Biosynthesis of Kitasamycin(Leucomycin) by Leucine Analog- Resistant Mutants of Streptomyces kitasatoensis

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1979, p /79/ /0$02.00/0 Vol. 15, No. 5 Biosynthesis of Kitasamyin(Leuomyin) by Leuine Analog- Resistant Mutants of Streptomyes kitasatoensis CLAUDE V1AZINA,'* CIgCILE BOLDUC,' ALICIA KUDELSKI,' AND PIERRE AUDET2t Department of Mirobiology, Ayerst Researh Laboratories,' St. Laurent, Quebe H4R IJ6 and Department of Mirobiology and Immunology, Shool ofmediine, Universite de Montreal, Montreal, Quebe H3C 3J7, Canada2 The biosynthesis of kitasamyin in Streptomyes kitasatoensis B-896 was profoundly influened by the addition of preursors to omplex and defined media: L-valine and L-leuine direted biosynthesis towards the pairs A4/A5 (R2 = butyryl) and A1/A3 (R2 = isovaleryl), respetively, and total kitasamyin titers were doubled and quadrupled, respetively. S. kitasatoensis B-896 was very resistant (>20 mg/ml) to a-aminobutyri aid, an analog of L-vahne, but very suseptible to L-leuine analogs 5', 5', 5'-trifluoroleuine and 4-azaleuine (5 to 10,tg/ml). The inhibition by 4-azaleuine ould be reversed by L-leuine, but by none of the other amino aids of the pyruvate family or the amino aids of the aspartate pathway. 4-Azaleuine-resistant mutants were isolated whih in the absene of any preursors overprodued L-leuine and a kitasamyin omplex mainly onsisting of the pair A1/A3. These 4-azaleuine-resistant mutants are presumed to be regulatory mutants in whih a-isopropylmalate synthase, the first enzyme of the L-leuine pathway, has beome either derepressed or desensitized to leuine feedbak inhibition. L-Leuine-regulatory mutants have eonomi value: in the absene of expensive preursors, they produe a kitasamyin omplex in whih the most potent pair Al/A3 is dominant and the least ative omponents are absent. Kitasamyin (leuomyin [10]) is a family of 14 antibiotis whih differ by the ayl substituent in position 4" (R2) of the myarose moiety and by the alternation of an hydroxyl and an aetyl group on arbon 3 (RI) of the 16-membered latone (17). The strutures and speifi antibioti ativities of kitasamyin members are reported in Table 1: the most potent pair is A1/ A3 (R2 = isovaleryl), followed by A4/A5 R2 - butyryl); moreover the hydroxylated member (R1 = OH) of eah pair is more ative than its aetylated partner. The omposition of the kitasamyin omplex produed by Streptomyes kitasatoensis is about 10% A1/A3, 60% A4/A5, and 30% other omponents. All kitasamyin members exhibit the same mode of ation, and the lipophili ayl group (R2) merely aids transport into target ells (24); thus, ompound All (R2 = H) inhibits protein synthesis by ell-free extrats of Esherihia oli as effiiently as ompound A1 (R2 = isovaleryl); yet, A1 is 30 times more ative than A11 in inhibiting growth. The biosynthesis of kitasamyin an be inferred from studies on magnamyin (8), a losely related marolide, and the work of Omura et al. (18) on kitasamyin. Myaminose and myarose would originate from gluose; N-methyl groups t Present address: Laboratoire Oto, Ville de Laval, Quebe, Canada H75 2A4. on myaminose and 0-methyl groups on the latone would derive from the S-methyl of methionine. 13C preursor studies (18, 21) have shown that arbons 1 and 2 and 9 to 16 are from aetate, arbons 5, 6, 17, and 18 are from butyrate, and arbons 7, 8, and 19 are from propionate. The origin of arbons 3 and 4 remains unknown. Carbons 20 and 21 in 3-aetylated ompounds (R1 = aetyl) also are from aetate; Omura et al. (15, 16) have reently observed that the 3-aetylated ompound A3 is derived from its 3-hydroxylated partner, Al, and that this aetylation is indued by gluose and valerate and repressed by butyrate. The biosynthesis of ayl groups at R2 is not firmly established. However, it has been known for many years (6) that isovaleryl and butyryl groups, present in the most desirable pairs A1/A3 and A4/A5, are derived from leuine and valine, respetively, in miroorganisms, and it is known that the isovaleryl side hain of magnamyin is derived from leuine (8). From the foregoing, it appears feasible to modify kitasamyin biosynthesis for improved omposition of the omplex and inreased poteny by favoring the formation of A1/A3 or A4/A5 in the produing organism, and by repressing the synthesis of the aetylated partner in either pair through the addition of butyrate. This paper mainly deals with the first objetive: the ation of leuine and valine an'd the overprodution of 738

2 VOL. 15, l TABLE 1. KITASAMYCIN BIOSYNTHESIS IN S. KITASATOENSIS 739 Struture' and poteny of kitasamyin Kitasamyin RI R2 Potenyh (U/mg) A, H COCH2CH(CH3)2 1,701 A:3 COCH:3 COCH2CH(CH:3)2 1,104 A4 COCH3 COCH2CH2CH3 984 A5 H COCH2CH2CH:, 1,528 A6 COCH3 COCH2CH:3 872 A7 H COCH2CH3 881 A8 COCH:, COCH3 233 As H COCH:3 288 Alo1(U)d COCH:3 H 86 A,1=(V)d H H 167 A12'? COCH:, COCH2CH2CH2CH2CH:, <528 A13e H COCH2CH2CH2CH2CH:3 528 A14e COCH:3 COCH2CH2CH(CH:3)2 Al5 H COCH2CH2CH(CH3)2 OH 1' CH:, CH:3 / N 4 A Myarose Myaminose 144 CH:, Latone b Components were separated by ounterurrent distribution, and their poteny was determined by omparison to a standard of 1,204 U/mg (D. Kluepfel and G. Shilling, personal ommuniation). 'Idential to josamyin (14). d Omura et al. (19). 'D. Kluepfel and G. Shilling (unpublished data). leuine and preferential formation of A1/A3 by leuine analog-resistant mutants of Streptomyes kitasatoensis is reported. The ation of butyrate is also desribed. A preliminary aount of this work was presented by Vezina (31). MATERIALS AND METHODS Kitasamyin-produing strains. S. kitasatoensis B-896 is the anestor of all the mutants desribed in this study. It was obtained by two rounds of mutagenesis (ultraviolet irradiation) and seletion from the original strain NRRL Strains B-896 produed a. 500 U of kitasamyin per ml in hemially defined medium (DM) and up to 3,000 U/ml in omplex medium (CM), whereas anestor strain NRRL 2486 yielded 100 U/ml under optimal onditions (CM). All ultures were propagated on solid medium at 30 C and 70% relative humidity for 12 to 14 days on tomato paste-oatmeal agar (TPO; 22). Conservation was by lyophilization (25) and by drying onidial suspensions in sterile soil (7). The inoulum was prepared as follows: surfae growth (spores and myelial fragments) on TPO from a Roux bottle ulture was suspended in 30 ml of sterile, distilled water; the suspension was homogenized by agitation for 15 min at 250 rpm in the presene of glass beads in a 250-ml Erlenmeyer flask OR2 and standardized by adding sterile water until a 1/10 dilution displayed a transmittane (1-m uvette, Coleman Junior 6A spetrophotometer) of 65%. Kitasamyin fermentation. Fermentations were done in 500-ml Erlenmeyer flasks ontaining 50 ml of medium; flasks were plugged with nonabsorbent otton, sterilized at 121 C for 20 min, ooled to 30 C, and inoulated with 1 ml of the inoulum. Inubation was at 30 C on a rotary shaker at 250 rpm (2-inh [a m] stroke) for 6 to 8 days. For growth determination during fermentation, 500-ml Erlenmeyer flasks equipped with optial glass side arms (light path, 1 m) were used. DM onsisted of the following (in grams per liter): L-methionine, 2.5; L-arginine, 2.5; (NH4)2SO4, 5; "erelose" (Com Produts In., New York), 10; soluble starh, 70; K2HPO4, 5. MgSO4..7H20, 0.5; NaCl, 0.5; CaCl2, 0.2; ZnSO4..7H20, 0.05; MnSO4. *7H20, 0.01; FeSO4.. 7H20, 0.02; distilled water to 1 liter; ph 7.2 to 7.4. CM was made by adding the following (in grams per liter) to DM medium: N-Z Case (Sheffield Chemials, Memphis, Tenn.), 0.5; maltose, 5; and sperm oil, 10, ph 7.0 to 7.2; erelose was inreased to 15, and CaCl2 was replaed by CaCO3, 3; tap water was used instead of distilled water. Stok solutions of L-leuine, L-valine, isoamyl alohol, and sodium butyrate were sterilized by membrane filtration (Millipore, 0.45-,um porosity) and added to the media before inoulation to final onentrations of

3 740 VRZINA ET AL. 1, 1, 0.2, and 0.1%, respetively. In preliminary experiments (not reported), these onentrations were found to give maximal effets. Suseptibility to leuine and valine analogs. The valine analog tested was L-a-amino-n-butyri aid (General Biohemials, Chagrin Falls, Ohio); the leuine analogs were 5', 5', 5'-trifluoroleuine, and 4-aza- DL-leuine (2-amino-3-dimethylaminopropioni aid) (ICN Pharmaeutials In., Cleveland, Ohio). Crystals of the analog were deposited 1 m from the edge of petri plates, 10 m in diameter, ontaining 20 ml of modified Hopwood (11) minimal medium (MMHM) previously inoulated by spreading 0.5 ml of the inoulum (above) by means of a sterile glass rod. MMHM onsisted of the following (in grams per liter): L-asparagine, 0.5; sodium itrate, 1; dextrose, 10; K2HPO4, 0.5; MgSO4. 7H20, 0.2; FeSO4 *7H20, 0.01; agar (Difo), 20; distilled water to 1 liter; ph 7.4 to 7.6 (KOH). Dextrose was sterilized separately as a 50% solution and added to the medium after sterilization. Inhibition by amino aid analog ould be deteted after 3 to 4 days of inubation at 30 C. Reversibility of inhibition was determined by depositing test ompounds (rystals) in the enter of plates. For preise determination of suseptibility, inhibitory analogs at various onentrations were inorporated into the medium. Isolation of 4-azaleuine-resistant mutants. A searh was made for 4-azaleuine-resistant mutants in S. kitasatoensis B-896. These mutants were very rare; olonies developing after prolonged inubation in the inhibition zone around rystals of the analog and those present on gradient plates (the Szybalski-Bryson method [29]) were generally found to have reverted to suseptibility on re-testing. Mutagenesis (ultraviolet irradiation to 99% kill) inreased mutant frequeny somewhat. The following method yielded satisfatory results: 250-mil Erlenmeyer flasks with optial side arm, ontaining 45 ml of liquid MMHM medium, were inoulated and inubated as usual. At early stationary phase (a. 48 h), 5 ml of a 4-azaleuine solution was added to a final onentration twie as high as that to whih the parent was resistant; 0.1-ml samples taken daily were spread on solid MMHM ontaining the same onentration of 4-azaleuine as that present in the flask. Resistant olonies developed after 1 to 2 weeks of inubation; they were purified twie and preserved on MMHM ontaining the same onentration of the analog. Resistant mutants were retested by repliation (Steers devie [27]) on MMHM ontaining various onentrations of 4-azaleuine. Kitasamyin determination. The prodution abilities of resistant mutants and parent B-896 were ompared by ulturing on MMHM (10 ml/10-m petri plate) at 30 C for 5 days, utting out 7-mm-diameter ylinders of agar, and transferring to glass plates (12 by 12 inhes [a by m]) ontaining 100 ml of Myin assay agar (Difo Laboratories, Detroit, Mih.), previously inoulated with Baillus subtilis ATCC 6633 (9). Inhibition zones were measured after 18 h of inubation at 37 C. For quantitative determinations, the standard ylinder plate assay was used. Standard solutions ontaining 0, 5, 20, 30, and 40 U of kitasamyin per ml in ph 7.6 potassium phosphate buffer (0.01 M) were used to onstrut standard urves. Dilutions of the "unknown" samples were made in the same buffer. ANTIMICROB. AGENTS CHEMOTHER. Kitasamyin omposition. Approximate omposition was determined by thin-layer hromatography. Broth samples were adjusted to ph 9 with NaOH and extrated with butyl aetate to give an extrat of a. 0.4 mg of kitasamyin per ml. A 10-pl amount was applied to silia gel plates. Kitasamyin omplex (0.5 mg/ml of methanol) and pure Al, A3, A4, and A5 (1 mg/ml of methanol) were similarly applied. Thinlayer hromatography plates were developed in hloroform: methanol:glaial aeti aid:water (79:11:8:2), dried, sprayed with sulfuri aid (10%), and heated at 160 C for 15 min. Quantitative determination of kitasamyin omposition was made by HPLC by the method of Omura et al. (20). Leuine determination. L-Leuine exretion was determined auxanographially by the method of Calvo (2). Salmonella typhimurium leubcd39, the test organism, bears a deletion whih prevents reversion to prototrophy and is resistant to kitasamyin, whih allows the determination of L-leuine in the presene of the antibioti. The test organism was grown on Calvo medium (2) enrihed with 50 mg of L-leuine per ml. After 24 h of growth at 37 C, ells of strain leubcd39 were washed thrie with saline by entrifugation at 9,000 rpm for 15 min and resuspended in saline. The washed suspension was finally standardized to read 70% transmittane (1-m uvette, Coleman Junior 6A spetrophotometer). It was prepared fresh daily. Calvo medium was inoulated with S. typhimurium leubcd39 inoulum and poured into petri plates or glass plates (12 by 12 inhes). Agar ylinders (7 mm diameter) from growth plates of streptomyetes under test were deposited on the surfae of the inoulated medium. After 18 h of inubation at 37 C, growth ould be deteted around the ylinders of leuine exreters. A rystal of leuine deposited on eah plate served as a positive ontrol. For the quantitative determination of leuine exretion, a ylinder plate assay (1) was first tried. The method was found of limited use beause of the diffiulty of preisely reading the diameters of growth. In subsequent experiments, a turbidimetri method (26) was used. Double-strength Calvo medium (without agar) was distributed into ulture tubes (5 ml/tube [16 by 150 mm]). A standard solution of L-leuine was distributed into a series of tubes to give final onentrations of 0, 10, 20, 40, 60, 80, and 100 mg/10 ml (per tube). Dilutions of "unknown" samples (1, 3, and 5 ml) were similarly distributed into another series of tubes. Volume was ompleted to 10 ml, and all tubes were sterilized at 121 C for 5 min, and then rapidly ooled to 37 C. Eah tube was inoulated with one drop of S. typhimurium leubcd39 inoulum and inubated at 37 C for 12 h. Turbidity was determined at 660 nm in a Coleman Junior6A spetrophotorameter. A standard urve was drawn, and the onentration of leuine in "unknown" samples was read by interpolation from the standard urve. RESULTS Effet of L-valine and L-leuine on kitasamyin titer and omposition. The addition of L-valine or L-leuine to DM had a profound effet on titer and omposition of the kitasamyin omplex. In preliminary experiments (31),

4 VOL. 15, 1979 the maximal effet was found at a onentration of 1% of either amino aid: L-valine, added to DM, inreased the proportion of A4/A5 mainly at the expense of minor omponents A6 to A15; addition of L-valine also had the effet of doubling poteny (1,000 instead of 480 U/ml). On the other hand, addition of L-leuine shifted the ratio of omponents in favor of Al/A3 and inreased titer to 1,700 U/mil. The ombined addition of 1% L-leuine and 0.2% isoamyl alohol, a produt of L-leuine degradation in many miroorganisms (6), led to further inrease in poteny (2,500 U/ml). Other presumptive preursors of kitasamyin ayl side hains had some effet, but their toxiity preluded their use in subsequent experiments. a-ketoisovalerate (0.2%), the branh point intermediate of the valine and leuine pathways, favored the prodution of Al and A5, but had no influene on poteny. a-ketoisoaproate (0.1%), the immediate preursor of leuine, favored the formation of Al and slightly inreased titer. L-Isoleuine (1%) greatly dereased titer. Leuine degradation produts, added singly to DM, only showed a modest effet (isoamyl alohol at 0.2% or isovaleri aid at 0.1%), or no effet at all (isovaleraldehyde at 0.1%); higher onentrations were toxi, and antibioti prodution was dereased. The time ourse of fermentation in omplex and defined media is illustrated in Fig. 1 and 2, respetively. In CM (Fig. 1), the effet of L- leuine was twie as large as that of isoamyl alohol; when added together, they showed an additive inrease in prodution. In DM (Fig. 2), L-leuine was also more effetive than isoamyl alohol, but a synergi effet was observed when s000 F 4000 *+l-leuine+iaa 3000/ / 4EL-Ieuine E AA 100 addition o-f{ 0 4 S Inubation (days) FIG. 1. EffetofL-leuine (1%) and isoamyl alohol (IAA, 0.2%), added to CM, on prodution of kitasamyin by S. kitasatoensis B-896. KITASAMYCIN BIOSYNTHESIS IN S. KITASATOENSIS E u 1000' soo E le200- o -i/ I nubation (days) myin by S. kitasatoensis B-896. FIG. (IAA, %o), Effet of L-leuine (1%) and isoamyl alohol added to DM, on prodution of kitasa- both ompounds were added. DM and CM gave similar distributions of kitasamyin omponents. The foregoing results prove the feasibility of produing kitasamyin omplexes of high poteny by adding to the prodution medium either L-valine, whih mainly leads to (A4)/A5, or L-leuine and isoamyl alohol, whih mainly give A1/(A3). The high ost of these amino aids, however, preludes their use in the prodution plant. The preparation of amino aid regulatory mutants, a onept used suessfully by Elander et al. (5) to produe pyrrolnitrin with tryptophan overproduers, was attempted in order to irumvent this eonomi diffiulty. Suseptibility of S. kitasatoensis B-896 to analogs of L-valine and L-leuine. The suseptibility of S. kitasatoensis B-896 to two L- leuine analogs, trifluoroleuine and 4-azaleuine, was tested auxanographially (Fig. 3): the organism was muh more suseptible to 4-azaleuine than to trifluoroleuine. When inorporated into agar (data not shown), 4-azaleuine inhibited growth ompletely at 5 to 10,ug/ml, whereas more than 100,ug of trifluoroleuine per ml was neessary to observe the same effet. On the other hand, the organism was very resistant (>20 mg/ml) to a-aminobutyri aid, a speifi inhibitor, or L-vahne in Serratia maresens (12). The inhibition of the kitasamyin produer by 4-azaleuine was reversed by L-leuine, as shown in Fig. 4; L-valine, L-isoleuine, L-threonine, and L-methionine had no effet. Beause the organism is more suseptible to 4-azaleuine than to trifluoroleuine and beause the former analog

5 742 VRZINA ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 3. Inhibition ofs. kitasatoensis B-896 by 4-azaleuine and 5',5',5'-trifluoroleuine on medium MMHM. Crystals of inhibitors were deposited 1 m above the lower edge ofplates. Plate on top ontained no inhibitor (ontrol). FIG. 4. Reversal of 4-azaleuine inhibition of S. kitasatoensis B-896 on medium MMHM. Crystals of 4- azaleuine were deposited on eah plate I m below the upper edge. Crystals of L-leuine (+LEU), L-isoleuine (+ILE), L-valine (+VAL), L-methionine (+MET), and L-threonine (+THR) were then deposited in the enter of indiated plates. has been shown in S. typimurium (B. I. Stieglitz, Ph. D. thesis, Cornell University, Ithaa, N.Y., 1971) to be more speifi to leuine than the latter, it was deided to searh for 5-azaleuineresistant mutants whih might overprodue L- leuine whih might diret biosynthesis to the pair Al/A3. Isolation of 4-azaleuine-resistant mutants. 4-Azaleuine-resistant mutants are very rare, and their expression and isolation require the presene of itrate in the medium (J. M. Calvo, personal ommuniation). This was onfirmed in S. kitasatoensis B-896. Moreover, olonies that developed in the inhibition zone around a 4-azaleuine rystal and those appearing on gradient plates were all unstable and reverted to suseptibility upon retesting. The organism was thus grown in shake flasks ontaining liquid MMHM, and 4-azaleuine was added after 48 h of inubation; therafter, daily samples were spread on solidmmhm ontaining the same onentration of the inhibitor. Only

6 VOL. 15, 1979 after 2 weeks did the first resistant olonies appear; altogether, 66 resistant mutants were isolated and purified. The mutants (and parent B-896) were tested auxanographially for L-leuine exretion; 29 of the 66 mutants exreted L- leuine, whereas parent B-896 yielded no zone. The 29 mutants were grown in DM in shake flasks, and the exretion of L-leuine and prodution of antibioti were determined quantitatively; their resistane to 4-azaleuine was also tested in MMHM plates. One mutant, designated 10/6, was seleted whih was more resistant to the inhibitor, exreted more L-leuine, and produed more kitasamyin than the other mutants and the parent (31). Mutant 10/6 was inhibited by 500,ug of azaleuine per ml. On plates ontaining the latter onentration of the inhibitor, some olonies developed on prolonged inubation (15 days). One hundred resistant olonies were isolated, purified, and retested for their resistane to azaleuine inorporated into agar. They were also grown in DM medium in shake flasks, and L-leuine exretion and kitasamyin prodution were determined. One mutant, designated 250/7, was superior to all others for the three parameters studied (31). As resistane to 4-azaleuine inreased in the mutants seleted, L-leuine exretion and kitasamyin prodution also inreased; furthermore, the omposition was shifted in favor of the pair A1/A3 (R2 = isovaleryl). The hromatogram in Fig. 5 A A5AI As A\ A5 A1 KITASAMYCIN BIOSYNTHESIS IN S. KITASATOENSIS 743 shows that azl 10/6 produed less of the minor omponents than did parent B-896 and that mutant azl 250/7 yielded the pair Al/A3 almost exlusively. The time ourse of fermentation by azl 250/7 in DM and CM is reported in Fig. 6. Kitasamyin was extrated after 6 days of inubation and found to onsist mainly of A1/A3. Isolation of highly resistant mutants from azl 250/7. Mutant 250/7 was grown in shake flasks for 48 h in MMHM, and 4-azaleuine was added to give final onentrations of 3.75 and 20 mg/ml. Inubation was ontinued, and mutants were isolated as desribed above. Twelve mutants were isolated, and all 12 upon retesting had lost the apaity to sporulate, exrete L-leuine, or produe kitasamyin. All attempts to improve their growth by enrihing DM and CM media were unsuessful. The severe seletion imposed by abrupt inrease in 4- azaleuine onentration was probably responsible for the "rippled" nature of these resistant mutants. Therefore, the experiment was repeated with more gradual inreases in 4-azaleuine onentration. Plates of MMHM ontaining 1.0, 1.5, 2.0, and 3.0 mg of 4-azaleuine per ml were inoulated by spreading on the surfae 0.1 ml of azl 250/7 inoulum. Another series of plates were inoulated with the survivors of ultraviolet irradiation (>99% kill). The resistant olonies that developed after several days of inubation were tested as done previously (Table 2). Several mutants exreted more L-leuine than parent 250/7. The highest exreter, whih yielded more than twie as muh L-leuine as did the parent, was resistant to 3 mg of 4-azaleuine per ml. However, none of the high leuine exreters (spontaneous or indued) yielded as muh kitasamyin as the parent. I-- E Comp ex mediuu m FIG. 5. Composition (thin-layer hromatography) of kitasamyin produed by S. kitasatoensis B-896 and its azl mutants 10/6 and 250/7 in DM in the absene ofpreursors. Fermentation broths were extrated after 6 days of inubation. Pure A, and pare A5 were added to the broth extrat, as indiated, to learly identify ertain omponents. Mixt., Commerial produt ontaining all kitasamyin omponents ' u E 500 DetnedAeduA O - o nu bat ion ( days) FIG. 6. Time ourse of kitasamyin fermentation by azl mutant 250/7 in CM and DM in the absene of preursors.

7 744 VtZINA ET AL. Resistant mutants were also isolated from liquid MMHM in shake flasks. Flasks were inoulated as usual, and 4-azaleuine was added at 0, 4, 18, and 42 h of inubation at various onentrations. Added at time 0, 4-azaleuine inhibited growth ompletely; it had no effet when added at 42 h (early stationary phase). The effet of various onentrations of 4-azaleuine added at 4 and 18 h is shown graphially in Fig. 7 and 8, respetively. In Fig. 7, growth was severely inhibited by all onentrations, but ould resume after 125 to 150 h of inubation in the presene of the lowest onentration (0.5 mg/ml). Added at 18 h (Fig. 8), 4-azaleuine was also inhibitory, but growth resumed muh earlier (50 to 75 h) and at all onentrations. At the end of inubation, samples from all flasks were spread on solid MMHM ontaining orresponding onentrations of 4-azaleuine; resistant mutants were isolated, purified, and tested as usual for resistane, L-leuine exretion, and kitasamyin prodution. All the 70 mutants tested were more resis- TABLE 2. Exretion of L-leuine and prodution of kitasamyin by spontaneous and ultraviolet-indued mutants from azl 250/7. Relative prodution" Resistane of mutants musstants (/ Spontaneous mutants Indued mutants (mg/ P n1l). Kitasa- Kitasa- L-Leuine myin L-Leuine myin a DM; inubation for 6 days at 30 C; 100 mutants per lass. Prodution of parent azl 250/7 taken as E o O _ 0.4. OS a. -o I --- _. No addition - o.s g/l a-@ 1.0 g/l 4C- 2.5 gig0' 0-0 g/l -S e T so I _ 1 50 looo Azoleuine Inu bation (ho urs) FIG. 7. Effet of 4-azaleuine, added after 4 h of inubation at the onentrations indiated, on growth of azl mutant 250/7 in medium MMHM. ANTIMICROB. AGENTS CHEMOTHER. E g,59/1 x C, st 4-Azoleuine Inubation h o u rs) FIG. 8. Effet of 4-azaleuine, added after 18 h of inubation at the onentrations indiated, on growth of azl mutant 250/7 in medium MMHM. tant than azl 250/7 and most exreted more L- leuine; 3 mutants exreted five times more L- leuine than the parent. One mutant, resistant to 5 mg of 4-azaleuine per ml, exreted 4 times as muh L-leuine and produed 1.5 times as muh kitasamyin as the parent. Effet of butyrate on kitasamyin omposition. Mutant azl 250/7 was ultured in shake flasks in DM medium in the presene and absene of sodium butyrate (0.1%). Kitasamyin was extrated after 4 and 6 days of inubation, and its omposition was determined by thinlayer hromatography (Fig. 9). The effet of butyrate was deteted only after 6 days of inubation (olumn S2. in Fig. 9): A1 was dominant over A3; however, A3 was not ompletely eliminated. DISCUSSION The omplex nature of the kitasamyin family and the unequal poteny of its omponents require us to define the omposition as well as the speifi ativity of the fmal produt to assure its uniformity. These requirements ompliate strain improvement programs whih aim for isolation of strains whih produe a omplex with an inreased proportion of desirable ompounds suh as A1/A3 or A4/A5. The physial separation of these pairs by ounterurrent distribution and the elimination of the others are tedious and uneonomial. Therefore, to simplify the mixture, the best solution lies in direting the biosynthesis towards desirable omponents either environmentally or genetially in the produing miroorganisms. Mutation and random seletion an lead to the isolation of strains with altered spetra of antibiotis (13, 31). Among other methods for altering the spetrum of metabolites (4, 23), the addition of preursors often is useful.

8 VOL. 15, 1979 KITASAMYCIN BIOSYNTHESIS IN S. KITASATOENSIS 745 isopropylmalate by a-isopropylnalate synthase, the first enzyme of the leuine pathway; a-isopropylmalate is isomerized to,8-isopropylmalate by a-isopropylmalate isomerase, and 1L-isopropylmalate is transformed into a-ketoisoaproate by,8-isopropylmalate dehydrogenase. The three enzymes are exlusive to the leuine pathway; finally, a-ketoisoaproate is onverted to L-leuine by transamination. On the other hand, degradation of L-leuine (6) would take plae A3_ through a-ketoisoaproate, isovaleraldehyde, af $^*^ _ and isoamyl alohol, the immediate preursor of A4_ isovaleryl group. Therefore, it was not surprising that the addition of a-ketoisovalerate A1 ^ ^ inreased titers v ^ofs A1 + A5, whereas that of a-ketoisoa- A _ 9 * proate greatly favored A1 at the expense of A5, and inreased total titers. Isovaleri aid and isoamyl alohol inreased total prodution but had no signifiant effet on the ratio A1:A5; isovaleraldehyde had no effet. L-Isoleuine, whih belongs to the aspartate family and is synthesized by a pathway parallel to that of L-valine, inhibited the formation of kitasamyin. In S. kitasatoensis, the valine pathway seems to be derepressed, beause (i) the organism is very resistant to a-aminobutyri aid, an inhibitor of aetohydroxy aid synthase (the first enzyme of the L-valine and L-isoleuine path- M S1 S2 M Sla S2a - M way), and (ii) it makes suffiient valine for the aumulation of the A4/A5 pair in the omplex. FIG. 9. Composition (thin-layer hromatography) The leuine pathway, however, is probably unof kitasamyin produed by azl mutant 250/7 in ab- der more stringent regulation, beause the orsene (S) and presene (Sz) of butyrate (0.1%). Fer- ganism is suseptible to trifluoroleuine and 4- mentation broths were extrated after 4 (S1 and S2) azaleuine, two inhibitors of a-isopropylmalate and 6 days (S,. and S2) of inubation. M, Mixture of synthetase (28). The orgamsm also makes only pure A3, A4,'A1,A5. limited amounts of leuine, and the omponents A1/A3 onstitute only a minority pair. The ation of added leuine appears to involve not only an Foster (6) reported that isovaleryl and butyryl inrease in the onentration of isovaleryl groups groups, present in kitasamyins A1/A3 and A4/ for the formation of A1/A3, but also repression A5, respetively, are derived from L-leuine and or inhibition of the valine pathway, beause the L-valine, and Grisebah and Ahenbah (8) pair A4/A5 is very strongly dereased in the proved that L-leuine is the preursor of the presene of L-leuine. isovaleryl side hain of magnamyin. The same The 4-azaleuine-resistant (azl) mutants of S. pathways seem to operate in S. kitasatoensis, kitasatoensis expetedly overprodued L-leubeause L-valine or L-leuine added to defined ine and yielded a kitasamyin omplex whih or omplex media drastially shifted kitasamy- mainly onsisted of the pair A1/A3. The onenin omposition in favor of the pairs A4/A5 and tration of L-leuine exreted inreased with the A1/A3, respetively, and appreiably inreased inrease in resistane up to a. 3 mg/ml; kitasathe total titer of antibioti. Beause the addition myin prodution inreased similarly (Table 2). of these amino aids is too expensive in the The relationship failed above this onentration. prodution plant, we deided to searh for reg- In onlusion, knowledge of the biosynthesis and ulatory mutants whih would overprodue regulation of the branhed-hain amino aids either L-vahne or L-leuine (see Demain [3] for was used empirially to modify the biosynthesis a review). L-Valine and L-leuine belong to the of kitasamyin. The interpretations given here, pyruvate family of amino aids (30). The branh however, are mainly speulative, and their orpoint intermediate is a-ketoisovalerate whih is retness must await the isolation and assay of transaminated to L-vahne or aetylated to a- the responsible enzymes.

9 746 VRZINA ET AL. ACKNOWLEDGMENTIS We are deeply indebted to Arnold L. Demain, Massahusetts Institute of Tehnology, for advie during pursuit of this study and for orretions of the manusript. We thank T. Hata, Kitasato University, for allowing us to obtain Streptomyes kitasatoensis NRRL 2486; H. E. Umbarger, Purdue University, for his gift of 5', 5', 5'-trifluoroleuine; and J. M. Calvo, Comell University, for S. typhimurium leubcd39. LITERATURE CITED 1. Bolinder, A. E Large plate assays for amino aids, p In F. Kavanagh (ed.), Analytial mirobiology, vol. 2. Aademi Press In., New York. 2. Calvo, J. M., M. Freundlih, and H. E. Umbarger Regulation of branhed-hain amino aid biosynthesis in Salmonella typhimurium: isolation of regulatory mutants. J. Bateriol. 97: Demain, A. L Overprodution of mirobial metabolites and enzymes due to alteration of regulation. Adv. Biohem. Eng. 1: Demain, A. L Mutation and the prodution of seondary metabolites. Adv. Appl. Mirobiol. 16: Elander, R. P., J. A. Mabe, R. L Hamill, and M. Gorman Biosynthesis of pyrrolnitrins by analogue-resistant mutants of Pseudomonas fluoresens. Folia Mirobiol. (Prague) 16: Foster, J. W Chemial ativities offungi. Aademi Press In., New York. 7. Green, H. C., and E. B. Fred Maintenane of vigorous mold stok ultures. Ind. Eng. Chem. 26: Grisebah, H., and H. Ahenbah tber die Herkunft der Isovaleriansaure im Magnamyin. Experientia 19: Grove, D. C., and W. A. Randall Assay methods of antibiotis-a laboratory manual. Antibiot. Monog. 2: Hata, T., Y. Sano, N. Ohki, Y. Yokoyama, A. Matsumae, and S. Ito Leuomyin, a new antibioti. J. Antibiot. Ser. A 6: Hopwood, D. A., K. F. Chater, J. E. Dowding, and A. Vivian Advanes in Streptomyes oeliolor genetis. Bateriol. Rev. 37: Kisumi, M., S. Komatsubara, and L. Chibata Valine aumulation by a-aminobutyri aid-resistant mutants of Serratia maresens. J. Bateriol. 106: Kluepfel, D., S. N. Sehgal, and C. Vezina Antimyin A omponents. I. Isolation and biologial ativity. J. Antibiot. 23: Omura, S., Y. Hironaka, and T. Hata Chemistry of leuomyin. IX. Identifiation of leuomyin A3 with josamyin. J. Antibiot. 23: Omura, S., J. Miyazawa, H. Takeshima, C. Kitao, and M. Aizawa Indution of the bioonversion ANTIMICROB. AGENTS CHEMOTHER. of leuomyins by gluose in a produing strain. J. Antibiot. 30: Omura, S., J. Miyazawa, H. Takeshima, C. Kitao, K. Atsumi, and M. Aizawa Bioonversion of leuomyins and its regulation by butyrate in a produing strain. J. Antibiot. 29: Omura, S., and A. Nakagawa Chemial and biologial studies on 16-membered marolide antibiotis. J. Antibiot. 28: Omura, S., A Nakagawa, H. Takeshima, K. Atsumi, J. Miyazawa, F. Piriou, and G. Lukas Biosyntheti studies using ':'C enrihed preursors on the 16-membered marolide antibioti leuomyin A3. J. Am. Chem. So. 97: Omura, S., A. Nakagawa, N. Yagisawa, Y. Suzuki, and T. Hata Chemistry of leuomyin. X. Conformational studies on leuomyin. Tetrahedron 28: Omura, S., Y. Suzuki, A. Nakagawa, and T. Hata Fast liquid hromatography of marolide antibiotis. J. Antibiot. 26: Omura, S., H. Takeshima, A. Nakagawa, J. Miyazawa, F. Phriou, and G. Lukas Studies on the biosynthesis of 16-membered marolide antibiotis using arbon-13 nulear magneti resonane spetrosopy. Biohemistry 16: Pridham, T. G., P. Anderson, C. Foley, L, A. Lindenfelser, C. M. Hesseltine, and R. G. Benedit A seletion of media for maintenane and taxonomi study of Streptomyes. Antibiot. Annu Queener, S. W., 0. K. Sebek, and C. Vezina Mutants bloked in antibioti synthesis. Annu. Rev. Mirobiol. 32: Rakhit, S., and K. Singh Struture-ativity relationship in sixteen-membered marolide antibiotis. J. Antibiot. 27: Raper, K. B., and D. F. Alexander Preservation of molds by the lyophyl proess. Myologia 37: Shokman, G. D Amino aids, p In F. Kavanagh (ed.), Analytial mirobiology, vol. 1. Aademi Press In., New York. 27. Steers, E., E. L. Foltz, B. S. Graves, and J. Riden An inoula repliating apparatus for routing testing of baterial suseptibility to antibiotis. Antibiot. Chemother. 9: Stieglitz, B. I., and J. M. Calvo Distribution of the isopropylmalate pathway to leuine among diverse bateria. J. Bateriol. 118: Szybalski, W., and V. Bryson Geneti studies on mirobial ross resistane to toxi agents. I. Cross resistane of Esherihia oli to fifteen antibiotis. J. Bateriol. 64: Umbarger, H. E Amino aid biosynthesis and its regulation. Annu. Rev. Biohem. 47: V6zina, C Mutation to hange the spetra of metabolites, p In D. Shlessinger (ed.), Mirobiology Amerian Soiety for Mirobiology, Washington, D. C.