi h 0.5-mm-diame e glass beads. Cell deb is was emo ed by wo consecu i e cen i uga ions a 4°C, he i s o 20 min a 8,000 x g and he second one o 2 h a 105,000 x g. P o amine sul a e (5 mg/g [we weigh ]), neu alized o pH 7 wi h NaOH, was added o he c ude ex ac , and, a e 1 h o s i ing a 4°C, he pelle was emo ed by cen i uga ion. Ammonium sul a e was added, and he ac ion p ecipi a ing be ween 30 and 45% sa u a ion ha con ained he aspa a e kinase ac i i y was edissol ed in BT bu e (iden ical o AT bu e excep ha he phos- pha e concen a ion was 20 mM), dialyzed ex ensi ely agains he same bu e , and s o ed a -80°C. Aspa a e kinase assay. Aspa a e kinase ac i i y was assayed as p e iously epo ed (16) by moni o ing ADP p oduc ion by use o he py u a e kinase-lac a e dehyd oge- nase coupled assay desc ibed o he E. coli homose ine kinase (2, 21), excep ha 10 mM ATP and 4 mM L-aspa a e we e used as he subs a es. One uni o ac i i y was de ined as he amoun o pa ially pu i ied ex ac equi ed o con- e 1 ,umol o ATP o ADP pe min a 30°C. The s anda d assay mix u e con ained he componen s desc ibed p e i- ously (19), wi h mino modi ica ions. The eac ion was ini ia ed by adding L-aspa a e o he mix u e. P o ein con- cen a ions we e de e mined by he me hod o Low y e al. (9). Amino acid de e mina ion. Amino acids we e analyzed by e e se-phase liquid ch oma og aphy by he me hod o Ma - inez-Fo ce and Beni ez (10). The sys em consis ed o a Wa e s high-pe o mance liquid ch oma og aph wi h a No apack C-18 column (18 by 100 mm), wo pumps, and a Wa e s abso bance de ec o . An IBM AT compu e wi h a Baseline 810 ch oma og aphy wo ks a ion was used as he con olle , in eg a o , and da a collec o . The g adien p o- g am consis ed o wo sepa a e sol en mix u es. Sol en A consis ed o 20 mM sodium phospha e (pH 7.2), 1.5% e ahyd o u an, and 93.5% dis illed wa e (by olume); sol en B consis ed o 10 mM sodium phospha e (pH 7.2), 55% ace oni ile, and 42.5% dis illed wa e (by olume). Amino acid samples we e p epa ed as desc ibed p e i- ously (5). Samples (40 ml) o a la e-exponen ial-phase cul u e (op ical densi y a 660 nm, app oxima ely 0.7), we e cen i- uged. The supe na an was eeze-d ied and esuspended in 1 ml o deionized wa e , and he suspension was il e ed h ough a 0.45-,um-po e-size Millipo e il e and used o measu e exc e ed amino acids. To de e mine in acellula amino acids, he pelle was esuspended in 2 ml o deionized wa e and boiled o 15 min. A clea supe na an was ob ained a e a b ie cen i uga ion and a il a ion as desc ibed abo e. Amino acids we e de i a ized by mixing 25 ml o each suspension wi h 75 ml o he ollowing mix u e: 0.4 M sodium bo a e (pH 10), 4.5 ml; 54 mg o o-ph halaldehyde pe ml o me hanol, 0.5 ml (10). RESULTS Condi ions o hyd oxyno aline oxici y es . P elimina y expe imen s showed ha hyd oxyno aline is oxic in media wi h a poo ni ogen sou ce such as p oline bu no in media wi h a good ni ogen sou ce such as ammonium sul a e (da a no shown). This is p obably due o he ac ha his compound en e s he yeas cell mainly h ough he gene al amino acid pe mease (11), whose ac i i y is bo h inhibi ed and ep essed by ammonium (6). Consequen ly, all o he media in which hyd oxyno aline oxici y was o be es ed ca ied p oline as he sole ni ogen sou ce. I was also ound (da a no shown) ha he addi ion o di e en amino acids such as h eonine, homose ine, aspa - agine, and ci ulline o media con aining hyd oxyno aline coun e ac ed i s oxici y. The deg ee o his e ec was a iable, p obably depending on he mechanism in ol ed in each case. As expec ed om he p oposed hyd oxyno aline mode o ac ion (4), h eonine and homose ine, a h eonine p ecu so , imp o ed he s ain g ow h. I is e y likely ha he h eonine o homose ine addi ion bypasses he need o he enzymes ac ing in his biosyn he ic pa hway, some o which a e inhibi ed by his d ug (16). Consis en wi h his iew, i was ound ha , e en hough me hionine alone had no e ec , he simul aneous addi ion o h eonine and me hi- onine comple ely es o ed g ow h. Besides, hese and o he amino acids may exe hei in luence a he anspo le el. Ci ulline, o ins ance, may compe e wi h hyd oxyno aline o he gene al amino acid pe mease, while o he s ha se e as good ni ogen sou ces (e.g., aspa agine) may a ec he APPL. ENVIRON. MICROBIOL. THREONINE OVERPRODUCTION BY S. CEREVISIAE MUTANTS 1679 0- *1 la V 2 V IC ._ S E 21 a o_ 0- 0 AHV2 AUV3 AHVS S ain FIG. 1. Quan i ies o amino acids accumula ed (inside) and exc e ed o he medium (ou side) by di e en Ah R Th E s ains. Symbols: _, h eonine inside; Eli, h eonine ou side; , isoleucine inside; W, me hionine inside. gene al amino acid pe mease ac i i y, hus hinde ing i s en ance in o he cell. Isola ion o hyd oxyno aline- esis an (AhVR) mu an s. Cells o he wild- ype s ain XCR13-5A we e pla ed, ei he di ec ly o a e ni osoguanidine ea men , on o SDP me- dium con aining 10 mM hyd oxyno aline, and he pla es we e incuba ed o 5 days. Spon aneous and NTG-induced mu an s a ose among he su i o s a equencies o 1.5 x 10-6 and 5.0 x 10-5, espec i ely. In S. ce e isiae, he exc e ion o an amino acid by a ce ain s ain usually indica es o e p oduc ion o his amino acid. This c i e ion was applied o selec candida es o become h eonine o e p oduce s om he Ah R s ains. Thus, all Ah R colonies ob ained we e subjec ed o he h eonine c oss- eeding es desc ibed in Ma e ials and Me hods. While 60 o he 128 NTG-induced Ah R mu an s es ed we e h eonine exc e o s (Th E), none o he 60 spon aneous Ah R colonies we e Th E. We do no ha e a plausible explana ion o his di e ence. The Ah R Th E s ains (designa ed by AHV ollowed by a numbe ) we e used in u he s udies. Dominance o ecessi eness es . The dominance o eces- si eness o he alleles esponsible o he Ah R and Th E pheno ypes wi h espec o he wild- ype alleles was in es- iga ed by c ossing he mu an s ains wi h s ain MMY1. All o he esul ing diploids we e able o g ow on SDP medium con aining 10 mM hyd oxyno aline, a e 4 days o incuba- ion a 30°C, p o ing he dominance o he mu an alleles. All o hem we e also able o c oss- eed he Th - s ain, i.e., o exc e e h eonine, when incuba ed o 4 days a 37°C. Howe e , when incuba ed a 22°C, hey showed a a ie y o esponses in he c oss- eeding es : no diploid exc e ed be o e 4 days o incuba ion, and while some p esen ed an exc e ion halo a e 7 days, o he s did no , e en a e 11 days o incuba ion. Fi e AH-V s ains whose espec i e AHV/ MMY1 diploids showed di e en c oss- eeding imes a 22°C we e chosen o u he cha ac e iza ion. The selec ed s ains we e AHV1 (c oss- eeding ime, 7 days), AHV2 and AHV3 (c oss- eeding imes, 9 days), AHV5 (c oss- eeding ime, 11 days), and AHV6 (no c oss- eeding obse ed a e 11 days o incuba ion). Accumula ion o amino acids by he mu an s ains. To es ablish whe he he Ah R Th E s ains we e, in ac , h eonine o e p oduce s, he quan i ies o his and o he amino acids accumula ed and exc e ed in o he medium by hese s ains we e in es iga ed. The esul s (Fig. 1) show ha all o hem accumula e 15 o 30 imes mo e h eonine and exc e e abou 3 imes mo e h eonine han he pa en al s ain. They also con ain mo e isoleucine, an amino acid ha de i es om h eonine, han he con ol does. Bo h in he mu an s and in he pa en al s ain, he accumula ed isoleu- cine seems o be p opo ional o ha o h eonine (abou 1:5 o 1:3). Howe e , he amoun o me hionine, whose biosyn- he ic pa hway sha es se e al s eps wi h ha o h eonine, is simila o ha o he con ol in all mu an s. Gene ic cha ac e iza ion. The ollowing expe imen s we e designed o de e mine how many genes a e esponsible o he Ah R and Th E pheno ypes and o y o es ablish wha gene(s) is esponsible o he h eonine o e p oduc ion phe- no ype. P e ious kine ic s udies demons a ed ha he as- pa a e kinase plays a e y impo an ole in he egula ion o he pa hway a he enzyma ic ac i i y le el (16). Thus, we i s ied o p o e i , a leas in some cases, h eonine o e p oduc ion depends on he p esence in he cell o a mu an allele o he HOM3 gene, which codes o his enzyme. This kind o es is usually ca ied ou by meio ic analysis o c osses be ween he mu an s ain and a s ain ca ying a known allele, in his case, hom3. Howe e , such a c oss p o ides only pa ial in o ma ion since he Hom3- spo es equi e he p esence o homose ine in he medium. This would in e e e se e ely wi h he hyd oxyno aline esis ance es . Consequen ly, only Hom3+ spo es a e sus- cep ible o such an analysis. As s a ed abo e, he p esence o amino acids in he medium in e e es wi h he hyd oxyno aline esis ance es . Thus, i was ound necessa y o elimina e as many gene ic ma ke s as possible om he c oss. AHV/MMY1 diploids we e spo ula ed, and he esul ing e ads we e VOL. 58, 1992 1680 RAMOS AND CALDERON TABLE 2. Gene ic analysis o Ah R Th E s ains No. o e adsa C oss Ma ke s PD NPD T YAHV2 x D273-11A Th E_HISJ 26 0 0 Ah - HIS1 26 0 0 Ah R-Th E 26 0 0 YAHV5 x D273-11A Th E_HISJ 19 0 0 AhVR-HIS1 19 0 0 Ah R -Th E 19 0 0 YAHV6 x D273-11A Th E_HISJ 13 0 1 Ah R HIS1 13 0 1 Ah R-Th E 14 0 0 a PD, pa en al di ype; NPD, nonpa en al di ype; T, e a ype. TABLE 3. E ec o L- h eonine and hyd oxyno aline on he aspa a e kinase ac i i y o he Ah R Th E s ainsa Inhibi ion (%) by: S ain Sp ac 0m (mU mg o p o ein-) 20 mM Hyd oxy L-Th eonine Hy oxy- no aline XCR13-5A 188 90 91 AHV1 38 0 0 AHV2 45 00 AHV3 135 0 32 AHV5 37 0 0 AHV6 44 50 21 a Each alue ep esen s he mean o a leas wo expe imen s; he s anda d de ia ion was, in all cases, a ound 15%. DISCUSSION dissec ed. Meio ic descendan s om hese diploids showing an Ah R Th E pheno ype and ei he a U a+ o U a- pheno- ype we e chosen and called YAHV ( ollowed by he same numbe as ha o hei pa en s). When analyzing his c oss, i could al eady be s a ed ha , in all cases, he exc e ion o h eonine seg ega ed in a 2+ :2- ashion, indica ing he monogenic na u e o his cha ac e . S ains YAHV1, YAHV2, and YAHV5 we e c ossed wi h s ain XCR28-4A (hom3). Abou 20 e ads de i ing om each c oss we e dissec ed and analyzed. In hese c osses, he exc e ion o h eonine also seg ega ed in a 2+ :2- ashion. Mo eo e , all o he Th E spo es we e Hom3+ and no ecombinan Hom3+ Th NE ( h eonine-nonexc e ing) spo e was obse ed. This esul sugges s ha hom3 and he h ee mu a ions a e allelic. The e o e, hese mu a ions ha e been designa ed HOM3R-1, HOM3R-2, and HOM3R-5. This conclusion was u he p o en when Ah R Th E mu an s we e c ossed wi h a s ain ca ying a hisl allele. The genes HIS] and HOM3 a e loca ed in he S. ce e isiae map a a dis ance o 2.5 cen imo gans (12). This c oss allowed us o ca y ou whole- e ad analysis. Table 2 p esen s he esul s o he gene ic analysis o he c osses be ween he s ains YAHV and D273-11A. I clea ly shows he close linkage be ween he ma ke s s udied. Thus, we can in e ha he o e p oduc ion o h eonine depends on he p esence in hese s ains o mu an alleles o he HOM3 gene. Aspa a e kinase ac i i y in he mu an s. S udies ca ied ou o de e mine he kine ic pa ame e s o he aspa a e kinase showed ha bo h subs a es, ATP and L-aspa a e, displayed no mal Michaelis-Men en sa u a ion kine ics, wi h Kms o 3.6 and 4 mM, espec i ely (16). I has also been es ablished ha h eonine e y e ec i ely inhibi s he aspa - a e kinase ac i i y, 3 mM being he concen a ion equi ed o hal -maximal inhibi ion (16). O o he amino acids es ed, only hyd oxyno aline inhibi s his ac i i y (16). These and o he s udies sugges ha he egula ion o he aspa a e kinase o S. ce e isiae akes place mainly h ough eedback inhibi ion by h eonine (16). Thus, an al e a ion in his enzyme, such as making possible he ac i i y o he enzyme bu no i s egula ion by h eonine, could cause o e p oduc- ion o his amino acid. To es whe he his was he case wi h he Ah R Th E mu an s, hei aspa a e kinase was pa ially pu i ied and he e ec s o h eonine and hyd oxy- no aline on he ac i i y we e assayed. The esul s (Table 3) show, in ac , ha all mu an s es ed ha e an al e ed pa e n o inhibi ion by bo h amino acids. In his a icle, we desc ibe he isola ion and cha ac e iza- ion o S. ce e isiae mu an s esis an o he h eonine analog hyd oxyno aline. Among hem, hose able o c oss- eed a h eonine auxo oph we e chosen as candida es o h eonine o e p oduc ion. This c i e ion has p o en o be use ul since all selec ed s ains a e, in ac , h eonine o e - p oduce s. Howe e , a nega i e esul in his biological es does no necessa ily mean ha he s ain p oduces he same amoun o h eonine as, o less han, he wild ype. I is possible ha e en i a la ge amoun o h eonine is p oduced pe cell, ei he i is no exc e ed in o he medium o he amoun p oduced and exc e ed is no enough o eed he es e s ain. I is also possible ha he g ow h a e o he s ain is so low ha he inal biomass, and hus he o al p oduc ion o h eonine, is small. I so, one would expec a con inuous dis ibu ion o he Ah R mu an s wi h espec o h eonine p oduc ion; only hose mu an s p oducing and exc e ing a ce ain le el o h eonine would be de ec ed as such in he es . In ac , when he c oss- eeding es was pe o med a 22°C, he he e ozygo ic Ah R Th /wild ype diploids beha ed e y di e en ly. Howe e , his beha io does no co ela e wi h he h eonine p oduc ion o he co esponden haploid s ain; in ac , he AHV6/MMY1 diploid is he slowes exc e o , despi e he ac ha AHV6 is he bes h eonine p oduce . Mo eo e , no co ela ion was ound be ween he amoun o h eonine p oduced and he amoun exc e ed by he mu an s: he amoun o accumula ed h eonine a ied be ween 15 and 30 imes mo e han ha in he wild ype, while he quan i y o exc e ed h eonine was, in all o he mu an s, only abou 3 imes highe han ha in he wild ype (Fig. 1). Thus, we can in e ha he a ie y ound among he he e ozygo ic diploids in he c oss- eeding es is no due o ue di e ences in exc e ion bu is due o di e ences in o he ac o s such as g ow h a e o lysis o he s ain a ha empe a u e. Since one o he pa en s o he diploids is, in all cases, s ain MMY1, such di e ences should e lec di e ences be ween he AHV mu an s. These mu an s may di e no only in hei HOM3 allele bu also in he gene ic backg ound due o he indisc imina e ac ion o he ni osoguanidine. S. ce e isiae mu an s able o exc e e amino acids ha e been isola ed p e iously (15). In all cases s udied, exc e ion was shown o be a di ec consequence o o e p oduc ion. Two genes speci ically a ec ing h eonine exc e ion (TEX1 and TEX2) ha e been desc ibed (5). These genes a e linked nei he o each o he no o HOM3 (5). Labo a o y s ains seem o ha e di e en combina ions o mu an and wild- ype APPL. ENVIRON. MICROBIOL. THREONINE OVERPRODUCTION BY S. CEREVISIAE MUTANTS 1681 TEX alleles. The close ela ionship be ween o e p oduc ion and exc e ion ound in all o he gene ic c osses ca ied ou in his wo k sugges s ha all s ains used a e isogenic wi h espec o he TEX alleles, and, he e o e, ha his ac o is i ele an o he in e p e a ion o he esul s. All Ah R Th E mu an s examined so a ha e an aspa a e kinase ha is o ally o pa ially insensi i e o eedback inhibi ion by h eonine. O he au ho s, using di e en s a - egies, ha e ound simila esul s. Nass and Po alla (14) isola ed mu an s esis an o he mac olide an ibio ic bo e- lidin. Thei mu an s ell in o ou g oups. Only one o hem, BOR1, co esponds o s ains ha o e p oduce h eonine; all BOR1 s ains bea mu a ions in he HOM3 gene. Thus, bo elidin is no adequa e o ob ain h eonine-o e p oducing s ains, since mos o he esis an mu an s isola ed do no accumula e his amino acid. As expec ed, AHV mu an s a e also esis an o 20 ,uM bo elidin (da a no shown), and, con e sely, he BOR1 mu an s es ed (BOR1-1 and BORI- III [see e e ence 14]) a e esis an o hyd oxyno aline (da a no shown). Delgado e al. (5) showed ha i is possible o di ec ly isola e h eonine-o e p oducing s ains by e e ing a Hom3- mu an ; all o he isola ed s ains had a eedback- insensi i e aspa a e kinase. Gi en he mode o ac ion o hyd oxyno aline (4), one would expec o ind, among he Ah R Th E s ains, mu an s in he enzymes suscep ible o eedback inhibi ion by h eonine, namely, aspa a e kinase, homose ine dehyd ogenase, and homose ine kinase. How- e e , i has been epo ed ha yeas aspa a e kinase is much mo e sensi i e o inhibi ion by hyd oxyno aline han ho- mose ine kinase (16). The e o e, i is logical ha only mu an s in he aspa a e kinase we e isola ed. On he o he hand, no co ela ion was ound be ween he deg ee o sensi i i y o eedback inhibi ion by h eonine o hyd oxy- no aline o he aspa a e kinase and he p oduc ion o h eonine (Table 3). This esul could mean ha he condi- ions used o measu e he inhibi ion do no o ally e lec he "in i o" ac i i y o he enzymes. Table 3 also shows ha he speci ic ac i i y o he aspa a e kinase in c ude ex ac s o mu an s is smalle han ha o he wild ype. This e ec could be due o ep ession by h eonine o he HOM3 gene, bu o he causes canno be dismissed. Thus, i appea s ha he p esence in he cell o a h eonine-insensi i e aspa a e kinase o e comes o he cons ain s imposed on i s ac i i y, ha is, he egula ion akes place mainly a he enzyma ic ac i i y le el. AHV mu an s accumula e homose ine o abou he same le el as BORI mu an s do (18), i.e., abou 10 imes mo e han he wild ype (da a no shown). They also o e p oduce isoleucine bu o a lesse deg ee han h eonine does (Fig. 1). The egula ion o he pa hway leading om h eonine o isoleucine elies mainly on he ILV1 gene and i s p oduc (L- h eonine deaminase), which ca alyzes he con e sion o h eonine in o o -ke obu y a e (7). Isoleucine o e p oduc ion in he mu an s is p obably due o he induc ion by h eonine o he gene CHAI coding o a ca abolic L-se ine (L- h eo- nine) deaminase (17). This enzyme can ca y ou he same eac ion as he anabolic h eonine deaminase bu is no sensi i e o eedback inhibi ion. The mu an s do no accumula e mo e me hionine han he wild ype does, which indica es ha he me hionine-speci ic pa o he ou e is s ic ly con olled, as expec ed om he epo ed s ong inhibi ion and ep ession o he homose ine- O- ansace ylase exe ed by me hionine and S-adenosylme- hionine (8). Con e sely, and on he basis o he esul s p esen ed abo e, he las pa o he h eonine pa hway seems o play a seconda y ole in he egula ion o he biosyn hesis o his amino acid. The ac s ha all Ah R Th E mu an s isola ed a e h eo- nine o e p oduce s and ha he HOM3-R alleles a e domi- nan sugges ha his s a egy could be e icien ly used o he isola ion o h eonine-o e p oducing s ains o indus ial yeas species. ACKNOWLEDGMENTS This wo k was suppo ed by he Spanish CICYT (g an BT87- 0010) and by he Jun a de Andalucia. We hank M. A. Delgado and E. Ma in-Rend6n o hei many help ul discussions, K. Po alla, Uni e si a Tubingen (Tubingen, Ge many) o kindly supplying he bo elidin and BOR mu an s, M. J. Fa an o collabo a ing in some expe imen s, and I. Ma inez o co ec ing he manusc ip . REFERENCES 1. Aida, K., I. Chiba a, K. Nakayama, K. Takinami, and H. Yamada (ed.). 1986. P og ess in indus ial mic obiology, ol. 24. Else ie , Ams e dam. 2. Bu , B., J. Walke , P. T u a-Bachi, and G. N. Cohen. 1976. Homose ine kinase om Esche ichia coli K12. Eu . J. Bio- chem. 62:519-526. 3. Calde 6n, I. L., and E. Ce da-Olmedo. 1983. 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