Further improvement of D -biotin production by a recombinant strain of Serratia marcescens

Process Biochemisny, Vol. 30, No. 6. pp. 553-562, 1995 Copyright 8 1995 Elsevier Science Ltd Printed in Great Britain. All tights reserved 0032.9592/95 $9.50 + 0.00 0032-9592(94)00060-3

ELSEVIER

Further Improvement of D-Biotin Production by a Recombinant Strain of Serratia marcescens M. Masuda,a* K. Takahashi/9b N. Sakurai,“,b K. Yanagiya,b S. Komatsubara” b & T. Tosaa “Research Laboratory Japan *Research laboratory 335, Japan

of Applied Biochemistry,

Tanabe Seiyaku Co., Ltd., 16-89

of Applied Biochemistry

at Toda, Tanabe Seiyaku Co., Ltd., 2-50

(Received

1994; revised manuscript received and accepted 26 November

14 October

Kashima-3-chome,

Yodogawa-ku,

Kawagishi-2-chome,

Osaka 532,

Toda-shi, Saitama

1994)

We have previously reported that a recombinant strain of Serratia marcescens was constructedfrom a host releasedfrom feedback repression of biotin biosynthesis and a recombinant plasmid carrying the mutated biotin operon. To improve Dbiotin production, we constructed a recombinant plasmid pLGM.304PA having the mutated biotin operon, a plasmid-stabilizing element and the ampicillin resistant gene, and introduced it into the o-biotin producing strain ETA23K-8 which contained an additional copy of the mutated biotin operon and the kanamycin resistant gene, resulting in ETA23K-S(pLGM304PA). Studies of the growth conditions for this recombinant strain indicated that high concentrations of sulphur and ferrous iron were required for good production of n-biotin. A batch culture fed by continuous addition of sucrose gave a maximum production of 600 mg litre-‘.

analogue, were isolated from the wild strain, yielding SB304 and SB412.’ Furthermore, resistances to both ethionine and S-aminoethylcysteine, sulphur-containing ammo acid analognes, were added to SB412 to generate ETA23, which produced 33 mg of D-biotin per litre of the medium containing sucrose and urea in a shake flask.s The mutated biotin operon was also cloned from SB304 on to a low-copy-number vector pLG339,” yielding ~LGM304.l~ This recombinant plasmid was further improved by introducing a plasmid-stabilizing element pm-B,” generating pLGM304P.n The above host was then improved by the addition of another copy of the mutated biotin operon on the chromosome, resulting in ETA23K-8.12 No recombinant strains have been obtained from this improved host.

INTRODUCTION D-Biotin is a vitamin required for human health care and as an animal feed additive. Some microorganisms require this compound for the industrial production of such metabolites as r_.-lysine and other L-amino acids. n-Biotin is industrially produced by chemical synthesis but several research groups have been working on developing more economical manufacturing methods using microbial production of D-biotin.lm6 Recently, we have constructed biotin-hyperproducing strains of Serratia marcescens Sr41. resistant to acidomycin, a biotin Mutants

*To whom correspondence

should be addressed.

553

554

M. Masuda, K. Takahashi, N. Sakurai, K. Yanagiya, S. Komatsubara,

This paper deals with the modification of pLGM304P, the construction of a recombinant strain using ETA23K-8 as a host, the optimization of the culture conditions, and the production of D-biotin in a fed-batch culture.

MATERIALS

AND METHODS

Strains and plasmids Escherichia coli C60013 and S. marcescens ETA23K-812 were used as host strains. S. marcestens ETA23K-8 was a o-biotin-producing strain with multiple mutations releasing feedback control and activating sulphur metabolism. Plasmids pKT24014 and pLGM304P* were used as sources of the ampicillin resistant (A@) gene and the mutant-type biotin operon, respectively. Plasmid pLGM304P carried the mutant-type bioABFCD genes and parB locus.’ ’

Medium The seed medium contained 10% sucrose, 1% urea, 0.1% K,HPO,, 0.2% MgSO,.7H,O, 0.01% FeSO, .7H,O, 0.6% corn steep liquor (CSL), and 1% CaCO,. The basal medium for batch cultures, using 500 ml Sakaguchi shake flasks and 2-l stirred fermentors, and the starting medium for fed-batch cultures contained 15% sucrose, 1.5% urea, 0.1% K2HP04, 0.2% MgS0,*7H20, 0.01% FeSO,. 7H,O, 0.1% CSL, 3% CaCO,, and 0.2% anti-foam DISFOAM CC220 (Nippon Oils and Fats Co., Ltd., Tokyo, Japan). Anti-foam was not added to the medium in shake flasks. The feed medium contained 67.5% sucrose, 5% urea, 0.15% K,HPO, and 0.1% CSL. Nutrient agar medium, containing 0.5% glucose, 1% peptone, 0.3% meat extract, 1% yeast extract, and 0.5% NaCl, was used for slant cultures. Nutrient agar plates containing ampicillin were used for the selection of transformants.

Genetic procedures and recombinant DNA techniques Standard procedures were used for plasmid preparation, restriction enzyme digestion, ligation, and agarose gel electrophoresis.‘s Transformation of E. coli and S. marcescens was carried out according to Maniatis et al.,15 and Takagi et a1.,16 respectively.

T. Tosa

Modification of pLGM304P and its introduction to ETA23K-8 To prepare the Ap’ gene, the 3-kb BarnHI-BstPI fragment carrying the Ap’ gene was isolated from pKT240 and was filled in with T4 DNA polymerase (Fig. 1). To construct pLGM304PA, plasmid pLGM304P, carrying the mutated biotin operon cloned from S. marcescens SB304 and the parB locus, was digested with EcoRI and XhoI sequentially and was blunted with T4 DNA polymerase. The digested pLGM304P was then ligated with the above DNA fragment carrying the Ap’ gene and introduced into E. coli C600. Colonies showing the Ap’ phenotype were selected and the plasmid was prepared from one of them, yielding pLGM304PA. Recombinant plasmid pLGM304PA was introduced into ETA23K-8, and a recombinant strain was obtained. o-Biotin production Seed cultures for D-biotin production in 2-l stirred fermentors (model MBW-2, Iwashiya Bioscience Co., Ltd., Saitama, Japan) were started by inoculating four loopsful of cells grown at 30°C for 20 h on nutrient slants into shake flasks containing 30 ml of the seed medium. Flasks were shaken at 30°C for 24 h at 140 rpm on a reciprocal shaker (70 mm stroke). Twenty-four ml and 28 ml of seed culture were inoculated into the 2-l fermentors for batch and fed-batch cultures, respectively. Batch and fed-batch cultures were carried out by using stirred fermentors containing 1.2 1 of the basal medium and 1 1 of the starting medium, respectively. Feeding was initiated 1 day after inoculation and continued until 8 days at a constant rate of 2.4 ml h-‘. In addition, 0.67 ml of 20% MgSO,.7H,O and 2 ml of 2% FeS04*7H,0 were added at one day intervals during 2-8 days. The concentration of dissolved oxygen (DO) was maintained at about 0.8 ppm by changing the rate of agitation as required. The culture temperature and aeration were maintained at 30°C and 0.5 volume per volume of liquid per minute, respectively. Analytical methods Cell mass was determined spectrophotometrically at 660 nm and expressed as biomass per litre of the medium, calculated by comparison with a conversion curve. o-Biotin was standard measured by bioassay with Lactobacillus plantarurn ATCC8014.” The amount of D-biotin plus

Improvement of o-biotin production

3kb BarnHI-BslPI

555

fragmenl

(Xho I) -

Fig. 1. Construction of the recombinant plasmid containing the Ap’ gene. (EC&I) and (Xhol) indicate the EcuFU and XHoI sites which were lost during construction of the derivative of pLGM304PA, respectively.

dethiobiotin was assayed with Candida tropicalis IF00058 and expressed as the amount of biotinrelated compounds. ’ Sucrose and urea were measured by the method of Dubois ef aLIs and the diacetyl monoxim method,r9 respectively. Ammonium was measured with an ammonia gas electrode (model 7 16 1, Denki Kagaku Keiki Co., Ltd., Tokyo, Japan). pH was continuously monitored by using a sterilizable pH electrode (model DPAS/120, INGOLD Co., Ltd., Zurich, Switzerland) attached to a pH controller (model FC-1, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). Chemicals D-Biotin was a product of Tanabe Seiyaku Co., Ltd. (Osaka, Japan). Dethiobiotin was prepared from D-biotin. Restriction endonucleases, T4 DNA polymerase and T4 DNA ligase were purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). Other chemicals were also obtained commercially and not purified further.

RESULTS Construction of pLGM304PA and ETA23K8(pLGM304PA) Since S. marcescens ETA23K-8 contains the kanamycin resistant (Km’) gene, it was difficult to introduce pLGM304P into ETA23K-8 using the Km’ marker. Plasmid pLGM304PA was therefore constructed by inserting the Ap’ gene from pKT240 into pLGM304P (Fig. 1). The resulting plasmid, pLGM304PA, was introduced into cells of ETA23K-8, yielding a recombinant strain, ETA23K-8(pLGM304PA ), which was used for further studies. Optimization production

of culture conditions

for o-biotin

(1) Effects of carbon and nitrogen sources High D-biotin production was obtained by using disaccharides such as sucrose and maltose as a carbon source (Table 1). When glycerol was used

556

M. Masuda,

K. Takahashi,

N. Sakurai, K. Yanagiya, S. Komatsuhara,

Table 1. Effect of various carbon and nitrogen sources on o-biotin production

T. Tosa

by 5. marce~ren~ ETA23K-8(pLGM304PA

)

Curbon source

Nitrogen source V@

n-Biotin produced (ma l- ‘)

Biotin-related compounds producedh (mg 1- ‘)

Culture time’ (d)

Glucose Maltose Fructose Glycerol Sucrose Sucrose Sucrose Sucrose

Urea (0.5%) Urea (0.5%) Urea (0.5%j Urea (0.5%) Urea (0.5%) (NH&SO, (1.1%) NH&I (O-89%) Peptone ( 1%)

22 47 31 458

55 68 61 76 62 52 55 50

2 2 3 >4 2 2 2 2

:: 28

OBacteria were cultured in 15 ml of the medium containing 5% carbon source, MgS0,.7H,O, O~Ol%FeSO,~7H,O, 0.1% CSI,, and I% CaCO, in shaking flasks. *The biotin-related compounds wcrc o-biotin and dethiobiotin. ‘Culture time was the day when the amount of o-biotin reached a maximum.

Table 2. Effect of concentrations

of sucrose and urea on o-biotin production

Sucrose @)

Urea cw

n-Riotin produced Img 1~‘)

5 1:

0.5 I.0

1.0 1.5 1.0 1.5

10 15 15 20 20

0.5

a nitrogen

source,

0.1%

K,HPO,,

0.2%

by S. marcescens ETA23K_8(pLGM304PA)O

YPiC-$

Biotin-related compounds produceB (mg I- I)

Culture time c ki)

(mgs-9

58 33 83

120 120 130

3 43

1.2 ;:;

19 21 11

100 73 200 210 210

150 160 230 340 370

4

0.7 0.7

5 8 8

1.3 1.0 1.1

;: 40 26 26

OBacteria were grown in 15 ml of the medium containing sucrose, urea, FeSO,.7H,O, 01% CSL and 1% CaCO, in shakingflasks. “The biotin-related compounds were o-biotin and dethiobiotin. “Culture time was the day when the amount of D-biotin reached a maximum. d Y,c, D-biotin produced relative to consumed sucrose. pY,,., , o-biotin produced relative to culture time.

as a carbon source, the amount of biotin-related compounds, presumably D-biotin plus dethiobiotin, was the highest but that of D-biotin was the lowest. Although maltose was very similar to sucrose in D-biotin production, maltose is more expensive than sucrose. Accordingly, sucrose was used as a carbon source for the cultures described below. The effect of nitrogen sources was examined in the medium containing sucrose as a carbon source. With the exception of peptone, each of the nitrogen sources was added at the concentration corresponding to 1% urea. The highest production of both o-biotin and biotinrelated compounds was obtained when urea was used as a nitrogen source (Table 1). The ratio of sucrose to urea was also examined. When the medium contained 15% sucrose and 1.5% urea,

0.1%

K2HF0,,

0.2%

(mgyT;- ,)

MgSO,7H,O,

0.01%

the productivities ( YPjC and Y,, ) were the highest on the basis of the amount of sucrose consumed and the culture time required for maximum production (Table 2). (2) Effect of phosphorous sources The use of K,HPO, produced higher D-biotin levels than other sources of phosphorus (Table 3). The optimum range of the K,HPO, concentration was very narrow and maximum production was found at 0.1% (Fig. 2A). When the concentration was outside the optimum range, the culture time was shortened but D-biotin production decreased. (3) Effect of sulphur sources Because a D-biotin molecule contains a sulphur atom, the supply of a sulphur source was expected

Improvement Table 3. Effect of various phosphorus

Phosphorus source K,HPO, HzPLO,

HPO, H,P,O,,

of u-biotin production

sources on o-biotin production

557

by S. murcescens ETA23K+(pLGM304PA

Concenrrarion VW

n-Biofin produced (mg lm ‘)

Biotin-related compounh” m I- 9

0.10 0.07 0.09 0.11 0.03 0.05 0.07 0.03 O-05 0.07

230 185 200 110 11 25 15 11 24 16

360 310 160 140 45 95 86 44 83 79

“Bacteria were cultured in 15 ml of the medium containing 15% sucrose. 1.5% urea, a phosphorus 0.01% FcSO,‘7H,O, 0.1% CSLand 1% CaCO, in shaking flasks. ae biotin-related compounds were o-biotin and dethiobiotin. “Culture time was the day when the amount of D-biotin reached a maximum.

Table 4. Effect of various sulfur sources on o-biotin production

Sulphur source

None L-Cystine r-Methionine Na,S.9H20 NaSH.XH,O Na,S,O,.SH,O Na&O, MgSO,.7H,O MgSO,.7H,O

(0.1%)” (0.2%)”

)” Culture

source, 0.2% MgSO,.7H,O,

by .Y marcescens ETA23K-8(pLGM304PA Cell mass

)”

I>-Biotin produced Pngl ‘)

Biorin-reIated compounds producedb (mgl-‘)

84 220 210 170 180 130 190

190 280 310 10 300 300 310

22 26 26 28

2 6 6

z!: 26

67 6

220 200

330 330

29 27

4

(g dry ceil I- I)

Culrure lime’ Gfl

“Bacteria were grown in 15 ml of the medium containing 15% sucrose, 1 .S% urea, 0.1% KZHPO,, 0.1% MgSO,, 7H,O, 0.01% FeSO,’ 7H,O, 0.3% sulphur source, 0.1% CSL and 1% CaCO, in shaking flasks. ‘The biotin-related compounds were o-biotin and dethiobiotin. ‘Culture time was the day when the amount of D-biotin reached a maximum. dMgS0,.7H,0 was added to the basal medium containing 0.1% MgS0,.7H,O, resulting in total concentrations of 0.2% and 0.3%, respeciively.

to be important for production. Sulphur sources were examined in a medium containing 0.1% MgSO,-7H,O and 0.01% FeSO,.7H,O. Although S. marcescens ETA23K-S(pLGM202PA) grew without an additional sulphur source, D-biotin production was only 80 mg I-’ (Table 4). The addition of various sulphur sources, such as L-methionine, L-cystine, Na,S-9H,O and NaSH. XH,O, markedly stimulated D-biotin production. The increase of the MgS0,.7HZ0 concentration to 0.2% also led to the production of more than 200 mg l- ’ of D-biotin.

(4) Efect of iron sources Since an increase in D-biotin production by the addition of metal ions had been reported,*‘)**’ the addition of metal ions was examined in using the medium containing 0.2% MgSO,- 7H,O and 0.000 1% FeSO,. 7H,O. D-Biotin production was not increased except in case of the addition of ferrous ions (data not shown) and hence the effect of a ferrous iron source in the medium was examined. When various ferrous compounds such as FeSO,. 7H,O, FeCl, and ferrous citrate were added at 0.03%, D-biotin production increased (Table 5). The concentration of FeSO,.7H,O

5.58

M. Masuda,

K. Takahashi,

N. Sakurai, K. Yanagiya, S. Komatsubara,

added to the basal medium was examined, the high D-biotin production was obtained at more than 0.0 1% in the medium (Fig. 2B). (5) Effect of temperature and dissolved oxygen Maximum D-biotm production was obtained at 30°C and O-751.50 ppm (Fig. 3). At 33°C the culture time was shortened but D-biotin production decreased. D-Biotin production cultures

was O-25 g 1-l at the beginning of the incubation, gradually decreasing during growth and was barely detectable at 1 d. Ammonium was detected only at 2 days. (2) Fed-batch culture fedIn order to increase D-biotin production, batch cultures were studied as follows. Cultures Table 5. Effect of various ferrous sources on production by S. marcescens ETA23K_8(pLGM304PA

by batch and fed-batch

(1) Batch cultures The time course of D-biotin production by ETA23K-8(pLGM304PA ) was investigated in the medium containing sucrose, urea and other nutrients in a 2-l stirred fermentor under the optimal culture conditions indicated above. Growth was accompanied by an increase of D-biotin production, which reached a maximum of about 250 mg l-l, and the cell mass reached a maximum of 25 g 1-l (Fig. 4). Sucrose and urea were gradually consumed as growth proceeded and sucrose was barely detectable at 6 d. Urea was gradually consumed but remained at 8 g 1-l at 6 d. The pH of the medium decreased to 6-O after 1 day and increased to 7.5 after 2 days. It then remained in the range of 7.5-8.0 until sucrose disappeared. The concentration of ammonium, which might be formed from urea through the autoclave sterilization of the medium,

4

T Tosa

D-biotin

)

Ferrous source

n-Biotin produced (mg I- 9

Biotin-related compounds produce& (mg I- ‘)

Culture time’ (4

None FeSO,.7H,O Fe2(S0,),KHz0 FeCl, FeCI, F~SO,.(NH,),SO, K,Fe(CW K,Fe(CN, Fe.citrate Fe. EDTA FeS

130 200 220 180 200 220 70

310 300 300 280 240 290 200 190 210 250 270

6

2;: 170 110

: 5 5 5 5 : 5 5

“Bacteria were grown in 15 ml of the medium containing 1.5% urea, K,HPO,, 0.2% 15% sucrose, 0.1% MgS0,.7H,O, 0.0001% FeSO,.7H,O, 0.03% ferrous source, 01% CSL and 1% CaCO, in shaking flasks. ‘The biotin-related compounds were D-biotin and dethiobiotin. ‘Culture time was the day when the amount of D-biotin reached a maximum.

*‘I*.,,~ : _:. ‘i--”

7 0.08 0.00 0.10 0.11 0.12 0.1

K2HP04 (%)

MOO1

0.001

0.01

FeSO4.7H20 (%)

Fig. 2. Effect of concentrations of K,HPO, and FeSO,-7H,O on u-biotin production by S. marcescens ETA23Kg(pLGM304PA). Bacteria were grown in 15 ml of the medmm containing 15% sucrose, 1.5% urea, KrHPO,, @2% MgSO,.7H,O, FeSO,.7H,O, 0.1% CSL, and 1% CaCO, in shaking flasks. The medium containing 0.01% FeSO,.7H,O and 0.1% K,HPO, was used for cultures of A and B, respectively. Symbols: 0, D-biotin produced; 0, biotin-related compounds produced; a, culture time when the amount of o-biotin reached a maximum.

Improvement of o-biotin production

.fi

120

z

80

‘i

27

33

30 Temperature

559

(‘C)

00 (ppm)

Effect of culture temperature and dissolved oxygen (DO) on o-biotin production by .S. marcescensETA23KFig. 3. 8(pLGM304PA ). Bacteria were grown in 2-1 stirred fermentors. The concentration of DO and culture temperature of cultures of A and B were maintained at about 0.8 ppm and 3o”C, respectively. Symbols: 0, n-biotin produced; 0, biotin-related compounds produced, n, culture time when the amount of o-biotin reached a maximum.

Culture time(d)

Fig. 4. Batch culture for n-biotin production by S. marcescem ETA23KS(pLGM304PA ) in the medium containing sucrose and urea in a 2-1 stirred fermentor. Bacteria were cultured in 1.2 1of a medium containg 15% sucrose, 1.5% urea, @l% K,HPO,, 0.2% MgSO,.7H,O, 0.01% FeSO,.7H,O, 0.1% CSL, 1% CaCG,, and 0.2% anti-foam DISFOAM CC220 in a 2-1 stirred fermentor. Symbols: A, growth; 0, D-biotin produced; 0, biotm-related compounds produced; Cl, sucrose; A, urea; 0, ammonium; ~1 PH.

were initiated with the starting medium and thereafter the feed medium containing sucrose, urea, K,HPO,, and CSL was added continuously to a stirred fermentor during the period of 1 day to 8 days (Fig. 5). The concentration of MgSO,. 7H,O was maintained at 0.2% by adding O-67 ml of 20% MgSO,*7H,O to the medium at one day intervals during 2-8 days. The concentration of FeS04.7H,0 was increased from O-01% to 0.0 15% by adding 2 ml of 2% FeSO, .7H,O solution to the medium at the same intervals described

above. The cell mass increased rapidly until 2 days and reached 40 g 1-l. Growth rate then decreased and cell mass reached a maximum of 50 g l- * after 10 d of cultivation. D-Biotin and biotin-related compounds were detected at 1 day and increased almost linearly until 10 days. As a result, the maximum concentration of D-biotin and biotin-related compounds reached 600 mg 1-r and 800 g I-‘, respectively. The total amount of sucrose consumed was 300 g 1-t. Plasmid pLGM304PA was stably maintained in the cell

560

M. Masuda, K. Takahashi, N. Sakurai, K. Yanagiya, S. Komatsubara,

T. Tosa

Culture time(d)

Fig. 5. Fed-batch culture for o-biotin production by S. marcesr‘ens ETA23K-8(pLGM304PA) in the medium containing sucrose and urea in a 2-1 stirred fermentor. The feeding was initiated at 1 day after inoculation and continued until 8 days at a constant rate of 2.4 ml h-t. In addition, 20% MgSO,.7H,O and 2% FeSO,.7HrO were added at intervals of one day during 2-S days. Symbols: A, growth; 0, o-biotin produced; l, biotin-related compounds produced; 0, sucrose; A, urea; 0, ammonium.

during the entire period of the fed-batch (data not shown).

culture

DISCUSSION Various r_-amino acid-hyperproducing strains of S. marcexens have been constructed and culture conditions for the production of L-amino acids have been improved.‘6~22-24 Sucrose was the most suitable carbon source for all of the ~-amino acidproducing strains. This sugar was also the most suitable carbon source for n-biotin production by S. marcescens ETA23K_8(pLGM304PA ). As a nitrogen source, urea has been used for the production of the above r,-amino acid. Ammonia (liquid) was effective as nitrogen source for L-amino acid production and the shortening of culture time in L-threonine and L-proline production.22,23 Since ammonium ion was not detected after 2 d during batch culture for D-biotin production, the supply of ammonium ion was expected to be short during the period for the maximum production. However, urea was more suitable for b-biotin production than ammonia (liquid) and ammonium sulphate. Several studies on the effect of amino acids, nucleotides, nucleosides, metal ions, yeast extract, and peptone on microbial

D-biotin productions have been reported.2*20*21~25-30 L-Alanine is introduced into pimeloyl-CoA, yielding 7-keto-&aminopelargonic acid, one of D-biotin intermediates. D-Biotin production by E. cali and Bacillus sphaericus26 were reported to be increased by the addition of L-alanine to the medium but the addition of this amino acid did not increase D-biotin production by ETA23K+J(pLGM304PA ). Increase in Dbiotin production by the addition of L-lysine, guanine, guanosine, uracil and thymine have also been reported2n~2’~2s~27-“0but the addition of these compounds did not increase D-biotin production by ETA23K_S(pLGM304PA ). Iwahara and Ogum ‘21 have reported that yeast extract and peptone induced bacterial D-biotin production and that this effect depended on ferrous ion contained in those compounds. Although D-biotin production using ETA23K_S(pLGM304PA) was not increased by the addition of yeast extract and peptone, the addition of ferrous iron compounds markedly increased n-biotin production. L-Amino acid synthesis by 5’. marcescens strains required 0~0001% FeS04.7H20 for maximum production but concentrations greater than O-01% were needed for the highest D-biotin production by ETA23K_8(pLGM304PA). A high demand for ferrous ion therefore characterizes the D-biotin

Improvement of o-biotin production production by ETA23K_8(pLGM304PA ). Ifuku et ~1.~’ reported that the addition of ferrous ion stimulated the conversion of dethiobiotin to Dbiotin the cell extracts of E. coli harbouring the bioB recombinant plasmid. Sanyal et uL3* have reported that the 82-kDa protein, which is the bioB product and a main factor for the biotin synthase reaction, has two [2Fe-2S] clusters per dimer and that the cluster is unstable because of the loss of sulphur from it during catalysis. Balavoine et ~1.~~ have demonstrated that iron catalyses the insertion of sulphur into the non-activated C-H bond. These findings and hypotheses are possibly related to the fact that D-biotin production by ETA23KS(pLGM304PA) required a high concentration of a ferrous compound. The introduction of a sulphur atom into dethiobiotin is important for high o-biotin production and therefore the effect of sulphur compounds on D-biotin production has been studied previously. 25,2’)The addition of L-methionine and inorganic sulphur such as compounds, Na,S*9H,O and Na,SO,= lOH,O, increased Dbiotin production by Succharomyces cerevisiae.25 Izumi et al. 29 have reported that a sulphur atom of L-methionine was involved in o-biotin synthesis from dethiobiotin in Rhodotorula using L-[%]methionine but that sulphur atoms in t-cystine and sodium sulphate were not. Ifuku et aL3’ and Sanyal et aL3* have indicated that S-adenosylL-methionine stimulated the conversion of dethiobiotin to D-biotin in a cell-free system. In contrast, Demo11 and Shive34 and Whitej5 have reported that S-adenosyl-L-methionine was not the sulphur source of n-biotin in the biotin synthase reaction of E. coli and that S-adenosyl-r-methionine might activate this enzyme. Because o-biotin production by ETA23K_8(pLGM304PA ) was increased by the addition of not only sulphur-containing amino acids but also inorganic sulphur compounds such as Na2S.9H20 and NaSH.XH,O, this strain is likely to have the ability of producing a specific sulphur intermediate which is required for the biotin synthase reaction. The optimum temperature of D-biotin production was 30°C. When the temperature was maintained above the optimum value, culture time was shortened but n-biotin production was decreased. When the concentrations of K,HPO, were high, a similar phenomenon was observed. Although the reason for the decrease in o-biotin production was not known, a slight delay in growth seemed to

561

lead to high D-biotin production. Therefore, in order to delay growth, the concentrations of urea were kept high in fed-batch cultures. As a result, a D-biotin concentration of about 600 mg l- ’ was achieved. The strain constructed and used in this work, ETA23K_8(pLGM304PA), produced with 20% more biotin than the conventional strain ETA23(pLGM304P), which produced 500 mg 1-l. This increase appeared to depend on the additional copy of the mutated biotin operon onto the chromosome of ETA23. Generally the productivity in fed-batch cultures, relative to the amount of consumed carbon source and culture time, can be higher than that of batch cultures but D-biotin productivity in fedbatch cultures using ETA23K_8(pLGM304PA) scarcely increased. The cessation of growth after 4 days appeared to prevent production from increasing. In order to improve productivity, the continuous growth without a decrease in the D-biotin production rate may be needed. When 600 mg 1-l of D-biotin was produced in fed-batch cultures, dethiobiotin was calculated to be accumulated at 200 mg l- *. If this dethiobiotin could be converted to o-biotin, then overall production would reach 800 mg 1-l. The complete removal of dethiobiotin from crude biotin crystals, recovered from the culture medium containing a large amount of dethiobiotin complicates the process of product purification. The efficient conversion of dethiobiotin to D-biotin may be expected to simplify the recovery process and to increase the amount of D-biotin accumulated.

ACKNOWLEDGEMENTS We are grateful to Dr I. Chibata, Dr M. Senuma and Dr S. Nagao for encouragement. We also thank Dr N. Nishimura and Dr S. Takamatsu for helpful discussion.

REFERENCES Yamada, H., Osakai, M., Tani, Y. & Izumi, Y., Biotin overproduction by biotin analog-resistant mutants of Bacillus sphaericus. Agric. Biol. Chem., 47 (1983) 101 l-6. Fisher, E. F. US Patent (1986) 86-01759. Ifuku, 0. & Yanagi, M., Cloning of the biotin operon from ITscherichia cob and construction of o-biotinproducing bacteria (in Japanese). Hakko to Kogvo, 46 (1988) 102-l 1. Gloeckler, R., Ohsawa, I., Speck, D., Ledoux, C.,

562

5. 6.

7.

8.

9. 10.

11.

12.

13. 14.

15.

16.

17.

18.

19.

M. Masuda, K. Takahashi, N. Sakurai, K. Yanagiya, S. Komatsubara, Bernard, S., Zinsius, M., Villeval, D., Kisou, T., Komogawa, K. & Lemoine, Y., Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin. Gene, 87 (1990) 63-70. Komatsubara, S., Omori, K., Imai, Y. & Sakurai, N., Jpn. Patent (1990) 90-27980. Sabatie, .I., Speck, D., Reymund, J., Hebert, C., Caussin, L.. Weltin. D.. Gloeckler. R.. O’Reean. M.. Bernard. S.. Ledoux, d., dhsawa, I., Komogaw& K., Lkmoine, Y. & Brown, S. W.. Biotin formation bv recombinant strains of Escherichia coli: influence of
20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34. 35.

T. Tosa

Sasakawa, T.Kimura & K. Kuratomi. Nannkodo, Tokyo, Japan, 1958, pp. 53-7. Eisenberg, M. A., Biotin biosynthesis. I. Biotin yields and biotin vitamer in cultures of Phycomyces blukesleeanus. J. Bacterial., 86 (1963) 673-80. Iwahara, S. & Oguni. K., Studies on the bacteria1 production of biotin-vitamer. Part 111. Effect of yeast extract on the production of biotin-vitamer (in Japanese). Nippon Nogeikagaku Kaishi, 47 (1973) 535-40. Komatsubara, S., Amino acids: genetically engineered Serratia marcescens. In Recombinant Microbes for Industrial and Agricultural Applications, ed. Y. Murooka & T. Imanaka. Marcel Dekker Inc., New York, USA, 1994, pp. 467-84. Masuda, M., Takamatsu, S., Nishimura, N., Komatsubara, S. & Tosa, T., Improvement of nitrogen supply for L-threonine production by a recombinant strain of Serratia marcescens. Appl. Biochem. Biotechnol., 37 (1992) 255-65. Ma&la, M., Takamatsu, S., Nishimura, N., Komatsubara, S. & Tosa, T., Improvement of culture conditions for t.-proline production by a recombinant strain of Serratia marcescens. Appl. Biochem. Biotechnol., 43 (1993) 189-97. -Niimura, T., Suzuki, T. & Sahashi, Y., Studies on the formation of biotin from desthiobiotin and sulfate in Saccharomyces cerevisiae. II. On sulfur sources of biotin formation bv washed cell susnension of vcast. J. VitaminoI., 10 ( (964) 224-30. a Iwahara, S., Tochikura, T. & Ogata, K., Studies on biosynthesis of biotin by microorganisms. Part VI. Biosynthesis of biotin vitamer by resting cell system of Bacillus spharricus. A@. Biol. Chem., 30 (1966) 1076-82. Ogata, K., lzumi, Y. & Tani, Y., Glutaric acid, a new nrecursor of biotin biosvnthesis. Agric. Biol. Chem., 34 (1970)1870-1. Ogata, K., Izumi, Y. & Tani, Y., Biosynthesis of biotinviamer from glutaric acid, a new biotin precursor. Agrir. Bioi. Chem., 37 (1973) 1087-92. lzumi, Y., Sugisaki, K. & Ogata, K., Incorporation of the sulfur of t-[%]methionine into the biotin molecule by intact cells of Rhodotorula glutinis. Biochim. Biophys. Acta, 304 (1973) 887-90. Iwahara. S. & Kancmaru. Y.. Biosvnthesis of biotin from dethiobiotin by intact cells of biotin-producing bacterium. Agric. Biol. Chem., 39 (1975) 779-84. Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M. & Fukushima. S.. Conversion of dethiobiotin to biotin in cellfree extracts of Escherichia coli. Biosci. Biotech. Biochem., 56 (1992) 1780-5. Sanyal, I., Cohen, G. & Flint, D. H., Biotin synthasc: purification, characterization as a [2Fe-2Slcluster protein, and in vitro activity of the Escherichia coli bioB gene product. Biochemistry, 33 (1994) 3625-31. Balavoine, G., Barton, D. H. R., Gref, A. & Lellouche, I., Iron catalyzed insertion of sulfur into the non-activated C-H bond. Tetrahedron, 48 (1992) 1883-94. Demoll, E. & Shive, W., The origin of sulfur in biotin. Biochem. Biophys. Res. Commun., 110 (1983) 243-9. White, R. H., Metabolism of L-[su[fane-“%]thiocystinc by Escherichia cob. Biorhemi~try, 2 l(l982) 4271-5.