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3,?3l,382 Patented Apr. 24, 1962 1 2 3,031,332 FERMENTATION PROCESS U?iNG SPORE FORMENG FUNGI Stanley G. Knight, Madison, Wis., assignor to Wisconsin Alumni Research Foundation, Madison, Wis., a cor poratlon of Wisconsin No Drawing. Filed Sept. 15, 1958, Ser. No. ‘760,840 ing free fatty acids was converted to the desired highly ?avored composition much more rapidly by the old spore 7 Claims. (Cl. 195-101) containing vegetative inoculum, in which the cells were relatively inactive, than by the young substantially spore free inoculum, in which cells were known to be relatively active. With this unexpected discovery it was then de cided to try the following experiment. Milk fat was ?rst modi?ed with lipase to release free fatty acids. Coni dia or conidiospores of Penicillium roqueforti were then The present invention relates to the microbiological 10 added to the modi?ed fat (free fatty acid) mixture and production of chemical compounds by conidia or conidio the mixture subjected to fermentation under submerged spores (commonly called spores) produced during fer mentation of a vegetative inoculum in an aqueous nu aerobic conditions. In a matter of minutes it was noted that the spores (heretofore considered inert) were con trient medium, and more speci?cally to the improvement verting the free fatty acids to ketones. Instead of days, which consists in the production of the compounds with 15 the desired ?avor was obtained in this manner in 24 the spores substantially free from vegetative inoculum in hours. a substantially nutrient-free aqueous medium. The ‘Investigations based on the above have shown that the spores, unlike vegetative inoculum which is made up of young active vegetative inoculum provides enzymes for vegetative growth material, i.e. mycelium, are substan~ splitting the fat and that the free acids are then con tially free from vegetative growth material. Also, un 20 verted to ketones when spores are produced in the old like vegetative inoculum which requires a special fermen relatively inactive vegetative inoculum. In this action tation nutrient medium, the spores do not require a nu the spores introduce a ketone (C=O) group on the num trient medium. ber 2 carbon atom and then decarboxylate the fatty acid. Heretofore, microbiological production of chemical compounds was carried outwith vegetative inoculum in a 25 fermentation medium containing nutrients such as corn~ This can be illustrated as follows: spores ll steep liquor, soybean oil, soybean meal, molasses, sucrose, Romolnomooon ——> RCHz-C-CHs etc. to provide assimilable‘ sources of nitrogen and car bon. See Fried et al. Patent 2,753,290‘ and Thoma et a1. carbon atoms. Where R is butyl (C4H9) the reaction where R is an alkyl group, e.g. containing 1--18 or more Patent 2,793,462. The vegetative inoculum used was 30 can be illustrated as follows: also made up of strong, young mycelium cells known to be substantially spore-free but metabolically very active. Recovery of the modi?ed chemicals from a medium con taining vegetative growth material, nutrients, particularly the complex organic nutrients, by-products of the same, 35 etc. obviously posed problems. Vegetative inoculum' spores OHaOH2CH2CHzCH2GH2CHiG O OH ——> H CH3CHtOH2CHzOHz—C-CH3 (Methyl, amyl ketone) Free fatty acids can be used as such where available and organic nutrients, however, were used as they were considered’ essential in this art. ‘and where not available they can be made readily by the fermentation period using vegetative inoculum (mold not essential where the process is carried out with agita tion or agitation accompanying aeration, the use of ho mogenized fat such as in homogenized milk fat is pre ferred both for the conversion of the fat to free fatty acid by the enzymes and the conversion of the fatty acid to the ketone by the spores. The overall action of the enzymes and spores is materially speeded up when the fat and fatty acid are dispersed in minute particles to pro-\ vide large areas of surface. acid hydrolysis ‘of fats or by modi?cation of the corre During my investigations of the conversion of fats, e.g. sponding fats by use of fat splitting enzymes such as milk fat, to highly ?avored compositions with molds of 40 lipase. While homogenization of the fat or fatty acid is the Penicillium type, e.g. P. roqueforti, it was noted that cells‘ in mycelium form with nutrient growth medium) was relatively short, e.g. 2 days, compared to the fer mentation, e.g. 4 days, using mold conidia or could io-spores. See copending Knight application, Serial No. 672,557, ?led July 18, 1957, now abandoned. It was also noted that the fermentation period using e.g. 7 day old, spore containing vegetative inoculum or spores alone was longer than that required with young, eg 2 day old, sub stantially spore-free vegetative inoculum. In view of this it was assumed that the spores as such were rela tively inert and that vegetative inoculum ‘and preferably The spores used in the present invention can be readily obtained from vegetative inoculum which hasbeen al lowed to grow for 4-6 days in submerged culture with aeration (e.g. shaking) in an aqueous cornsteep liquor young inoculum should be used. Investigations also dis~ lactose or other nutrient medium in accordance with closed that the fermentation mechanism apparently pro 55 standard practices (except for culturing period) used to ceeded ?rst by the hydrolysis or splitting of the fats and prepare vegetative inoculum. The spores present in the then by the conversion of the resulting free fatty acids old vegetative inoculum are harvested by ?rst straining to the desired highly ?avored aromatic ketones. It was through cheesecloth to remove most of the mycelium and also noted that the development of the desired ?avor then by ?ltering through glass wool to remove the re could be hastened by the addition of small amounts of 60 maining mycelium. The spores in the ?ltrate are re lipase or like fat splitting enzyme. See Farnham Patent covered by centrifuging and are then washed with water 2,531,329 and Knight application, supra. Up to this to remove any retained nutrients. They may be stored in time, however, it appeared that the use of vegetative inoc ‘dry form or resuspended in distilled water and be kept ulurn with accompanying growth media (with or without at 4° C. The suspension can be standardized to contain, added‘ fat splitting enzymes) was necessary for short fer for example, about one billion spores/ml. The spores mentation periods, although it was appreciated that the can also be grown on surface culture, e.g. nutrient agar, use of spores was otherwise advantageous in that it was ‘and after scraping. off the spores and suspending in water, easier, using a spore inoculum, to avoid or control un the spores can be recovered in substantially pure form by wanted secondary reactions caused by bacteria, etc. 70 ?ltering, centrifuging, etc., as described above. In continued investigations it was noted, after lipase The spore containing old vegetative inoculum can also modi?ed the milk fat, that the resulting mixture contain be used as such and, While this introduces vegetative 3,031,332 3 4 manner as the processes in the references including the growth and some nutrients into the mixture, it represents patents noted below, for example, except that the fer a material improvement over the use of the nutrient mentation of the vegetative inoculum in the nutrient me dium, as used in the references and heretofore, is replaced fermentation mixtures. When the vegetative inoculum is used it can be recovered in fairly pure form free from by the spores of the speci?ed organism, substantially free from vegetative growth material, and the reaction is soluble nutrients by centrifuging and by washing the re covered solids with water. The use of spores, however, carried out in a substantially nutrient-free aqueous me substantially free from both vegetative growth and nutri dium. If necessary, the aqueous mixtures should be ents is preferred. buffered to provide a pH of about 6.5—7.5. The follow The following examples are illustrative. 10 ing microbial modi?cations of steroids are illustrative: Example I (1) Reduction of progesterone to A4-pregnene-20/3-o1~ 3-one with Streptomyces lavendulae. Fried, 1., et al., J. About 10 mg. of lipase is ?rst added to about 1000 ml. Am. Chem. Soc. 75:5764 (1953). of homogenized milk containing added cream to provide about 12% milk fat and the mixture shaken for about 4 hours at 37° C. The resulting fatty acid mixture is next 15 steamed for about 10 minutes to kill the lipase. After cooling to 25° C., 200 ml. of an aqueous dispersion of conidia or conidiospores of a white mutant of P. roque (2) Dehydrogenation of secondary alcohols: (a) A5-androstene-3?,17?-diol to testosterone with Pro ¢(zczin0myes erythropolis. Tur?tt, G. E., Biochem. J. 40:79 1946). (b) Estradiol to estrone with Streptomyces albus. Welsch, M., et al., Compt. rend. soc. biol, 142:1074 forti, prepared as described above, and containing about a billion spores/m1., substantially free from vegetative 20 (1948). (3) Hydroxylation in position 1: A4-androstene-3,17 inoculum and nutrients, are added to the fatty acid mix dione to M-androstene-lu-ol-3,l7-dione with Penicillium ture and the resulting mixture agitated with aeration at sp., Dodson, R. M., et al., J. Am. Chem. Soc. 79:3921 about 1 vol/min. at a temperature of about 25° C. for about 2.5 hours. The resulting highly ?avored composi tion containing aromatic ketones derived from the fatty 25 acids can be concentrated and used as such or be obtained (1957). (4) Hydroxylation in position 2: A4-pregnene-l7a,21 diol-3,20-dione to A4-pregnene-2l8,17a,2l-triol-3,20-dione with Streptomyces sp., Herzog, H. L., et al., J. Am. Chem. in dry form by spray drying in accordance with estab Soc. 7923922 (1957). lished practices in the drying art. The white mutant used (5) Hydroxylation in position 6: Progesterone to A4 in this example can be prepared as described in the refer 30 pregnene-6?-ol-3,20-dione with Streptomyces aureofaciens. ences cited in the Knight application, supra. Fried, 1., et al., Recent Progr. Hormone Res. 11:157 The 1 mg./ 100 ml. ratio of lipase to milk is merely (1955). See also Dulaney, E. L., et al., Mycologia illustrative as the amounts, depending on the percent of 47:464 (1955); Fried, J., et al., J. Am. Chem. Soc. fat, temperature, time, etc. can vary widely with optimum 74:3962 (1952); Meister, P. D., et al., J. Am. Chem. amounts for a particular composition being readily ascer tained by preliminary test. The same applies to the 35 Soc. 75:416 (1953) and Eppstein, S. H., et al., I. Am. Chem. Soc. 751408 (1953) involving the microorganisms amount of spores used as the optimum amount (which Aspergillus ochraceus, Aspergillus niger, and Rhizopus can also be ascertained by preliminary test) can vary arrhizus. under different reaction conditions, different kind of (6) Hydroxylation in position 7: Progesterone to A4 spores, etc. Example II 40 pregnene-7a-ol-3,20-dione with Phycomyces blakeslecanus. Fried et al., US. Patent 2,753,290. See also Meystre, C., et al., Helv. Chim. Acta 38:381 (1955) using a spe To a 1% aqueous mixture of octanoic acid bulfered with phosphate buffer to pH 6.5 is added conidia or cies of Peziza for a similar type reaction on deoxycorti costerone. conidio-spores of P. roqueforti, substantially free from vegetative inoculum and nutrients, in a ratio of about 45 (7) Hydroxylation in positions 10, 11 or 12: 100,000,000 spores/3 ml. of the aqueous octanoic acid mixture. The mixture is then subjected to agitation with (a) l9-nor-progesterone to l0e-hydroxyl-l9'nonproges terone with Rhizopus nigrlcans. Pederson, R. L., et al., aeration for about 2 hours at about 25° C. The result J. Am. Chem. Soc. 7821512 (1956). ing methyl, amyl ketone can be recovered by distillation or like means known in the art. Example III This example follows Example II using hexanoic acid, nonanoic acid and decanoic acid, respectively, for the preparation of methyl, propyl ketone; methyl, hexyl ke tone, and methyl, heptyl ketone. By selecting the ap (b) Progesterone to A‘Lpregnene-lla-ol-3,20-dione with 50 Rhizopus arrhizus. Peterson, D. H., et al., I . Am. Chem. Soc. 74:1871 (1952). See also Peterson, D. H., et al., J. Am. Chem‘. Soc. 752412 (1953). (c) Reichstein’s compound S (A4-pregnene 17a,21-diol 3,20-dione) to hydrocortisone (A4-pregnene-ll?,l7a,2l 55 triol-3,20-dione with Cunninghamella blakesleeana. Han son, F. R., et al., I. Am. Chem. Soc. 75:5369 (1953). See also Shull, G. M., et al., I. Am. Chem. Soc. 772763 other ketones can be prepared in a similar manner with (1955), and Thoma et al., US. Patent 2,793,162 involv out vegetative inoculum or nutrients. ing similar reactions ‘with Curvularia lunata, Trichotlze 60 cium roseum, and Coniothyrium hellebori. Example IV (d) Progesterone to A4-pregnene-12/3-ol-3,20-dione and This example is in accordance with the above examples the related 12?,15B-diol with Calonectria decora. Schu propriate fatty acid containing at least 4 carbon atoms but employs 4-—5 day old spore containing vegetative bert, A., et al., Ber. 90, 2576 (1957). (8). Hydroxylation in other positions are described in soluble nutrients, dried and then ground to a powder and 65 Meister, P. D., et al., Absts. 123rd meeting Am. Chem. resuspended in water. The ketones can be recovered by Soc. (1953); Fried et al., US. Patent 2,753,290; Cam distillation, extraction with water immiscible solvents, de erino, B., et al., Gazz. Chim. ital., 86:1226 (1956); cantation where the ketone is insoluble in water, etc. Meystre, C., et al., Helv. Chim. Acta 38:381 (1955); Separation of the ketones is not required when making a Perlman, D., et al., J. Am. Chem. Soc. 74:2126 (1952); ?avoring composition as in Example I. 70 Thoma, R. W., et al., J. Am. Chem. Soc. 79:4818 (1957); Dulaney, E. L., et al., Appl. Microbiol. 3:372 (1955); In addition to converting fatty acids to ketones the im Meystre, C., et al., Helv. Chim Acta 37:1548 (1954); proved process of the present invention employing conidia McAleer, W. J., et al., Arch. Biochem. Biophys. 62:109 or conidiospores can be used to advantage for the modi ?cation of other compounds including steroids or steroid (1956). inoculum which has been centrifuged to remove water intermediates. The process is carried out in the same 75 (9) Dehydrogenation: Hydrocortisone to A'-4-pregna :H5M. 5 3,031,382 6 diene-l1,3,17a,21-triol-3,20-dione with Streptomyces la tative growth material-free and nutrient-free aqueous me dium in which the spores cannot germinate. 2. The process of claim 1 where the compounds are vendulae. Fried et al., US. Patent 2,793,164. See also Mobile, A., et al., J. Am. Chem. Soc. 77:4184 (1955); Vischer, E., et al., Helv. Chim'. Acta 38:835 (1955). (10) Side chain degradation: Progesterone to A'-4~ fatty acids. 3. In the microbiological production of a chemical compound by fermentation of a fungi of the genus Strep androstadiene-3,20-dione with Streptomyces lavendulae. Peterson, G. E., et al., J. Bact. 742684 (1957). See also Vischer, E., et al., Experienta 9:371 (1953); Tur?tt, G. E., Biochem. I. 422376 (1948). See also US. Patents 2,602,769; 2,649,400; 2,649,401; 2,649,402; 2,695,260; 2,735,800; 2,753,290; 2,762,747; 2,768,928; 2,789,940; 2,802,775; 2,809,919; 2,812,285; tomyces, the improvement which consists in ?rst pro ducing spores and vegetative growth material by cultur ing the fungi in a nutrient medium, separating the spores 10 from the vegetative growth and nutrient materials, and and 2,830,937 which involve microorganisms forming spores which can be used in a nutrient-free medium in place of the vegetative inoculum in the described proc esses. Patents 2,602,769 and 2,735,800 listed above, use fungus of the order Mucorales. As noted above, the processes are carried out in ac cordance with the processes described in the references carrying out the production of the compound with the spores in a substantially vegetative growth material-free and nutrient-free aqueous medium in which the spores cannot germinate. 4. In the microbiological production of a chemical compound by fermentation of a fungi of the genus Peni cillium, the improvement which consists in ?rst producing spores and vegetative growth material by culturing the fungi in a nutrient medium, separating the spores from except (1) that the vegetative inoculum employed in the 20 the vegetative ‘growth and nutrient materials, and carry reference process is replaced by spores of the organism ing out the production of the compound with the spores substantially free from vegetative growth material and in a substantially vegetative growth material-free and nu (2) that the aqueous nutrient fermentation medium trient-free aqueous medium in ‘which the spores cannot employed in the reference process is replaced by a substan germinate. tially nutrient-free aqueous medium. The time required to 25 5. In the microbiological production of a chemical complete the conversion with spores (and which can be compound by fermentation of a fungi of the genus readily ascertained by preliminary tests in each case) Aspergillus, the improvement‘ which consists in ?rst is materially shortened and the yield materially increased producing spores and vegetative growth material by cul over that using a young vegetative inoculum. Aside from the fungi in a nutrient medium, separating the the time element, however, the recovery of the modi?ed 30 turing spores from the vegetative growth and nutrient materials, chemical in ‘good yield (which, here again, can be car and carrying out the production of the compound with ried out in accordance with the reference process) is the spores in a substantially vegetative growth material much easier from the relatively clean reaction mixture free and nutrient-free aqueous medium in which the of the present invention than the prior art fermentation cannot germinate. mixtures containing vegetative growth, organic nutrients, 35 spores 6. In the microbiological production of a chemical by-products of the same, etc. Puri?cation of the modi compound by fermentation of a fungi of the genus ?ed compound after isolation is also much easier in the Rhizopus, the improvement which consists in ?rst pro process of the present invention than is possible from the ducing spores and vegetative growth material by cultur highly contaminated culture ?ltrates resulting from the fermentation of vegetative inoculum in a nutrient me 40 ing the fungi in a nutrient medium, separating the spores from“ the vegetative growth and nutrient materials, and dium‘. carrying out the production of the compound with the As noted above lipase can be used to release free spores in a substantially vegetative growth material-free fatty acids from milk ‘fat. In the illustrative examples and nutrient-free aqueous medium in which the spores cannot germinate. acids are converted to ketones. Whereas in these ex 45 7. In the microbiological production of a chemical amples the reactions are conducted separately it should compound by fermentation of a fungi of the order Mu be understood that the enzymes and spores can be used spores are added to the resulting mixture and free fatty simultaneously. corales, the improvement which consists in ?rst produc ing spores and vegetative growth material by culturing I claim: 1. In the microbiological production of a chemical com 50 the fungi in a nutrient medium, separating the spores from the vegetative growth and nutrient materials, and pound by fermentation of a spore forming fungi in an carrying out the production of the compound with the aqueous nutrient medium containing assimilable sources spores in a substantially vegetative growth material-free of nitrogen and carbon in which the spores can germi and nutrient-free aqueous medium in which the spores nate and grow and produce vegetative growth material, the improvement which consists in ?rst producing the 55 cannot germinate. spores by fermenting the spore forming fungi in said nutrient medium, separating the spores from the result ing fermentation medium containing vegetative growth and nutrient materials and carrying out the production of the compound with the spores in a substantially vege 60 References Cited in the ?le of this patent Chem. Peterson Soc‘., et74,al.:5933-5936 DMS, March (1952). 20, 1961, Iourn. Dulaney et Appl. MicrobioL, 32336-340 (1955).