CA1172191A - Process for preparing mycarosyltylactone - Google Patents

Abstract

Abstract of the Invention A new microorganism, Streptomyces fradiae NRRL 12201, which produces mycarosyltylactone (5-O-mycarosyl-20-dihydro-20,23-dideoxytylonolide) and 2 process for preparing tylactone (20-dihydro-20,23-dideoxytylonolide) and mycarosyltylactone by submerged aerobic fermentation of this microorganism, or a mycarosyltylactone-producing mutant or recombinant thereof, are provided.

Description

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PROCESS FOR PREPARING MYCAROSYLTYLACTONE

Summary of the Invention This invention relates to a new strain of streptomYces fradiae and to a process for preparing tylac-tone and a new macrolide compound by submerged aerobic fermentation of this new strain. The new compound, which is 5-O-mycarosyl-20-dihydro-20,23-dideoxytylonolide, will be called mycarosyltylactone for convenience herein.
Mycarosyltylactone has structure 1:
lo R

~ CH
22 j~1 7~ fH
C~3~ 2 1~ 6~:H2-CH3 j 3~
~ {~------o ~ /~-OH

CH3-CHz~ 3 3 Mycarosyltylactone is disclosed in a co-pending Canadian patent application Serial No. 382,652 by Robert L. Hamill and Gene M. Wild entitled MYCAROSYLTYLACTONE.

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Tylactone has structure _:
R
-CH s 1 0 ~
CHs-ll t-CH2-CH3 -G~i CH3-t t 0 ~ ~ /

Tylactone is the subject of a co-pending Canadian patent application of Robert L. Hamill, Gerald L. Huff, Richard ~o H. Baltz and Eugene T. Seno, entitled TYLACTONE, Serial No. 380,953. Another method for making tylactone is the subject of our co-pending Canadian patent application entitled PROCESS FOR PREPARING TYLACTONE, Serial No.
380,946.
Tylactone and mycarosyltylactone are useful inter-mediates from which 16-membered macrolide antibiotics can be prepared. Although no stereochemical assignments are indicated in the structures given herein, the stereo-chemistry of the compounds is identical to that of the corresponding portion of tylosin.
Description of the Drawinq The infrared absorption spectrum of mycarosyl-tylactone in chloroform is presented in the accompanying drawing.

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Detailed Descri~ion .--This invention relates to a new microorganism and to a new process which uses this microorganism. --The new microorganism is a mutant strain of _ repto~
myces rradiae. The new process is a method of making ...
mycaros.yltylactone and tylactone by fermentation of ...
this ~ E~ fradiae strain under submerged .-aerobic conditions until a substantial amount of these compounds are produced. .-The following paragraphs describe the proper- .
ties of mycarosyltylactone, the new macrolide compound produced by the process of this invention. .
Mxcaxosxltxl--act-on-e The structure of mycarosyltylactone is shown ..
in formula 1. Mycarosyltylactor.e is a white solid -.
which crystallizes from heptane, hexane or ethyl -.
acetate-hexane~and which melts at about 1~2-184C. I~
has the following approximate percentage elemental composition: carbon, 67%; hydrogenr ~%; and oxygen, 24%. It h~s an empirical ~ormula of C30H50O8 ~nd a molecular weight of about 538O .-The inf~ared absorption spectrum of mycarosyl-tylactone in chloro~orm is shown in the accompanying 25 drawing. Observable absor~tion maxima occur at the following frequencies ~cm 1): 3640 ~medium), 2941 and 2907 [doublet (strong)], 2421 Ivery small), 1712 (strong), 1678 (medium), 1523 (small), 1590 ~st~~ong), 1456 (medium), 1404 (sm~ll), 1374 ~small), 1359 ~houlder), 1314 (small), 1234 (small), 1263 (very small), 1229 . .

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( mall), 1178 (strong), 1157 (medium) r 1134 (very small), 110~ (small), ].078 (ve~y small), 1050 (medium), --1025 (very small), 1000 (strong), 984 (strong), 962 (medium), 920 (very small), 911 (very small), 887 (small), 867 (small), 848 (shoulder)~ 836 (small), and -799 (small). -The ultraviolet absorption spectrum of -mycarosyltylactone in ~eutral ethanol exhibits an -~-absorption maximum at about 2~2 nm tElCm = 568). --_ ]O ~ycarosyltylactone i5 nearly insoluble in water, but is soluble in organic solvents such as acetone, methanol, ethanol, dimethyl~ormamide, chloro- -form, diethyl ether, petroleum ether, benzene and dimethyl sulfoxide.
~5 One important use o~ mycarosyltylac~one is as -an intermediate to make tylactone and tylactone deriva- -tives. The following paragraphs describe the proper~ies of ty}actone.
lactone `
The structure of tylactone is shown in formula 2. Tylactone is ~ white solid which crystal-lizes ~rom heptane, hexan~ or ~thyl acetate-hexan and --which melts at about 162-163C. It has the following 2~ approximate percentage elemental composition: carbon, --70%; hydrogen, 9.7%; oxygen, 20.3~. It has an emp~ric~l -LGrmula of C~3E13805 and a molecular weight of about 394. --....

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,, The infrared absorptior. spectrum of tylactone .--in chloroform has observable absorption maxima at the following frequencies (cm 1~ 3~34 (medium), 2924 (strong), 2398 (weak), 2353 (weak), 1709 (ve~ strong), 1678 (very strong), 1626 (small), 1592 (very strong), 1458 (strong), 1441 (shoulder), 1404 (strong), 1379 .....
~small),. 1316 (strong~, 1284 (medium), 1181 (very ... ~.
strong), 1143 (strong), 110~ ~medium), 107~ (medium), -1049 (very small), 1025 (medi~m), 984 (very strong), .. ~
958 (strong), 923 (medium~, gll (shoulder), 8~9 (small), .-868 (medium), 840 (medium), 820 (very small) and 561 -.. -.
(small). .-.
The ultraviolet (W) absorptio~ spectrum oS -tylactone in neutral ethanol exhibits an abso-ption l; maximum at about 282 nm (El%m = 560). ...
Tylactone has the 40110wing specific ro- :
tation: -[a]25 -55.23 (c 1, CH OH). .--

- 3 .:.
Electrometric titration of ~ylactone in 66% .-.-20 aqueous dimethylformamid~ indicates i~ has nc titrata- .-ble groups. -.---. ;:
Tylactone i5 nearly insoluble in water, but ..
is soluble in organic solven~s such as acetone, methanol, '.
etha~ol, dimethylformamide, chloroform, diethyl ether, 25 petroleum ether, benzene and dimethyl sul~oxide.
Chromatography of Mycarosyltxlactone ' Mycarosyltylactone can be distinguis~ed from ...
tylactone and tylosin by silica-gel thin-layer chroma- `
30 tog.raphy (TLC). Sulfuric acid spray, ei~ler concentra~ed -. .

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or diluted (50%), may be used for detection. With this datection system tylactone appears initially as a .--yellow-to-brown spot, and mycarosyltylactone appears as --a blue-purple spot. If silica-gel plates with a .--.
fluorescent background are used in the chroma~ography, .-W detection is convenient. The approximate Rf values ...
of mycarosyltylactone axe summarized in Table 1. .-Table 1 -:-:._ TLC OL MYCarOSY1tY1aCtCTIea --. .
Rf Value__ -Compound Ab B -Mycarosyltylactone 0.17 0.44 Tylactone 0.50 0.62 Tylosin 0 D O O ~ O ~'''' aMedium: Silica gel --bSolvent: A - benzene: ethyl acetate (4:1) ..
B = benzene: ethyl acetate (3:2) :-20 Preparation of Mycarosyltylactone and Tylactone -. . .
Mycarosyltylactone and tylactone are prepar~d ..
by culturing a strain of Streptomyces fradiae which ..
produces these compounds under submerged aerobic con~
ditions in a suitable culture medium until a substantial -amount of compound i5 produced.
The culture medium used to grow the Stre~to .
myces fradiae can be any one o~ a number of media. For ...
economy in production, optimal yield, and ease of product isolation, however, certain culture media are prererred. Thus, for example, preferred carbon sources ~72~9~. ai , ....

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in large-scale fermentation include carbohydrates such as dextrin, glucose, starch, ~nd corn meal and oils ~--such as soybean oil. Preferred nitrogen sources --include corn meal, soybean meal, fish meal, amino acid~
and the like. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding iron, --potas3ium, sodium, magnesium, caicium, ammonium, chloride, carbanate, sulfate, ~itrate, and like ions.
Essential trace elements necessary for the ---growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of --the medium in amoun~s suficient to meet the growth 15 requirements of the organism. lt may be necessary to ---add small amounts ~i.e. 0.2 ml/L) of an antifoam agent such as polypropylene glycol IM~W~ about 2000) to l~rge-scale fermentat on media i foaming becomes a problem.
As is customary in aexobic submexged ulture processes, sterile air is bubbled through the culture ---medium. Yor efficient antibiotic produciion ~he per- --cent of air saturation for tank production should be ~ .
about 30~ or above (at 2~C and one atmosphere of :::
pressure).
~or pxoduction of su~stantial quantities of these compo~nds, submerged aerobic fermentation in --tanks is preferred. Small quantities may be obtained by shake-flask culture. 3ecause of the time lag in production com~only ~ssociated with ino~ulation of .

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., large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume o culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture. The vegetative inoculum is then transferred to a larger tank. The medium used for the vegetative --inoculum can be the same as that used for larger fermen- -tations, but othex media can also be used.
~o Production o~ mycarosyltylactone and tylactone can be followed during the fermentatio~ by testing --samples of the broth, using T~C or high-performance liquid ch~omatography with a. W detection system. --Following their production under submerged aerobic fermentation conditions, mycarosyltylactone or tylactone can be recovered from the fer~entation medium -:
by methods used in the fermentation art. Because of the limited solubility of these compounds i~ water, they may not be altogether soluble in the medium in which they are produced. Recovery can be accomplished by 1) extraction of the fermentation broth or 2) fil- --tration of the fermentation broth and e~traction of --both the filtered broth and the mycelial cake. A
variety of techniques may be used in the ex~raction Z5 processeC. A preferred technique for purification of the filtered broth involves extracting the bro~h (generally without pH adjustment) with a suitable solveni such as amyl ~cetate or petroleum ether, concentrating the organic phase under YacUum to give crystals or an oil. The crystals or oil thus obtained may be purified by adsorption chromatograp~y to give mycarosyltylactone and tylactone.

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,, , ~-5659 _g_ ", . ., The Microorganism ---The new microorganism of ~his i~vention was obtained by chemical mutagenesis of a Streptomyces -:-fradiae strain which produces tylosin. The new micro~
S organism produces only minimal amounts of tylosin, but ---produces mycarosyltyl~ctone and tylactone as major ---compo~ents. The new micxooxganism is also classified as a strain of ~ fradiae. A culture of this microorsanism has been deposited and mzde part o~ the 10 stock culture collection of the Northern Regional --Research Center, Agricultural Research, North Central --Resion, 1815 North University Street, Peoria, Illinois, ---61604, from which it is available to the public under the acce sion nu~er NRRL 12201.
As is the case with other~organism3, the char~cteristics of Stre~tomyces fradiae NRRL 12201 are __ .
subject to variation. Recombinants, mutants or variants of ~he NRRL 12201 strain may be obtained by --methods known in the ar~. For example, mutants c~n be ---o~tained by treatmant with various known physical and chemical mutagens, such as ultraviolet light, X-r~ys, gamma rays, and N~methyl-N'-nitro~N nitrosoguanidine.
All na~ural and inducad variants, mutants and recom-binants of Strept~yces fradiae NRRL 12201 which retain the characteristic of mycarosyltylactone production are a par~ of this invention.
S. fradiae NRRL 12201 can be grown at tem-peratures between about 10 and about 40~C. Optimum production of m~carosyltylactone appears ~o oçcl~r at temperatures of about 28 .

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Tylactone and mycarosyltylactone are useful inter-mediates from which 16-membered macrolide antibiotics can be prepared. Mycarosyltylactone (1) can be hydrolyzed using mild acid conditions to give tylactone (2). Mild acid hydrolysis conditions are known in the art. Approp-riate solutions having a pH of about four or below can be used to accomplish the hydrolysis. A polar organic cosolvent, such as an alcohol ~for example, ethanol) should be included to keep the reactants in solution.
Temperatures of about 20 to about 100C can be used in this method. The reaction time needed to carry out the hydrolysis varies, depending upon the pH of the reaction mixture and the temperature used. At higher pH levels the reaction rate is slower, and at higher temperatures the reaction rate is faster. The reaction is carried out by treating mycarosyltylactone with a mild acid solution for a time sufficient to effect removal of the mycarosyl group to give tylactone.
Alternatively, and sometimes preferably, tylactone can be prepared by treating mycarosyltylactone in the fermentation broth in which it is produced, using mild acidic conditions as above described for a time sufficient to convert the mycarosyltylactone to tylactone. Tylactone thus prepared can be isolated from the fermentation broth using techniques known in the art.
Tylactone can be bioconverted to tylosin or tylosin-related compounds as described by Hamill et al.
in Serial No. 380~953. The bioconversion is accomplished B~

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by adding tylosin to a growing culture of a biocon- -verting microorganism. The bioconverting microorganism can be a Streptom~-ces strain which either produces tylosin itself or ls capable of produclng tyLosin except that it i~ blocked in tylactone formation.
A strain which is capable of producing -;-tylosin except that it is blocked in tylactone for- --mation can be obtained by treating a tylosin-producing ---strain with a mutagen and screening survivors ror tho~e which axe unable to produce tylosin. Those survivors which are unable to produce tylosin are further screened to determine which strains are unable to produce tylactone but are still capable of bioconverting tylactone to tylosin. These strains are identifiDd by adding tylactone to small shake-flask cultures of the selected su-vivors to determine if they bioconvert ---tylactone to tylosin. ---Strept~myces fradiae stxains NR~L 2?02 and NRRL 2703 are examples of Streptomyoes strains which are capable of producing tylosin. A typical mutagen which may be used to obt~in the selected ~trains is N-methyl-N'-nitrQ-N-nitro oguanidine.
Tylactone is especially useful in the prep- --aration of labeled compounds for biosynthetic or 25 metaboli~ studies. By labeling either the tylactone --portion or the added sugar moieties, qpecifically labeled tylosin useful for biosynthetic or metab~lic ..
studies can be obtained. `-In order to illustrate more fully the opera~
tion o~ this invention, the following examples are provided:

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,--_ Example l ..
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A. Shake-flask Fermentation of Mycarosyltylactone .,, A lyophilized pellet of Strep~omyces fradiae -.~
NR~L 12201 is dispersed in 1-2 ml of sterilized water. .-.-A portion of thi~ solution (0~5 ml) is used to in- --oculate a vesetative medi-~m (150 ml~ having the fol- .~
lowing composition: --~ Amount (%) ---...
Corn steep liquor l~0 -Yeast ex~ract 0.5 -._ Soybean grit~ 0.5 CaCO3 0.3 :
Soybean 31l (cxude) 0.45 ....
Deionized water 97.25 . . --.
, . . .
Alternatively, a vegetative culture of S. --fradiae NRRL 12201 preserved, in 1-~1 volumes, in ..
liquid nitrogen is rapidly thawed a~d used to inoculate --the vegetative medium. The inoculated vegetative ... -medi~m is incubated in a 50G-ml Erlenmeyer flask at 29C. for about 48 hours on a closed-box shaker at = .~
about 300 rpm.
This incubated vegetative medium (0.5 ml) is 25 u~ed to inoculate 7 ml of a production medium having .-~
the following composition: `

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In~redient Amount ~%) .,._.
Beet molA.sses 2.0 .. -.
Corn meal 1.5 ..
Fish meal 0.9 Corn gluten 0.9 ....
NaCl 0.1 ..
( 4)2 4 0 04 CaCO 0.2 .--3 ....
Soybean oil (crude) 3~0 .-Deionized water 91.36 .--.-. -:
The inoculat~d fermentation medi~m is incu~
bated in a 50-ml bottle at 29C. for about 6 day~ on a ...
closed-box shaker at 300 rpm.
15 B. Tank Fermentation of ~ycarosy--ylactone -and_~lactone . ., In crder to provide a la~ger volume o~ inocu- .. --.
lum, 60 ml of vegetative culture prepa~ed in a manner .-20 similar to that described in section ~, ls used to -.. -inoculate 38 L of a second-stage vegetative growth ..
medium having the folIowing composition~
Ingredient Amount (~
Corn steep liquor 1.0 ~.-Soybean meaI 0.5 Yeas~ extract 0.5 -CaCO3 0.3 Soybean oil (crude) 0.5 .. -.
Lecithin ~crude) 0.015 .
Water 97.185 ..
Adjust pH to 8.5 with 50% NaOH solution. ...

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This second-stage veget~tive medium is incu~
bated in a 68-liter tank for about 47 hours at 29CC. -.
Second-stage culture (4 L~ thus prepared is ..
used to inoculate 40 liters of sterile production ~-medium having the following composition:
Amount ( ~ ) Fish meal 0.92 .. -Corn meal 1.;7 Corn glu~en 0.~2 -.
CaC~3 0.21 NaCl 0.10 -.
~NH4)2HPO4 0 04 --~eet molasses 2.10 Soybean oll (crude) 3.15 -Lecithin 0.09 Water 90 90 .-Adjust pH ~o 7.2 with 50% NaOH 501ution.
The inoculated production medium is allowed ....
20 to ferment in a 68-liter tank for about 5 days at a .-temperature of 28C. The fermentation medium is j-aerated with sterile air to keep the dissol~ed oxygent.,, level ~etween about 30~ and 50% and is stirred with conventional agitators at about 300 rpm. ~:
.
ExamE~2 `~
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Fermen~a~ion broth (900 ml),.obtained as described in Example 1, Section A, is extracted with ...
30 petroleum ether (900 ml). The petroleum ether extract `.

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is concentrated under an air stream ~.o give an oil. --The oil is dissolved 7 n a small amount o ethyl -acetate (about 15 ml). Heptane (about 15-20 ml) is --added. The ethyl acetate is slowly allowed to evap- --orate to permit crystallization. The crystals are --separat~d to give 450 mg of a crystalline mixture of -tylactone and mycarosyltylactone. --Additional material can be obtained by adding an equal volume of methanol to the re~aining 10 whole broth, flltering the resulting solution, and ---extracting the filtrate with methylene chloride. ~-The crystalline mixture (400 mg) is sep- --arated by dissalving it in ben~ene. The benzene ;~
solution is chromatographed over a siLica-gel (Woelm) column, packed with benzene. Elution i5 monitored by silica-gel thin~layer chromatography, using a benzene~
ethyl acetate (3:2) solvent system and conc. sulfuric acid spray for detection. The column is first eluted with benzene to remove lipid substances, then with one liter of benzene:ethyl acetate (9:1), 1400 ml of benzene:ethyl acetate (6:1) and 900 ml o~ benzene:- -ethyl acetate ~3:1~ to separate and isolate tylactone and mycarosyltylactone. Fractions having a volume of a~out lS0 ml are collected. Tylactone is eluted first (fractions 14-19), and mycarosyltylactone fs elu~ed later (fractions 22-26). Fractions containing each are combined, evaporated under vacuum, and crys~al-lized from heptane to give 150 mg of ~ylactone and 120 mg of mycarosyltylactone.

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Example 3 ----. .
Preparation of TYlactone from Mycarosyltylactone --Mycarosyltylactone, prepared as descri~ed in Example 2, is dissolved in a methanol-aqueous hydro- - -chloric acid solution (pH 1.8). The resulting solution i5 allowed to stand until hydrolycis is complete (about 48 hours) at room temperature and then i5 -adjusted to pH 7.0 by the addition of sodium hydroxide. ---lG This solution is extracted with ethyl acetate, dichloro- -me~hane or chloroform. The extract is dried under vacuum to give tylactone.
Example ~ -Alternate ~re aration of T lacton~ from Mvcaros 1-LJ tylactone --Mycarosyltylactone, prepared a~ ~escrib~d in Example 1, is treated using the procedure of Example 3 --to give tyla~tone in the fermentation bro~h. The - -.-:
tylactone is isolated according to the procedure of Example 2.
.
Exam~le S
Preparation of Tylosln from Tylac~one .;
A Streptom~ces ~radiae strain which formerly --produced tylosin but which is blocked in macrclide ring clo ure is fermented according to the procedure described in Example 1, Section A. A temperature of 28C is used. Tylactone is added to the fermentation

4~ hours after inoculation. The fermentation is then continued until a substantial amount of tylosin is 1~7~

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produced, i.e. about three additional days. The presence of tylosin is determined by testing samples ',--of the broth agairlst organisms known to be sensitive ,',',-, to tylosin. One useful assay"organism is ~ -coccus aureus ATCC 9144. Bioassay is conveniently '-performed by an automated turbidometric method. -,,-', Alternative assay methods include thin-layer chro- ,-',',~-matography and high-performance liquid chromatography with UV detection. '~
Example 6 __ Pre~aration of Labeled Tvlosin ,-' Mycarosyltylactone is prepared by the method '~
of Examples 1 and 2 except that a labeled acetate, 15 propionate, or butyrate is incorporated into the -fermentation medium. Labeled mycarosyltylactone thus ---., . -produced is used to prepare labeled tylactone by the method of Example 3. Using the procedure of Example --

5 the label~d tylactone i~ converted to tylosin which --is labeled on the macrolide ring.
Example 7 Alternate Preparation of Labeled Ty_~osin --' .
Tylactone, prepared by the method of Example 3, ,,' 25 is used to prepare tylosin according to the method of "''-Example 5 ~xcept that a labeled sugar moiety such as glucose is added to the ~econd fermenta~ion to provide '',,, tylosin which is labeled o~ the sugar moieties. "''' .......

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Claims (7)

We claim:

1. A process for preparing mycarosyltylactone, which has the formula and tylactone, which has the formula:

which comprises cultivating Streptomyces fradiae NRRL
12201 or a mycarosyltylactone-producing mutant or recombinant thereof in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of compound is produced.

2. The method of claim 1 which comprises cultiv-ating Streptomyces fradiae NRRL 12201.

3. The method of claim 1 or 2 which includes the additional step of isolating mycarosyltylactone.

4. The method of claim 1 or 2 which includes the additional step of treating the fermentation broth with mild acidic conditions to convert the mycarosyltylactone to tylactone.

5. The method of claim 1 or 2 which includes the additional step of isolating tylactone.

6. The microorganism Streptomyces fradiae NRRL
12201 or a mycarosyltylactone-producing mutant or recombinant thereof.

7. The microorganism of claim 6 which is Streptomyces fradiae NRRL 12201.