Folia Microbiol. 46 (I), 17-20 (2001)

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Steroid Biotransformation by Different Strains of Micrococcus sp. N. DOGRAa*, G.N. QAZ1b alnstitute of Microbial Technology, Chandigarh- 160 014. India bRegtonal Research Laboratory, Council of Scient~c and Industrial Research. Jammu - 180 001, lndia fax +91 172 690 632, +91 172 690 585 e-mail [email protected] Received 2 October 2000

ABSTRACT. A strain of Micrococcus sp. was isolated for its capability of side chain degradation of cholesterol. This strain was characterizedand identified as Micrococcus roseus. It was

found to be the best strain for the production of androsta-l,4-diene-3,17-dione and androst-4-ene-3,17-dione compared with other Micrococcus strains.

Biotransformations of sterols and/or steroids are widely used for the production of pharmacologically active steroids. One such economically important biotransformation has been the side chain degradation of cholesterol. This yields androsta-l,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AED), important starting materials for the synthesis of anabolic drugs and contraceptives. The objective of the study was to isolate a variety of bacteria for the degradation of cholesterol to study steroid biotransformation and increase the production of ADD and AED. This can help in probing the process of cholesterol degradation under different biological conditions. After screening a variety of microorganisms a promising strain of Micrococcus was isolated in Regional Research Laboratory (Jammu, India) for its capability to utilize cholesterol as the sole carbon source (Vadalkar et al. 1980). It carries a 10 kb plasmid designated pMQV10 (Verma et al. 1988). In a previous study, curing of this strain and transformation experiments revealed that the plasmid codes for cholesterol degradation and streptomycin resistance (Dogra and Qazi 1999). Isolate RJ6 was identified as Micrococcus roseus. It possesses a higher activity of cholesterol side-chain degradation to yield ADD and AED compared with other Micrococcus strains, viz. M sodonensis, M luteus and M. flavus. Characterization of this strain as well as a genetic study may provide information that would provide information on this industrially important strain in detail (cf. Zohri 2000). There is scant published literature available on the biodegradation of sterols (Zohri and Abdel-Galil 1999; Zohri 2000, and references cited therein). This is the first report identifying M. roseus as an important steroid biotransforming organism which opens the way for the application of this strain for industrial purposes.

MATERIALS AND METHODS Microorganisms. Bacterial strains were isolated at the Regional Research Laboratory (Jammu, India) from rat faeces by an enrichment technique using cholesterol as the sole source of carbon. They were identified by morphological and other microbiological and biochemical characteristics. As control strains were used Micrococcus sodonensis strain NCIM2379, Micrococcus flavus strain NCIM2376 and Micrococcus luteus strain NCIM2169. Medium and growth conditions. Medium 2 • YT (Miller 1972) was used for the maintenance of Micrococcus isolate, M. sodonensis, M. flavus and M. luteus were maintained on LB agar medium (Berger and Kimmel 1987). Cholesterol was added into the medium in micronized form by sonicating in 0 . 1 % Tween 80 in a 20-kHz ultrasonicator for 10 min. All experiments were carried out at 30 ~ Liquid cultures were grown in 25 mL of LB broth in 100-mL Erlenmeyer flasks each by shaking at 2.9 Hz on an orbital incubator at 30 ~ Growth was measured spectrophotometrically (A550) after 2 d. Biotransformation o f steroids. The strains M sodonensis, M. luteus and M. flavus and the isolate RJ6 were cultured separately in 50 mL of biotransformation medium in 500-mL flask each. Degradation of cholesterol was determined as described previously (Dogra and Qazi 1999). Briefly: After growth, liquid cul*Presentaddress: Depagmentof Pathologyand Microbiology,Facultyof VeterinaryMedicine, St. Hyacinthe,QuebecJZS 7Z6, Canada.

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tures were extracted with ethyl acetate and residues subjected to TLC. Spots were evaluated densitometrically (Gel Doc, Pharmacia-Biotech) (see Fig. 1). PlasmidDNA was isolated according to Verma et al. (1988). Morphological characteristics. Identification of the organism was made up to species level. Preliminary identification was based on Bergey's Manual o f Determinative Bacteriology (1974). Cell form and growth on LB agar were observed after 2 d of incubation. The method described by Conn et al. (1960) was employed for Gram staining. Motility was investigated by the hanging drop method. Biochemical characteristics of the organism were analyzed according to Harrigan and McCance (1966) and Cowan and Steel (1974). All the tests were performed by taking single colonies from the culture plate. Fatty acid composition of the R J6 strain was estimated according to the Microbial Identification System Operating Manual HP5898A, obtained from Hewlett-Packard (USA). Isolation ofgenomic DNA was done according to Anonymous (1990). The DNA base composition (GC content) was calculated from its thermal denaturation temperature (Tin) measured according to Mandel and Marmur (1968).

RESULTS AND DISCUSSION ADD and AED are two important starting materials for the manufacture of steroid drugs by side chain degradation of cholesterol and/or phytosterol. ADD is used to obtain estrogens and other antifertility drugs and AED is used in the synthesis of androgens and the diuretic drug spironolactone. A great effort has been put into devicing methods to increase the yields of ADD and AED. 9c~-Hydroxylase is an enzyme which breaks the steroid ring and makes the steroid available for complete oxidation. Several strategies have been employed to suppress 9c~-hydroxylase (Martin 1984; Nagasawa et al. 1970). Fermentation and microbiological techniques have already been developed in Regional Research Laboratory (Jammu) to obtain ADD and AED from cholesterol and sitosterol, respectively. One hundred samples of soil and animal feces were processed by Vadalkar et al. (1980). Cholesterol was converted to ADD by five bacterial isolates, of which R J6 was found to yield a higher amount of ADD. This bacterial strain has been isolated from rat faeces. It has an excellent cultural stability and good bioconversion yield (Vadalkar et al. 1980). It harbors the pMQV10 plasmid of 10 kb size (Verma et al. 1988). In a previous paper we reported that strain RJ6 is a steroid biotransforming strain. Curing and transformation studies indicate that the pMQV10 plasmid carries genes for cholesterol degradation and streptomycin resistance (Dogra and Qazi 1999). Bioconversion of sterols to ADD by different strains ofMicrococcus sp. Efficiency of the Micrococcus CHO ) isolate RJ6 for biotransformations and yields of ADD ~nd AED was compared with three other Micrococcus strains, viz. M sodonensis, M flavus and M luteus. All the four selected strains were able to convert cholesterol to ADD ) ADD and other degradation products. Bioconversion of cholesterol is 100 % in all the strains (Fig. 1). The yield of ADD after 3 d was about 63 % in the case of isolate RJ6 which was higher than the rest of the strains which is 60 % in M. sodonensis; 57 % in M. flavus and 50 % 1 2 3 4 5 in M. luteus by considering standard ADD as 100 % (Table I). Screening o f Micrococcus sp. for the presence ofplasmid DNA. Several methods were tried to isolate Fig. 1. HPTLCof the products of cholesterol bioplasmid DNA from M. sodonensis, M. flavus, M. luteus conversion by strains ofMicrococcus spp.; 1 - stanand Micrococcus isolate R J6 but these attempts were not dards ADD and cholesterol (CHO), 2 - M. roseus, successful. Plasmid DNA was detected in the RJ6 isolate 3 - M. sodonensis, 4 - k/Lflavus, 5 - A~ luteus. only by following the method of Verma et al. (1988).

STEROID BIOTRANSFORMATION IN Micrococcus sp.

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19

Curing and transformation studies indicate that cholesterol degradation and antibiotic resistance characters in the isolate RJ6 are plasmid encoded (Dogra and Qazi 1999) while absence of plasmid DNA in M sodonensis, M f l a v u s and M. luteus indicated that these characters are encoded in genome. Taxonomic properties o f steroid-biotransforming organism. T h e Table I. Biotransformation of cholesterola by strains of Micrococcus spp. after 3-d fermentation Strains Control c RJ6

M,flavus M. luteus sodonensis

ADD,% b 100 63 57 50 60

aAdded cholesterol was completely metabolized in all cultures. ' bRelative to cholesterol. CStandard ADD added.

morphological and physiological properties of bacterium RJ6 are summarized in Table II. Pigmentation and colony morphology were the tests for its presumptive identification. Cells were spherical in tetrad form, non-motile and non-sporulating. Colonies were Gram-positive, red, smooth, slightly convex with regular margins. The optimal growth temperature was 30 ~ but it could grow well at 37 ~ Optimal pH was 7.0. Strain R J6 showed growth on nutrient agar with 7.5 % NaCt. The strain was lysozyme-resistant and the GC content of its DNA was 72.2 % (mol/mol). The predominant fatty acids of the organism were stearic acid (42.8 %) and palmitic acid (35.3 %) (Table III). Percentage contents of fatty acids of isolate RJ6 show similarity to the fatty acids of M roseus (Brooks et al. 1980). From properties summarized in Table It, bacterium RJ6 seemed to fall into genus Micrococcus with reference to 8th edition of Bergey's Manual. The bacterium was found to possess the same physiological and biochemical characteristics as M roseus.

Table 11. Microbiological and biochemical tests for Micrococcus strain R J6

Test

Reagent

Result

Strain R J6

Hydrolysis of gelatin

mercuric chloride solution

zone formation

Hydrolysis of casein

mercuric chloride solution

zone formation

Production of indole from tryptophan

few drops of sulfuric acid

appearance of rose color

-

Production o f ammonia from peptone and arginine

Nessler's reagent

appearance o f brown color

-

Production of hydrogen sulfide

lead acetate paper

blackening of lead acetate paper

+

Production of ammonia from urea

phenol red

change in color from yellow to red

-

Reduction of nitrate

Griessilosvay's reagents

red color

+

Hydrolysis of starch

Gram's iodine solution

zone formation

+

Oxidation o f other carbohydrates

bromothymol blue

blue to yellow color

+

Acid production from glucose

neutral red

red color

+

Production of 3-hydroxy-2-butanone from glucose

5 or 10 % potassium hydroxide

pink color

Utilization of citrate as the sole source of carbon

bromothymol blue

green to blue color

-

Production ofpolypeptides from sucrose

-

growth of mucoid character

-

Hydrolysis of Tween compounds

Tween 80 nutrient agar

zone formation

Catalase

10 x H202

gas bubbles

Urease

Christensen's urea medium

red color

Aesculin hydrolysis

aesculin agar

blackening in and around the colony

Oxidase

1% N,N,N',N'-tetramethyl-

dark purple color

+ -

- 1,4-benzenediamine dihydrochloride

M. roseus has received considerable attention for pigmentation of bacterioruberins which bind to synthetic membranes of phosphatidylcholine with almost equal affinity (Chattopadhyay et al. 1997). It is also reported to have a characteristic feature of biodegradation of synthetic lubricant (Wright et al. 1993) but the steroid-biotransforming character of M roseus has not been reported so far. This study not only adds to the growing list of organisms involved in steroid biotransformation but helps us to consider M. roseus as a good strain for degradation of cholesterol to form ADD and AED when compared with other Micrococcus strains. With this study M. roseus is becoming one of the best characterized Gram-positive bacteria. Additional bio-

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chemical and genetic analysis of this strain will result in a better understanding of degradation of cholesterol in the microbial world. Table !1I. Fatty-acid profile of bacterium RJ6 Systematic name

Common name

Tetradecanoic acid cis-9-Hexadecenoic acid Hexadecanoic acid Octadecanoic acid Dodecanoic acid

myristic acid palmitoleic acid palmitic acid stearic acid eicosanoic acid

Short-hand formula C 14:0 C 16:149 C 16:0 C 18:0 C20:0

Rt, min

Percentage

7.27 9.16 11.25 14.39 18.29

15.6 12.7 35.3 42.8 1.2

This work was supported by grant from the Council of Scientifc and Industrial Research, India. REFERENCES

Anonymous: Selective Isolation and Identification of Actinomycetes. Laboratory Manual - Protocol of Experiments Carried Out by the Participants of the Workshop Organised by 1MTECH. IMT, Chandigarh (India) 1990. BERGER S.L., KIMMELA.R.: Guide to molecular cloning techniques. Meth.Enzymol. 152, 165-166 (1987). Bergey's Manual of Determinative Bacteriology, 8th ed. Williams & Wilkins, Baltimore 1974. BROOKS B.W., MURRAYR.GE, JOHNSON J.L., STACKEBRANDTE., WOESE CR., FOX G E : Red pigmented micrococci: a basis for taxonomy, lnternat.J.Syst. Bacteriol. 30, 627-646 (1980). CHATTOPADHYAY M.K., JAGANNADHAMM.V., VA[RAMAN]M., SHIVAJ[ S.B.: Carotenoid pigments of an Antarctic psychrotrophic bacterium Micrococcus roseus, temperature dependent biosynthesis, structure, and interaction with synthetic membranes. Biochem.Biophys.Res. Commun. 239, 85-90 (1997). CONN H.J., DARROW M.A., EMMEL V.M.: Staining Procedures Used by the Biological Stain Commission. University of Rochester Medical Center, Rochester, New York, 2rid ed., pp. 226-227. William and Wilkins, Baltimore 1960. Cowan & Steel's Manual for the Identification of Medical Bacteria, 2rid cal. Cambridge University Press, Cambridge 1974. DooRA N., QAZ1 G.N.: Biotransformation of cholesterol by Micrococcus species mediated by plasmid DNA. World J.MierobioL Biotech. 15, 41 I--415 (1999). HARRIGAN W.F., MCCANCE M.E.: Laboratory Methods in Food and Dairy Microbiology. Academic Press, London-New York-San Francisco 1966. MANDEL M., MARMUR J.: Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. MethEnzymol. 12B, 195-206 (1968). MARTIN CK.A.: Sterols, pp. 79-95 in H.J. Rehm, G. Reed (Eds): Biotechnology, Vol. 6A. K. Kieslich-Verlag-Chemie, Weinheim (Germany)-Deexfield Beach (Florida, USA) 1984. Microbial Identification System Operating Manual HP5898A. Hewlett-Packard, Newark (Delaware, USA) 1992. MILLER J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1972. NAQASAWAM., WATANABEN., HASHIBAH., MURAKAMIM., BAE M., TAMURAG., ARIMAK.: Microbial transformation of sterols. V. Inhibitors of microbial degradation of cholesterol. 34, 838-844 (1970). VADALKARK., QUPTA F.C., BHAT H.K., CHOPRA C.L.: Bioconversion of pregnenolone to 1,4-androstadiene-3,17-dione by Arthrobacter simplex. IndJ.Exp.Biol. 18, 245-248 (1980). VERMA V., QAZI G.N., PRASHADR., CHOPRA C.L.: An economical large scale procedure to purify Micrococeus plasmid DNA. Biotechniques 6, 936-940 (1988). WRIGHT M.A., TAYLORF., RANDLESS.J., BROWNDE., HIGGINSl.J.: Biodegradation of a synthetic lubricant by Micrococcus roseus. Appl. Environ. Microbiol. 59, 1072-1076 (1993). ZOHRI A.A.: Progesterone transformation as a biochemical aid in classification of the genus Emericella. Folia Microbiol. 45, 391-396 (2000). ZOHRI A.A., ABDEL-GALILMS.M.: Progesterone transformations by three species ofHumicola. Folia MicrobioL 44, 277-282 (1999).