Vol. 49, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1985, p. 338-3440099-2240/85/020338-07$02.00/0Copyright © 1985, American Society for Microbiology

Optimum Conditions for Ursodeoxycholic Acid Production fromLithocholic Acid by Fusarium equiseti M41

SONGSRI KULPRECHA,t TARO UEDA, TAKUYA NIHIRA,* TOSHIOMI YOSHIDA, AND HISAHARU TAGUCHIInternational Center of Cooperative Research and Development in Microbial Engineering, Japan, Faculty of Engineering,

Osaka University, Osaka 565, Japan

Received 6 June 1984/Accepted 23 November 1984

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions forursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41showed maximum conversion at 28°C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation.Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In thepresence of 0.5 M KCI, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) wereenhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7fi-hydroxylation oflithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled bydissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peakappeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygentension, respectively. After reaching the maximum, the 70i-hydroxylation activity in the mycelium declinedrapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations,such as pH shift (from pH 7 to 8), addition of 0.5 M KCI, and dissolved oxygen control, were applied to theproduction of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2g/liter) was produced within 96 h of cultivation.

For several decades, a great deal of attention has beengiven to the study of microbiological transformation ofsteroidal compounds, particularly of the corticosteroid type(4, 26). During the same period, however, the structurallyrelated bile acids have received much less attention. Morerecently, interest in the bile acids has increased considera-bly, principally due to the finding that a naturally occurringbile acid (chenodeoxycholic acid) possesses the therapeuticproperty of solubilizing gallstones (1, 6).

Lately ursodeoxycholic acid (UDCA; 3ot,7p-dihydroxy-5r-cholanic acid) (7p-epimer of chenodeoxycholic acid) hasbeen reported to have similar therapeutic properties (22),and with respect to side effects and dose response, UDCAhas attained an evaluation superior to that of chenodeoxy-cholic acid (7, 27). Because at present gallstone disease isone of the leading causes of hospitalization (15) and ca. 10%of human adults possess gallstones (19), the personal andeconomic impact of curing or preventing gallbladder diseasewould be immense. However, the amount of naturallyavailable UDCA is very low, and a seven-step chemicalsynthesis of UDCA has not solved the supply problem dueto its low yield (9 to 14%) (4, 20).

In a previous paper (25) we reported the formation ofUDCA, using one-step transformation from lithocholic acid(LCA; 3ot-hydroxy-5,B-cholanic acid), by Fusarium equisetiM41. In this paper we report the optimization of biotransfor-mation of LCA into UDCA.

MATERIALS AND METHODSChemicals. LCA, chenodeoxycholic acid (3a,7a-dihy-

droxy-53-cholanic acid), and cholic acid (3a,7a,12a-trihy-droxy-53-cholanic acid) were purchased from Sigma

* Corresponding author.t Present address: Department of Microbiology, Faculty of Sci-

ence, Chulalongkorn University, Bangkok 10500, Thailand.

Chemical Co., St. Louis, Mo. Hexafluoroisopropanol wasobtained from E. Merck AG, Darmstadt, West Germany.Trifluoroacetic anhydride was purchased from GasukuroKogyo Co. Ltd., Tokyo, Japan. Sabouraud dextrose broth(0382-15-1) was the product of Difco Laboratories, Detroit,Mich. Yeast extract was obtained from Daigo NutritiveChemicals, Ltd., Osaka, Japan. [Carboxyl-14C]LCA waspurchased from Amersham International, Amersham, U.K.(specific radioactivity, 55 mCi/mmol). All other reagentswere the highest grade available from Nakarai Chemicals,Ltd., Kyoto, or Wako Pure Chemical Industries, Ltd.,Osaka, Japan.Medium. Sabouraud dextrose broth was used to prepare

the stock and seed culture of F. equiseti M41 throughout thiswork. The basal supplement contained (in 1 liter) 0.5 g ofMgSO4 * 7H2O, 0.5 g of CaCl2 * 2H20, 1 g of yeast extract,and 0.01 g each of FeSO4 * 7H20, MnSO4 * H20, CuS04*5H20, Na2MoO4 * 2H20, and ZnSO4 - 7H20. Medium A,used for the survey of carbon and nitrogen sources, con-tained 10 g of carbon source, 5 g of nitrogen source, 3 g ofKCl, 2 g of KH2PO4, 3 g of CaCO3, 1 g of LCA, and basalsupplement in 1 liter of deionized water. Medium B con-tained 40 g of carbon source, 12 g of nitrogen source, 1 g ofLCA, and basal supplement in 1 liter of 0.1 M potassiumphosphate buffer (pH 7.0). Medium C, used for jar fermen-tation, was the same as medium B, for which dextrin andL-asparagine were chosen as the carbon and nitrogen sources,respectively.

Cultivation. For the inoculum preparation, 100 ml ofSabouraud dextrose broth in a 500-ml Sakaguchi flask wasinoculated with 5 ml of the stock culture (stored at -80°C)and incubated at 28°C on a reciprocating shaker (120 rpm)for 72 h. The main cultivation was performed with each100-ml portion of either medium A or medium B in 500-mlSakaguchi flasks, inoculated with 5 ml of a 72-h inoculum,and incubated at 28°C on a reciprocating shaker (120 rpm)for 7 or 14 days.

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MICROBIAL PRODUCTION OF UDCA 339

TABLE 1. Effect of carbon source on formation of UDCA byF. equiseti M41"

UDCA productionCarbon source (10 g/liter) (mg of UDCA per g

of dry cells)

D-Xylose ................................. 2.83L-Arabinose ............................... 3.14D-Glucose ................................ 2.60D-Fructose ............................... 2.87D-Galactose ............................... 2.98D-Mannitol ............................... 3.69Salicin ................................... 0Maltose .................................. 4.73Lactose .................................. 7.43Raffinose ................................. 7.05Sucrose .................................. 5.03Starch (soluble) ............... ............ 7.68Carboxymethyl cellulose ........ ........... 10.54Dextrin .................................. 18.46Malt extract .............................. 5.88Sodium acetate ............... ............ 7.60Glycerol.................................. 7.60Sodium succinate ............ ............. 2.07

Cultivation was performed with medium A containing the indicatedcarbon or nitrogen source in a 500-ml Sakaguchi flask on a reciprocatingshaker (120 rpm) for 7 days at 28°C. NaNO3 (5 g/liter) was fixed as the nitrogensource. UDCA production is expressed as UDCA formed from added LCA(100 mg per flask). Cell mass ranged from 0.4 to 0.8 g of dry cells per flask.

For cultivation with a jar fermentor, 1.5 liters of eithermedium C or medium D in a 2.5-liter jar fermentor (typeMD-250-3S; L. E. Marubishi, Tokyo) was inoculated with100 ml of a 72-h inoculum. The temperature was kept at28°C, the aeration rate was kept at 1 vol/vol/min (vvm), andthe pH was controlled at 7.0 unless otherwise indicated.Dissolved oxygen tension (DOT) was controlled at thedesired value ±5% deviation by automatically changing theagitation speed. DOT and pH during cultivation were mon-itored with a membrane-type dissolved oxygen electrode(type DX-26; L. E. Marubishi) and a pH electrode (typeD-26; L. E. Marubishi), respectively.

Assay. Bile acid content was determined gas chromato-graphically after derivatization with hexafluoroisopropanoland trifluoroacetic anhydride (17) as described before (25),using cholic acid as an internal standard. The relativeretension times of UDCA, LCA, and a by-product againstcholic acid were 0.67, 0.29, and 0.89, respectively.

Cell mass during cultivation was expressed as dry weight(grams per liter). Total sugar content was determined asglucose by the phenol-H2SO4 method (8).UDCA-producing activity with resting cells. Wet mycelium

(1 g), harvested by suction filtration with a Buchner funneland washed thoroughly with deionized water, was sus-pended in 20 ml of 0.1 M Tris-hydrochloride (pH 8.0) in a100-ml Erlenmeyer flask. The conversion reaction, initiatedby adding 20 mg of LCA, was performed at 28°C at a shakingspeed of 180 rpm (M-100-N; Taiyo Scientific Industries Co.,Ltd., Tokyo, Japan). At appropriate intervals, a sample (1ml) was withdrawn and submitted to bile acid analysis.Uptake of [carboxy-'4C]LCA. The mycelia used for the

measurements were cultivated in a 2.5-liter jar fermentorwith 1.5 liters of medium C, under the conditions of pH 7.0,28°C, aeration rate of 1 vvm, and 0% DOT. (The cultivationconditions, under which oxygen consumption by the funguswas equal to the oxygen supply from air at an aeration rateof 1 vvm with a varied agitation rate, are represented by theterm "DOT 0%." The agitation rate at DOT 0% was the

maximum agitation rate at which the indicator of the DOTmeter [type DX-26; L. E. Marubishil remained at 0%.) After40 h of cultivation, the mycelium was collected by centrifu-gation (15,000 rpm, 4°C) and washed three times withice-cold 0.01 M Tris-hydrochloride (pH 7.0) by centrifuga-tion (15,000 rpm, 10 min, 4°C). Finally, the mycelium wascollected by suction filtration with a Buchner funnel. Onegram of wet mycelium thus obtained was suspended in 20 mlof ice-cold 0.1 M Tris-hydrochloride (pH 8.0) with or with-out 0.5 M KCI. After preincubation for 5 min at 28°C, theincorporation was started by adding 20 ,ul of [14C]LCAdissolved in ethanol. At the indicated time, a 1-ml samplewas withdrawn, mixed with 50 ml of ice-cold 0.01 MTris-hydrochloride (pH 7.0), filtered through a 0.45-pummembrane filter (type TM-2; Toyo Kagaku Sangyo Co.,Ltd., Osaka, Japan), and washed five times with 10 ml eachof ice-cold 0.01 M Tris-hydrochloride (pH 7.0). After dryingthe membrane filter at 60°C for 20 min, the radioactivity wasmeasured in the presence of 10 ml of toluene scintillantcontaining 2,5-diphenyloxazole (9 g/liter) with a liquid scin-tillator (Beckman LS 7500; Beckman Instruments, Inc.,Irvine, Calif.).

RESULTS

Medium composition for the higher production of UDCA byF. equiseti M41. In a previous paper (25) describing F.equiseti M41 as a transformer of LCA into UDCA, we usedmodified Czapek-Dox medium for screening and oatmealmedium for UDCA production. However, in oatmeal me-dium the production yield was rather low (-35%). Further-more, due to the viscosity of oatmeal medium, culturalconditions, such as DOT, could not be determined satisfac-torily. Therefore, to find suitable medium compositions withwhich higher UDCA production and low viscosity would beobtained, we at first investigated the effects of carbon andnitrogen sources on UDCA production (Tables 1 and 2). Weobserved a ninefold difference in UDCA production pergram of dry cells among 18 carbon sources, i.e., from 18 mgof UDCA per g of dry cells with dextrin to 2 mg of UDCAper g of dry cells or no activity with sodium succinate andsalicin, respectively. Also, a greater than ninefold differencewas detected among eight nitrogen sources, e.g., 27, 21, and3 mg of UDCA per g of dry cells with NH4Cl, L-asparagine,and tryptone, respectively. Because the pH values of vari-ous media after cultivation were between 8.4 and 9.1 exceptwith lactose (pH 7.8), sodium succinate (pH 9.3), and meatextract (pH 6.8, Table 2), the nature of the carbon or

TABLE 2. Effect of nitrogen source on formation of UDCA byF. equiseti M41'

UDCA productionNitrogen source (5 g/liter) (mg of UDCA per g

of dry cells)

NH4C1 ................................... 27.68(NH4)2SO4 ................................ 20.17L-Asparagine ............................. 21.25Casamino Acids ........................... 4.59Peptone .................................. 4.67Tryptone ................................. 3.07Yeast extract ............................. 6.77Meat extract .............................. 0

a Same as footnote a, Table 1, except sodium succinate (10 g/liter) was fixedas the carbon source.

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340 KULPRECHA ET AL.

nitrogen source rather than the pH seemed to be the primaryreason for differing UDCA production.

Next, to determine the best combination of carbon andnitrogen sources for UDCA production, we investigated thecombined effect of some carbon and nitrogen sources (Table3). In this experiment we used 0.1 M potassium phosphatebuffer (pH 7.0) instead of 0.3% (wt/wt) CaCO3 to maintainthe pH during cultivation with Sakaguchi flasks. In thepresence of a higher concentration of potassium phosphate,UDCA production became higher and constant. This stimu-lating effect of potassium phosphate is described later. Withthe medium composed of dextrin and L-asparagine, a veryhigh conversion (39 mg of UDCA per g of dry cells) wasobserved after 14 days of cultivation (Table 3), whereas verypoor production was exhibited with inorganic ammoniumsalt as the nitrogen source. The low pH in these cultures mayindicate that the rapid pH decrease was the reason for poorUDCA production. However, even under pH control in jarfermentors (same control as described in Fig. 7a), NH4Clcombined with either dextrin or sodium succinate was foundto be ineffective (<5% UDCA production after 120 h com-pared with 80% conversion with dextrin and L-asparagine).Therefore, we excluded the use of inorganic ammonium saltas a medium constituent.

Thus, from the results obtained, dextrin and L-asparagineseemed to be good medium constituents for UDCA produc-tion and were selected for further study. As for the ratio ofL-asparagine/dextrin, 12 to 20 g of L-asparagine per liter with40 g of dextrin per liter resulted in similar UDCA produc-tion, whereas more or less L-asparagine was less effective.

Reaction conditions for UDCA production. To investigateseveral reaction conditions for UDCA production, it becamenecessary to assay 7p-hydroxylation activity of the myce-lium. Because we aimed to optimize UDCA production byliving mycelium and the activity measurements with cell-freeextract revealed the instability of 7p-hydroxylation activity(recovery, <1%), we developed an assay method for restingmycelia of F. equiseti M41. UDCA formation by restingmycelium proceeded linearly for up to 4 h (Fig. 1). The useof Tris-hydrochloride buffer was essential in this assaybecause with other buffers the pH during the assay changedrapidly and frequent pH adjustment became necessary. Thereaction rate (375 ,ug/g of wet cells per h) with restingmycelium agreed well with that during cultivation (330 ,ug/gof wet cells per h), indicating that the activity measured bythis method was a good indication of the UDCA-producingactivity of the mycelium.

First we investigated the effects of pH and temperature onUDCA production (Fig. 2). The optimum temperature for

TABLE 3. Combined effect of carbon and nitrogen sources onformation of UDCA by F. equiseti M41 during cultivation with

Sakaguchi flasks"UDCA production (mg of UDCA per g of dry cells)

Carbon sourceNH4CI (NH4)2SO4 L-Asparagine

Starch (soluble) 0.28 (3.6) 0 (3.2) 34.39 (7.9)

Dextrin 0.27 (3.0) 0 (2.9) 39.34 (7.7)

" Cultivation was performed with a 100-ml portion of medium B containingthe indicated carbon (40 g/liter) or nitrogen (12 g/liter) source in a 500-mlSakaguchi flask for 14 days at 28'C. The pH at the end of cultivation isindicated in parentheses; the initial pH was adjusted to 7.0. Other experimen-tal conditions are identical to those described in Table 1, footnote a. Cell massranged from 1.5 to 1.7 g of dry cells per flask.

100

0-

E1-.

(1~

-

o

0

So

Incubation time (h )

FIG. 1. Time course of UDCA formation by resting cells of F.equiseti M41. After 40 h of cultivation with medium C (1.5 liters) ina 2.5-liter jar fermentor, the mycelia used were collected andassayed as described in the text. During the cultivation, pH andDOT were controlled at pH 7.0 and 0% saturation, respectively.Other experimental conditions are described in the text.

UDCA production as well as for growth was found to be28°C. Higher or lower temperature caused a sharp decline inenzyme activity. The optimum pH for UDCA productionwas found to be pH 8.0 with a narrow range, i.e., fivefoldincrease in activity from pH 7.0 to 8.0, whereas the fungusgrew well between pH 5 and 7. Therefore, during jarfermentation, a pH shift to 8.0 (UDCA production phase)from pH 7.0 or lower (growing phase) seemed to enhanceUDCA production.

-

2

3

4p

120

100

a-

>80

60

Z 40

201

10 20 30 40 6.0 7.0 8.0 9.0

Temperature (IC) P

FIG. 2. Effects of temperature (a) and pH (b) on formation ofUDCA by resting cells of F. equiseti M41. Experimental conditionsare almost identical to those described in the legend to Fig. 1, exceptthat temperature or pH was varied. In (b), 0.1 M potassiumphosphate buffer (0), 0.1 M Tris-hydrochloride buffer (0), or 0.1 MPIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)]-hydrochloride(A) buffer was used to obtain the desired pH value.

b

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MICROBIAL PRODUCTION OF UDCA 341

-

-0-

._

_0-)

0e

0 25 50 75 100

Dissolved oxygen tension (o/%)FIG. 3. Effect of DOT on formation of UDCA by resting cells of

F. equiseti M41. Mycelia cultivated in medium C (6 liters) with a

10-liter jar fermentor were harvested at 40 h, washed, and sus-

pended in 0.1 M Tris-hydrochloride (pH 8.0) (50 g of wet mycelia per

liter). The conversion reaction was performed at 28°C with each1-liter portion of the suspension in a 2.5-liter jar fermentor equippedwith a membrane-type DOT electrode. The DOT was controlled atthe indicated value by changing the agitation speed (aeration rate, 1vvm).

I_

> _

`0

cn <

CL

<I

o (M

c]EZ _ .

000 0.25 0.50 0.75 1.0

K+ concentration (M )FIG. 4. Effect of potassium ion concentration on formation of

UDCA by resting cells of F. equiseti M41. Experimental conditionsare essentially identical to those described in the legend to Fig. 1,except that the indicated amount of potassium ion was added as

either KCl (0) or K2HPO4 (0).

As with other fungal hydroxylases (12, 13), it is probablethat atmospheric oxygen would be incorporated directly intothe substrate, and the oxygen concentration during conver-

sion may affect the reaction rate. Indeed, at up to 60%saturation we observed an increase in UDCA production(Fig. 3), whereas the reaction rate reached a plateau at a

higher DOT. From the results it is evident that the fungusrequired a rather high DOT, between 60 and 100% satura-tion, for maximal UDCA production.

In preliminary experiments with Sakaguchi flasks, we

observed that high concentrations of potassium phosphatebuffer (0.1 M, pH 7.0) facilitated UDCA production. Evenunder pH control during jar fermentations, a higher concen-tration of potassiunm phosphate was effective (1.8-fold UDCAin the presence of 0.1 M potassium phosphate, pH 7,compared with that in the presence of 0.04 M potassiumphosphate, pH 7), excluding the possibility that the buffereffect was the reason for higher production. Therefore, we

investigated the concentration dependence of either KCI or

K2HPO4 on UDCA production, using resting mycelia (Fig.4). Both compounds enhanced UDCA production by two-fold at a K+ concentration of 0.5 M, suggesting that K+rather than HP042- was effective in the activation. Toinvestigate this activation further, we tested the effect ofseveral ions on UDCA production (Table 4). Na+ and Rb+were about as effective as K+, whereas Cs+ was onlyeffective at higher concentrations. Li+ was most effective atlower concentrations but was inhibitory at 0.5 M, probablydue to its well-known toxicity (10). NH4+ as well as all of thedivalent cations were inhibitory. For testing anions, we usedsodium salt to keep the pH and maintain the Tris concentra-tion of the reaction mixture. In the presence of Na2SO4,Na2HCO3, or NaNO3, UDCA production was less than thatobtained in the presence of NaCl, indicating the inhibitorynature of those anions. As for HP042, the promoting effectseemed to come from coexisting Na+. Indeed, when Tris-H3PO4 instead of Tris-hydrochloride was used as the reac-

TABLE 4. Effect of several ions on UDCA formation by restingcells of F. equiiseti M41"

Relative activity (%7) at concn of:Addition

0.1 M 0.25 M 0.5 M

None 100Li+ 189 81Nat 156 211K+ 161 211Rb+ 147 280Cs+ 104 223NH4+ 85 0

Ca2+ 0 0Mg2+ 0 0Mn2t 0 0Zn2+ 0 0

HPO42t (2Na+) 162 202HP042- (Tris)* 74 70S022- (2Na+) 124 80HCO- (Na+) 105 44NO3- (Na+) 120 14

a Experimental conditions are essentially identical to those described in thelegend to Fig. 1. Cations were added as the chloride form at the indicatedconcentration. Anions were added as sodium salt; in the case of the phosphateion (indicated by asterisk) the pH of the reaction mixture was adjusted to pH8.0 by changing the concentration of Tris. Therefore, at 0.1 and 0.5 Mphosphate ion concentrations, the concentrations of Tris buffer were 0.06 and0.32 M. respectively, instead of 0.1 M as in the other cases.

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342 KULPRECHA ET AL.

tion buffer to check the sole effect of HP042-, the phosphateanion was found to be slightly inhibitory. Thus, it can beconcluded that the activating effect on UDCA productionwas monovalent cation specific.As for the mechanism of a K+-dependent increase in

UDCA production, the following possibilities were consid-ered: (i) increased solubility of LCA in the presence of K+;(ii) increased LCA uptake into the mycelium in the presenceof K+; and (iii) direct activation of LCA 7p-hydroxylation byK+. Regarding the first possibility, we found no remarkabledifference in the solubility ofLCA upon addition of K+, i.e.,13 and 10 mg/liter in the absence and presence of 0.5 M KCI.For the third possibility, 7,B-hydroxylation with cell-freeextract was not affected by the addition of 0.5 M KCl (datanot shown). Therefore, we investigated LCA uptake in theabsence and presence of K+ (Fig. 5). Apparently, theaddition of 0.5 M KCl increased LCA uptake. The initialuptake rate and equilibrium concentration of LCA of themycelia were enhanced by 5.7- and 1.7-fold, respectively.Thus, enhanced UDCA production in the presence of K+can be attributed to the increased LCA uptake into themycelia of F. equiseti M41.

Induction of enzyme activity by LCA. To investigatewhether LCA 73-hydroxylation was catalyzed by an induc-ible enzyme, we cultivated F. equiseti M41 in the presenceand absence of LCA and measured the appearance of7,-hydroxylation activity in the presence of cycloheximide(250 ,ug/ml), which did not much affect the activity. Asmentioned above, mycelium cultivated in the presence ofLCA catalyzed 7,-hydroxylation (375 p.g/g of wet cells perh), whereas mycelium cultivated without LCA possessed noactivity in spite of the same growth curve as that in thepresence of LCA. Therefore, it can be concluded that theenzyme(s) was induced by substrate LCA.

Effect of DOT on hydroxylating activity during cultivation.As mentioned before, the optimum DOT for conversion ofLCA was determined at between 60 and 100% saturation.

s 20

E15 + 0.5 M KClE

_ 5 -

C.)

0

1 0 1 2K5C 1

{L

D ~~Incubation time (mmn)

the presence (0) and absence (0) of 0.5 M KCl. Experimentalconditions are described in the text.

.' 0.6-

0.4.';0X0 O

0.40.2

0o%A,

20 40 60Cultivation time (h)

FIG. 6. Effect of DOT during cultivation on the amount of LCA7p-hydroxylating activity. Cultivation was performed with mediumC (1.5 liters) in a jar fermentor (2.5 liter) and at a temperature of280C, aeration rate of 1 vvm, and pH of 7.0. DOT was controlled atthe indicated value by changing agitation speed. At the indicatedcultivation time, a sample was withdrawn, and the 713-hydroxylatingactivity of the mycelia was assayed as described in the text. DOTduring cultivation: 0, 0%; A, 5%; M, 15%; 0, 30%; /, 40%; El,60%; V, 100% saturation.

However, DOT may also affect the synthesis or degradationof the enzyme. To clarify this point, we investigated theeffect of DOT during cultivation on 7p-hydroxylation activ-ity (Fig. 6). When the fungus was cultivated at a DOT of 15%or higher, the enzyme activity reached the maximum after 25h of cultivation, whereas the peak was delayed to 34 and 50h at 5 and 0% DOT, respectively. Cell growth was not thereason for this phenomenon because both the growth rateand cell mass at the stationary phase were identical under allDOTs (data not shown).

After the activity reached its maximum, the activity in themycelium decreased rapidly irrespective of the DOT (70%reduction in 10 h at pH 7.0). However, this decline inenzyme activity was retarded by increasing the pH to 8.0(15% reduction in 10 h). Also, the addition of 0.5 M KCIcould retard the decline in activity, although less remarkablythan that caused by a pH shift (data not shown).

Production of UDCA by jar fermentations. From the linesof evidence obtained with resting mycelium, optimum con-ditions for UDCA production were estimated as follows: (i)pH 7 or lower during growth phase and pH 8.0 duringproduction phase; (ii) addition of 0.5 M K+ during produc-tion phase; (iii) 60 to 100% DOT during production phase;(iv) different shifting time after different DOT control duringgrowing phase (i.e., production phase should be initiated at40 to 50 h by changing conditions when the fungus grew at0% DOT and at 25 h when it grew at 15% DOT or higher).

In Fig. 7 and 8 are shown the time courses of cultivationwith these shifts in condition. In the control experiment inwhich the pH was maintained at 7.0 throughout the cultiva-tion without KCl addition, UDCA remained at 0.2 g/litereven after 150 h of cultivation. In contrast, 0.8 g (80% ofyield) of UDCA per liter was produced within 72 h (Fig. 7a)when the DOT was controlled at 0% saturation during cellgrowth and condition shifts at 40 h (pH to 8.0, DOT to 100%saturation, and addition of 0.5 M KCl) were done. In thiscultivation, substrate LCA was almost consumed at 70 h,

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MICROBIAL PRODUCTION OF UDCA 343

201

z

tntn

=

I)

dolU, 10

_

o0

~0.

v

4<ii

0L

I

0

to0)

0

Cultivation Time (h)

30r

cw 20

01n

to1

0

Z

0)

4

40

OD

50 100 150Cultivation Time (h)

FIG. 7. UDCA production in jar fermentors (a) with pH slfrom 7.0 to 8.0 and addition of 0.5 M KCl at 40 h of cultivation a(b) with the same condition shift at 40 h and LCA addition at 60Cultivation conditions are described in the text. At the arrcproduction was initiated by changing conditions.

and this lack of substrate seemed to be the main reason wUDCA production ended at 0.8 g/liter. Indeed, by providiadditional LCA, UDCA production reached 1.23 g/liter146 h of cultivation (Fig. 7b). Next, to shorten the cultivatitime, we selected a higher DOT (40% saturation) during tgrowth phase and performed the same condition shifts ath (Fig. 8). As predicted, almost the same amount of UD((1.20 g/liter) was produced during the shorter cultivatitime (96 h).

DISCUSSIONAmong steroidal hydroxylases of microbial origin, almi

all are known to be inducible (3, 5, 11), and a few have bereported to be constitutive (27, 28). However, little kno'edge has been available on the hydroxylation of bile aciwhich possess a structure very similar to that of sternhormones. In this report we confirmed that LCA ,hydroxylation also shared the inducible nature, suggestithat the inducible nature may be common to bile ahydroxylation.By surveying several compounds as medium constituen

dextrin and L-asparagine were found to be very effectiveUDCA production (Tables 1, 2, and 3). We observed ti

slowly utilizable carbon sources, such as dextrin or solublestarch, tended to facilitate UDCA production. Because ahigh concentration of glucose was very inhibitory for UDCAproduction by resting mycelium (88% inhibition in the pres-ence of 20 g of glucose per liter), enzyme repression orinhibition by glucose may be one of the reasons. In clearcontrast, organic ammonium rather than nitrate was thepreferred nitrogen source. L-Asparagine is known to be oneof the highly required nutrients for the growth of Fusariumspecies (30), and well-balanced growth in the presence ofL-asparagine might be a cause of higher UDCA production.However, the actual mechanism by which dextrin or L-aspar-agine facilitated UDCA production remains unclear andneeds further investigation.By using resting mycelium we determined the optimum

conversion conditions to be pH 8.0, temperature of 28°C,and DOT of 60 to 100% saturation, and we found twofoldactivation by the addition of 0.5 M KCl (Fig. 4). Our results(Fig. 5) suggested that higher UDCA production by K+resulted from the increased uptake rate or equilibrium con-centration of LCA. On the absorption of bile acids by theliver (2, 9, 24) or small intestine (16, 21, 23), transport of bileacids has been reported to be Na+ dependent. This effect

, was explained by the cotransport of bile acids with Na+uptake catalyzed by Na+,K+-ATPase. A similar mechanism

cm may be operating in our system. Another possibility is thatmonovalent cations disturbed the membrane structure sothat passive transport was enhanced. Li' was the mosteffective of the monovalent cations at a lower concentration,whereas Rb+ or Cs' became more effective at a higherconcentration (Table 4). Ito et al. (18) reported that theentrance of plasmid DNA into Saccharomyces cerevisiaewas enhanced by treatment with a monovalent cation,especially Li' or Cs'. The optimum concentration of Cs'was rather high (1.0 M). Considering that a rather highconcentration (0.5 M) was also necessary for the optimumactivation of 7,B-hydroxylation activity, enhanced passive

hift transport of LCA due to membrane disarrangement seemedmnd) h.

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0 50 100 150Cultivation Time (h)

FIG. 8. UDCA production in a jar fermentor with 40% DOTduring growth phase and 100% DOT during production phase.Experimental conditions are essentially the same as those describedin the legend to Fig. 7, except that DOT was controlled at 40%saturation until 40 h of cultivation and another portion of LCA (1g/liter) was added at 60 h. The production phase was initiated at thearrow.

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344 KULPRECHA ET AL.

more likely, although further investigation is necessary toclarify the mechanism.

F. equiseti M41 converts LCA into UDCA, but UDCAwas not the sole product. The ratio of UDCA to a by-productwas 4:1 (Fig. 7 and 8). After UDCA production ended due tolack of LCA (Fig. 7), by-product formation continued with aconcomitant decrease in UDCA. From this phenomenon andthe behavior of the by-product on gas chromatography andthin-layer chromatography, the by-product seemed to be a3a,7P,12a-trihydroxy derivative of 5-cholanic acid, al-though it was not identified completely. UDCA productionby this fungus would be improved further by eliminating orinhibiting the pathway by which the by-product is formed.

ACKNOWLEDGMENT

This work was partially supported by a grant-in-aid (no. 58103001)from the Ministry of Education, Science and Culture of Japan.

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