Some Aspects of the Microbial Production of Biotin

Some Aspects of the Microbial Production of Biotin YOSHIKAZU IZUMIAND KOICHI OGATA* Department of Agricultural Chemistry, Kyoto Unizjwsity, Kyoto, lapan

..

..

..

11. Biosynthetic Pathway of Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pimelyl-CoA Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. KAPA Synthetase., . . . . . . . . . C. DAPA Aminotransferase . . . . . D. DTB Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Biotin Synthesizing Reaction . . . . . . . . . . . . . . . . . . . . . . . . . 111. Regulation of Biotin Biosynthesis . . . . . . . . . . . . . . . . . IV. Preparation of Biotin and Its Vitamers. .................... A. Chemical Processes for Preparation of Biotin . . . . . . . . . . . B. Microbial Synthesis of Biotin and Its Vitamers . . V. Biotin Antimetabolites and Their Actions on Biotin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Actithiazic Acid . . . . . .... .. B. a-Dehydrobiotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. a-Methylbiotin and a-Methyldethiobiotin . . . . . . . . . . . . . . D. Amiclenornycin and Stravidin . . . . . . . . . . . . . . . . . . . . . . . . . E. Adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Biodegradation of Biotin and Its Vitamers VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145 146 148 149 149 151 153 155 158 158 160 163 164 166 167 168 169 170 172 173

I. Introduction It is well known that biotin, vitamin H and coenzyme of carboxylase, plays a vital role in the process of microbial production of amino acids, as a control factor for the regulation of the amount of product. The action mechanism of biotin in glutamic acid production, which is closely related to the membrane permeability of glutamic acid-producing organisms toward glutamic acid, has been elucidated by extensive studies in a variety of ways. This information has led to the recent establishment of a unique new method for glutamic acid production from n-paraffin by a glycerol-requiring mutant of an nparsn-utilizing bacterium. Thus, in the field of industrial fermentation, biotin is closely linked with the microbial production of amino acids (Kinoshita and Tanaka, 1972). This is also indicated by the fact that, as described later, studies on the microbial production of glutamic acid have greatly contributed to the clarification of the biosynthetic pathway of biotin in microorganisms. *Professor Ogata is deceased

146

YOSHIKAZU IZUMl A N D KOICHI OGATA

One of the most common practical uses of biotin is as a supplement of culture media for amino acid fermentation. In addition, biotin has been attracting increasing interest recently as a food and fodder supplement. At present, the industrial preparation of biotin is carried out through a chemical process; a fermentative process remains to be established. Because of intricacy of the chemical process, an efficient and easy biotin fermentation is highly desirable for the supply of large quantities. Here we will review the biosynthesis and biodegradation of biotin from the viewpoint of industrial fermentation.

II. Biosynthetic Pathway of Biotin Studies on the biosynthesis of biotin in microorganisms started directly after the determination of its chemical structure (Melville et al., 1942) with stuhes on the nutritional requirements of microorganisms (Mueller, 1937a,b; du Vigneaud et al., 1942; Eakin and Eakin, 1942; Dittmer and d u Vigneaud, 1944; Dittmer et al., 1944; Lilly and Leonian, 1944; Stokes and Gunness, 1945)and contemporaneous studies on its total synthesis (Harris et al., 1943, 1944a,b, 1945). Subsequently, through radio-chemical and genetic biochemical studies (Tatum, 1945; Pontecorvo, 1953; Ryan, 1956), part of the biosynthetic pathway was proposed. Thereafter a hypothetical pathway was established by Okumura et al. (1962a-e) through their investigation of the microbial production of glutamic acid. In glutamic acid production using biotin-requiring organisms, Brevibacterium lactofmentum and B . flavum, they found that, besides dethiobiotin (DTB)and pimelic acid, a variety of chemically synthesized pelargonic acid derivatives including, 7,8-diaminopelargonic acid (DAPA), 7-keto-8-aminopelargonic acid (KAPA), 7-amino-8-ketopelargonic acid, and 7&diketopelargonic acid, and also oleic acid, satisfied the biotin requirement. They strongly suggested that these compounds might be precursors in the biotin biosynthesis, from the relation in the chemical structure. They also investigated the amount of each biotin vitamer’ which gives a maximum yield of glutamic acid, and the amount of intracellular biotin formed on incubation in the presence of each of these biotin vitamers. Subsequently, Ogata et al. and Iwahara et al. made extensive investigations of biotin biosynthesis and verified the main pathway of Okumura et al. using growing and resting cells. Their evidence was as follows: (1)Various microorganisms synthesized large amounts of biotin vitamers, mainly DTB, in the presence of pimelic acid (Ogataet al., 1965a,b; Iwahara et al., 1966a);(2)various bacteria ‘The term “biotin vitamer” is used in this review to indicate a compound that can satisfy the biotin requirement in the growth of biotin auxotrophs.

147

MICROBIAL PRODUCTION OF BIOTIN

synthesized KAPA from pimelic acid (Ogata et al., 1965b; Iwahara et al., 1967); (3) resting cells of Bacillus sphaericus synthesized DTB both from pimelic acid (Iwahara et al., 1965, 1966e)and from the KAPA obtained from the culture filtrate of Bacillus cereus (Iwahara et al., 1966b); (4) various microorganisms had the ability to synthesize biotin from DTB (Iwahara et al., 1966~). Using biotin auxotrophic mutants of Escherichia coli, Rolfe and Eisenberg (1968)and Pai (1969a) also verified the proposed pathway of Okumura et al. by means of cross-feeding tests, the identification of accumulated biotin vitamers and growth experiments with each vitamer. They confirmed the pathway of biotin biosynthesis to be pimelic acid 4 KAPA + DAPA + DTB + biotin. In this manner, studies of the biosynthetic pathway of biotin progressed using whole cells. The first demonstration of the enzymic system involved in biotin biosynthesis was made by Eisenberg and Star (1968).Since that time,

COAT L-Alan,ine

v+ ,

COOH CHz(CHd4COOH

Pimelic mid

ATP, Mg"

&-SCoA CHz(CH&COOH

Pimelyl -CoA

PLP

9

7-Keto-8-amino-

H V - 7 pelargonic acfd H3C CHz(CHz)4CWI-I (KAPA) tjHz NHz

H~-+H H3C , CHz(CH?)LCOOH HCO3ATP. Mg2'

7,B-Diaminopelargonic a (DAPA)

d

0

F,YH HY

Dethiobiotin

(DTBI

J

B Hy/'\yH

Biotin

I?&&3-l&COOH FIG. 1. Biosynthetic pathway of biotin in microorganisms.

148

YOSHIKAZU IZUMI AND KOlCHI OGATA

studies have used cell-free systems to determine the enzymic steps in biotin biosynthesis, and so far revealed all the steps from pimelic acid to DTB, as shown in Fig. 1. The final step from DTB to biotin has not been enzymically resolved yet. The following sections describe the four enzymes involved in the synthesis of DTB from pimelic acid, and the final step of biotin biosynthesis. A. PIMELYL-COASYNTHETASE

I t was assumed that pimelyl-CoA, the activated form of pimelic acid which condenses with L-alanine to form KAPA, may be formed from pimelic acid and CoA or acyl-CoA, and that an enzyme synthesizing pimelyl CoA from pimelic acid may be responsible for the first step in biotin biosynthesis by microorganisms. Eisenberg and Star (1968), using various microorganisms, reported that an enzyme activity catalyzing formation of pimelyl-CoA could not be detected by the hydroxamate method. They pointed out that one cause might be the high pimelyl-CoA deacylase activity in the cell extracts. Izumi et al. (1972a)were able to demonstrate enzyme activity catalyzing the formation of pimelyl-CoA from pimelic acid and CoA in microbial cell extracts, using the coupling reaction of the KAPA synthetase system described in Section II,B, where the KAPA formed from pimelyl-CoA was bioassayed with Saccharomyces cerevisiae as the test organism. On investigation the distribution of the activity of pimelyl-CoA synthesizing enzyme in cell extracts of about 100 strains of bacteria, Izumi et al. detected higher activity in Bacillus megaterium, Pseudomom jluorescens, and Micrococcus roseus. Izumi et al. (1972a)found that, other than pimelic acid and CoA, ATP and Mg2+ were necessary for this enzyme reaction. The enzyme was purified about 34-fold from the cell extract of B . megaterium by ammonium sulfate fractionation and DEAE-cellulose column chromatography (Izumi et al., 1974). K , values were 2.7 X lop4M for pimelic acid, 5.5 x M for CoA, 1.5 x lop3M for ATP, and 1.5 X M for M$+. Optimum temperature was 32"C, and optimum pH was 7.0 (phosphate buffer) or 8.0 (Tris-HC1 buffer). The pimelyl-CoA formed by this reaction was converted to its hydroxamate, and identified with the hydroxamate of authentic pimelylCoA by paper chromatography. Metal-chelating agents like EDTA, ophenanthroline, and a,a'-dipyridyl, and ferric ion, markedly inhibited this enzyme reaction. Further, Izumi et al. (1974) showed that Mnz+ and ADP could be substituted for Mg2+ and ATP, respectively. From these results, and the fact that dicarboxylic acids other than pimelic acid could not serve as a substrate, the pimelyl-CoA synthetase appears to be a new enzyme that should belong to EC 6.2.1 (acid-thiol ligases).

MICROBIAL PRODUCTION OF BIOTIN

149

B. KAPA SYNTHETASE Eisenberg and Star (1968), using crude cell extracts from a biotin auxotrophic mutant ofE. coli, reported that KAPA was synthesized from pimelylCoA and L-alanine through the participation of pyridoxal5’-phosphate (PLP). This was the first report to elucidate the biosynthetic pathway of biotin at the enzymic level. There were two main reasons which led them to consider the possibility of this reaction. One was the finding of Iwahara et al. (1966e) of the remarkable stirnulatory effect of L-alanine on the production of DTB from pimelic acid by resting cells of Bacillus sphaericus. The other was the close resemblance between the reaction of pimelic acid and alanine to form KAPA and the reaction of succinyl-Cob and glycine to form 6-aminolevulinic acid through the preparation of PLP (6-aminolevulinic acid synthetase involved in prophyrin biosynthesis) (Shemin et al., 1955). Izumi et al. (1972b, 1973a) partially purified the enzyme from the cell extract of B . sphaericus by ammonium sulfate fractionation, protamine treatment, and DEAE-cellulose column chromatography. This enzyme reaction required PLP as coenzyme and was strongly inhibited by various carbony1 reagents which are inhibitors peculiar to vitamin B6 enzymes. They investigated the amino acid specificity of the enzyme and established that the only amino acid that condensed with pimelyl-CoA was L-alanine; L-serine, which Eisenberg and Star (1968) demonstrated to replace Lalanine using a crude cell extract of E . coli, and L-cysteine, which Lezius et al. (1963) presumed to condense with pimelyl-CoA to form 9-mercapto8-amino-7-oxopelargonic acid, did not serve as substrate. The reaction suffered strong competitive inhibition by such amino acids as L-cysteine, D-alanine, L-serine, glycine, and D- and L-histidine, especially L-cysteine. Further, Izumi et al. (1973b) measured this enzyme activity in the cell extracts of about 100 strains of bacteria and found the enzyme present in many bacteria. Thus, KAPA synthetase, like 6-aminolevulinic acid synthetase, catalyzes the condensation reaction accompanying decarboxylation to form a ketoamino acid from acyl-CoA and amino acid with PLP as coenzyme. Therefore, Izumi et al. (1973b)proposed that KAPA synthetase should be regarded as a member of the acyltransferases (EC 2.3.1)and that it should be named pimelyl-CoA:L-danine 2-C-pimelyltransferase or L-danine pimelyltransferase. C. DAPA AMINOTF~ANSFERASE Pai (1971), using the cell extract from a biotin auxotrophic mutant ofE. coli which was deficient in DTB synthetase, found that DAPA was formed from

150

YOSHIKAZU IZUMI AND KOICHI OGATA

KAPA, and pointed out the existence of a DAPA aminotransferase. To measure the activity of this enzyme, DAPA was converted to DTB by the coupling reaction of the DTB synthetase system, as described in Section II,D, and DTB was bioassayed. He reported that methionine and PLP were amino donor and coenzyme, respectively, in this enzyme reaction. Later, Eisenberg and Stoner (1971) also found this enzyme activity using the crude cell extract of a biotin auxotrophic mutant of E. coli, but in this case the enzyme activity was measured directly by bioassay of the reaction product, DAPA. In the subsequent investigation of amino donors, L-methionine was found to be effective in the reaction with resting cells but required the addition of ATP and M 3 + with the cell extract. From this interesting result, they thought the true amino donor might be S-adenosyl-L-methionine (SAM). Izumi et al. (1975) investigated the activity of this enzyme in the cell extracts of about 100 strains of bacteria. Their method of measurement of the activity, like that of Pai (1971), was to convert the DAPA formed to DTB by the coupling reaction of DTB synthetase, and to determine DTB by bioassay with Bacillus subtilis. They found Brevibacterium divaricatum, Salmonella typhimurium, Bacillus roseus, Micrococcus roseus, and E . coli to have high activity. From the cell extract of B . divaricatum, which showed the highest activity, they purified the enzyme about 5000-fold by ammonium sulfate fractionation, acetone fractionation, DEAE-cellulose, and hydroxylapatite column chromatographies, and two applications to Sephadex G-100 gel filtration (Izumi et al., 1973c, 1975). The enzyme preparation gave a single band on disc gel electrophoresis. This enzyme was found to be a typical PLP enzyme, having absorption maxima in the region of 320 nm and 410 nm as well as 280 nm. It was confirmed to be a new aminotransferase showing specifcity only for SAM as amino donor. As amino acceptor, 7amino-8-ketopelargonic acid, an isomer of KAPA, as only one-hundredth of the activity of KAPA. Pyridoxamine 5'-phosphate (PMP), as well as PLP, can act as coenzyme. K , values were 0.69 X M for KAPA, 0.55 X M for SAM, and 0.83 X M for PLP. Stoner and Eisenberg (1975a,b)made a more detailed investigation of the properties of this enzyme purified 1000-fold from an extract of a regulatory mutant of E. coli. They were able to resolve the cofactor, PLP, from the enzyme in the presence of phosphate buffer after incubation with the amino donor, SAM. The molecular weight was estimated to be 94,OOO+- 10,OOOby gel filtration and sucrose gradient sedimentation. Furthermore, sodium dodecyl sulfate disc gel electrophoresis established the molecular weight of the enzyme subunit as 47,000 +- 3000, and therefore, the enzyme is considered to consist of two subunits of similar molecular weight. Since Stoner and Eisenberg (1975a) could not detect the expected keto product of SAM, S -adenosyl-2-oxo-4-methylthiobutyric acid, in the DAPA

MICROBIAL PRODUCTION OF BIOTIN

7-keto-&aminopdargonic acid (KAPA) PMP MenzymeP

I

151

7.8-diaminpdargonic acid

) PLPA enzyme

nonenzymic

2-0x0-3-butenoic acid

+

5'-methylthioadenosine

FIG. 2. Proposed mechanism for the DAPA aminotransferase reaction. From Stoner and Eisenberg (1975a).

aminotransferase reaction with the use of chemical reagents, they considered that the keto product might decompose nonezymically to yield 5'-methylthioadenosine and 2-0x0-Sbutenoic acid. This assumption was supported by the result that when the reaction mixture containing S-adenosyl-~-[2-'~C]methionine as amino donor was acidified and passed through a Dowex 50-H+ column, a radioactive neutral compound was detected in the effluent. The compound was confirmed to be enzymically produced in amounts equivalent to the DAPA produced. On the basis of this experimental evidence, the DAPA aminotransferase reaction was formulated as shown in Fig. 2 (Stoner and Eisenberg, 1975a). KAPA was found to show strong substrate inhibition (Pai, 1971; Eisenberg and Stoner, 1971; Izumi et al., 1975). The inhibition by KAPA is competitive with SAM (Stoner and Eisenberg, 1975b; Hotta et al., 1975). The enzyme activity was strongly inhibited by phenylhydrazine, semicarbazide, isoniazide, and hydroxylamine, which are typical inhibitors of vitamin B, enzymes (Izumi et al., 1975). Thus, because of the high specificity for SAM as amino donor, the enzyme should be classified as a new type of enzyme (S-adenosyl-~-methionine:7keto-8-aminopelargonate aminotransferase) of the group EC 2.6.1 (Izumi et al., 1975).

D. DTB SYNTHETASE Eisenberg and Krell (1969a) found that DTB was formed from DAPA by resting cells of a biotin auxotrophic mutant of E . coli. This reaction was accelerated 2- to 3-fold by addition of L-serine, NaHC03, and glucose. Sub-

152

YOSHIKAZU IZUMl A N D KOICHI OGATA

sequently, Eisenberg and Krell (1969b), Pai (1969b), and Cheeseman and Pai (1970), using E . coli, and Yang et al. (1970b), using Pseudmonas graueolens, observed the formation of DTB from DAPA by cell extracts, and clarified that HC03-, ATP, and M$+ as well as DAPA are necessary for this enzyme reaction. Krell and Eisenberg (1970) purified the enzyme (DTB synthetase or ureido ring synthetase) about 2OO-fold from the cell extract of E . coli and obtained an enzyme preparation of over 90% purity. The enzyme has a molecular weight of 42,000 and is composed of two subunits. Yang et al. (1970b, 1971c)also purified the enzyme about 2000-fold from the cell extract of P . graveolens by ammonium sulfate fractionation, DEAE-cellulose and hydroxylapatite column chromatographies, and Sephadex G-200 gel filtration, and obtained an enzyme preparation showing a nearly symmetric peak upon ultracentrifugation. The sedimentation coefficient (s,,,,~) was 3.496 x cm/sec. The optimum pH was 7.0-8.0 and the optimum temperature was almost 50°C. Yang et al. (1969a, 1971a) observed that biotin diaminocarboxylic acid (BDC), a compound lacking the ureido part of biotin, was converted to biotin by resting cells of Bacillus sphaericus and Rhodotorula rubra and proved that the reaction proceeds by means of DTB synthetase. However, the activity of BDC as substrate for this enzyme is about one-tenth that of DAPA, and pelargonic acid derivatives, KAPA and 7-amino-8-ketopelargonic acid, could not act as substrate (Yang et al., 1970b, 1971~). CO, had higher activity than HC03- (Krell and Eisenberg, 1970). Of metal ions tested, Mn2+ showed 95-136% and Fez+ 71-91% of the activity of M g + (Yang H2N NHz -COOH H3C

*I

c02

9-0H~&COOti

H3C

I

ATP

FIG.3. Proposed mechanism for DTB synthesis from DAPA by DTB synthetase. From Krell and Eisenberg (1970).

MICROBIAL PRODUCTION OF BIOTIN

153

et al., 1971~).CTP, UTP, GTP, and ITP showed 10-20% of the activity of ATP (Ogata et al., 1973b). Ogata et al. (1973b) demonstrated that the enzyme reaction was strongly inhibited by chelating agents, such as EDTA, a,a’-dipyridyl, and o-phenanthroline. Moreover, it was established that ADP shows competitive inhibition toward ATP (Krell and Eisenberg, 1970; Ogata et al., 1973b) and that the substrates DAPA and BDC are competitive with each other (Ogata et al., 1973b). Investigation of the enzyme reaction stoichiometry proved that equimolar amounts of DTB and ADP are formed (Krell and Eisenberg, 1970; Ogata et al., 1973b). Based on this observation, Krell and Eisenberg (1970) proposed a reaction mechanism for DTB synthetase as shown in Fig. 3. DTB synthetase is a new kind of carboxylase (EC 6.3.3.aa) because it catalyzes carboxylation accompanying the formation of ureido ring (Barman, 1974).

E. BIOTINSYNTHESIZING REACTION It has been recognized that DTB is converted to biotin during growth of Saccharomyces cerevisiae (Dittmer et al., 1944), Aspergillus niger (Wright and Driscoll, 1954), E . coli (Pai and Lichstein, 1965c), and other microorganisms including yeasts, molds, actinomycetes, and bacteria (Iwahara et al., 1966~).Tepper et al. (1966) confirmed this conversion with A . niger using [14C]DTB, labeled in either the carbonyl group or the carboxyl group. Biotin synthesis from DTB using resting cells was demonstrated with E . coli (Pai and Lichstein, 1965c, 1966), S. cerevisiae (Niimura et al., 1964a,b,c), and Rhodotmula glutinis (Izumi et al., 1973d). Furthermore, Niimura and Shimada (1967) observed the conversion using protoplasts of Bacillus megaterium. However, there have been no studies on enzymic synthesis of biotin from DTB. In the investigation of sulfur sources for biotin biosynthesis from DTB using resting cells of S . cerevisiae, Niimura et al. (1964b) found methionine sulfoxide and methionine to be most effective, and that NazS03, NazS, NaZSO4,homocysteine, SAM, methylmercaptane were also effective. Niimura et al. (1964~) next used [35S]-methionineand detected the reaction products by radioautography: radioactive biotin, biotin d-sulfoxide, biotin 1-sulfoxide, biocytin, and biocytin sulfoxide. Izumi et al. (1973d) tested the effect of various sulfur compounds using resting cells of R . glutinis which forms appreciable amounts of biotin from DTB. In the presence of DTB, this organism formed hardly any biotin on addition of inorganic sulfates and sulfites or L-cysteine, but formed considerable amounts of biotin on addition of DTB and methionine, in particular the

154

YOSHIKAZU IZUMI A N D KOICHl OGATA

L-form of methionine. Next, after reaction using ~-[~~S]methionine, they isolated radioactive biotin by cation and anion exchange column chromatography, avidin treatment, and dialysis, and identified it by radiochromatography and bioautography. They also confirmed that S contained in 1 molecule of L-methionine is incorporated into 1 molecule of biotin. Li et al. (1968b) carried out experiments with A . niger on the incorporation into biotin of car~nyl-['~C]DTBand carboxyl-[14C]DTB randomly labeled with 3H in order to measure a change of the number of hydrogen atoms during the conversion of DTB to biotin. The ratio of 3H/'4C for DTB and for the biotin formed showed that the 3H radioactivity of biotin was 15 to 20% lower than that of DTB. Thus, they considered that DTB might be converted to biotin with the loss of 3 of 4 hydrogen atoms. More recently, Parry and Kunitani (1976) have developed a new, stereospecific synthesis of DTB and reexamined the mechanism of the conversion of DTB to biotin by using specifically labeled [3H]DTB. The samples of tritiated DTB synthesized were each mixed with dZ-[IO-'4C]DTB and the doubly labeled precursors were then administered to cultures of A . niger. After incubation, the biotin synthesized from each doubly labeled precursor was isolated as d-biotin sulfone, and converted to biotin sulfone methyl ester. The methyl esters were purified by chromatography and then recrystallized to constant activity and constant ratio of 3H/14C.From the results shown in Table I, it appears that the introduction of sulfur at C-1 and C-4 of DTB takes place without the loss of hydrogen from C-2 or C 3 , suggesting that unsaturation is not introduced at C-2 or C 3 during the biosynthesis of biotin from DTB. However, they consider that the possibility of enzymic removal of hydrogen from C-2 or C 3 followed by replacement of the hydroTABLE I TFUTIATED DETHIOBIOTIN INTO BIOTIN"

INCORPORATION OF SPECIFICALLY

0

Expt. no. 1

2 3 4

precursor

3H/"C for biotin sulfone methyl ester

Percent 3H retention

6.05 2.89 6.88 5.88

5.74 3.04 4.81 3.10

95 105 70 53

3H/14Cfor Precursor

dl-[2,3-3H;10-'4C]DTBb d-[3-3H;IO-14C]DTE dl-[1-3H;IO-'*C]DTB dl-[4(RS)-3H;10-14C]DTB

"From Parry and Kunitani (1976). *Precursor had 58% 3H at C-2, 42% at C-3. 'Precursor had 17% 3H at C-2, 83% at C-3.

MICROBIAL PRODUCTION OF BIOTIN

155

gen without exchange cannot be excluded. Moreover, the result that the incorporation of dl-[1-3H]DTB into biotin proceeds with 30% tritium loss is consistent with the removal of one hydrogen atom from the methyl group of DTB. The result that dl-[4(RS)-3H]DTBis incorporated into biotin with 47% tritium loss suggests that the stereospecific removal of one hydrogen atom from C-4 of DTB may occur during the formation of biotin. Thus, these results clearly demonstrate that two hydrogen atoms are removed from d-DTB during its conversion to d-biotin. The next step in elucidating the mechanism of the biosynthetic conversion of DTB to biotin should be to investigate how the methyl group at C-1 and the methylene group at C 4 of DTB are converted and to determine the order of fimctionalization of C-1 and C 4 in DTB during its conversion to biotin.

111. Regulation of Biotin Biosynthesis Pai and Lichstein (1962, 1965a) cultured E . coli in medium supplemented with various concentrations of biotin, and measured the amounts of the biotin vitamers formed. In contrast to the constant intracellular content, the amount of biotin vitamers formed extracellularly decreased remarkably with added biotin. This inhibition of biotin vitamer biosynthesis was specific for biotin, hardly occurring with DTB, oxybiotin, and biocytin. Using resting cells of E . coli, Pai and Lichstein (196513) investigated whether this regulation occurred via repression, via feedback inhibition, or via both. In the presence of chloramphenicol, which inhibited the growth of the organism, the cells grown in a biotin-free medium synthesized as much p g of biotin vitamers per milligram of cells during a 90-minute as 80 x incubation period, whereas those grown in the biotin-supplemented (50 x p g m l ) medium synthesized only 5 x lop4 p g of biotin vitamers per milligram of cells. Furthermore, the cells grown in the biotin-free medium synthesized as much biotin vitamers in the presence of both chloramphenicol and biotin as they did in the presence of chloramphenicol alone. These facts indicated that biotin biosynthesis in E . coli might be controlled via repression rather than via feedback inhibition. Moreover, as no conversion of DTB to biotin occurred in the resting cells of either the mutant or wild-type strain which had been grown in the presence of exogenous biotin, the repression by biotin was also found to be operative in this conversion step ofE. coli (Pai and Lichstein, 1966).Pai and Lichstein (1966)pointed out that there is a critical level of exogenous biotin and that the biotin synthesizing enzyme system is almost completely repressed only when the biotin content of the medium was raised above 10 to 20 x lop4pg/ml. This coincides with the concentration of intracellular biotin (15 x lop4pg/ml culture) found in the organism after growth had ceased (Pai and Lichstein, 1965a) and

156

YOSHIKAZU IZUMI A N D KOICHI OGATA

TABLE I1 DTB-SYNTHESIZING ABILITY OF RESTING CELLSOF Bacillus sphaericus a DTB synthesized Cells harvested

(/ALg/ml)

From basal medium (cells: 97.1 rng/rnl)

45.0

From DTB-supplemented (100 pg/ml) medium (cells: 95.0 mg/ml)

42.3

From biotin-supplemented (0.2 pg/ml) medium (cells: 98.7 mg/ml)

0.05

“From Iwahara (1968)

is close to the level giving optimum growth of a biotin-requiring mutant ofE. coli (Ferguson and Lichstein, 1957). Iwahara (1W)also found the accumulation of biotin vitamers to be almost completely inhibited by the addition of biotin to the culture medium of various bacteria. However, this inhibitive action was not observed at all with fungal species. Whether this is due to the impermeability of the organisms toward exogenous biotin or to the insusceptibility of their biotin biosynthesis to repression by biotin has not been determined. As shown in Table 11, resting cells of B . sphaericus harvested from the basal medium without

Biotin added (pg/ml) FIG. 4. Effect of biotin on the synthesis of 7-keto-8-aminopelargonic acid (KAPA) synthetase of Bacillus sphaericus (-0) and 7,8-diaminoplargonic acid (DAPA) aminotransferase of Breoibacterium divaricaturn (0-0). From Izumi et al. (197313, 1975).

157

MICROBIAL PRODUCTION OF BIOTIN

0

01

02

03

04

Q5

06

"

Ib

Biotin added (pqlml)

FIG. 5. Effect of biotin on the synthesis of pimelyl-CoA synthetase (0-0) of Bacillus inegaterium. From Izumi et al. (1974).

(-0)

and DTB syn-

thetase

added biotin or from the DTB-supplemented medium readily synthesized DTB from pimelic acid, whereas cells obtained from medium supplemented with biotin synthesized very little DTB. These facts also bear out the suggestion of Pai and Lichstein (1965b)that the inhibitive action of biotin depends on repression. From progress in the enzymic solution of the biosynthetic pathway of biotin, the biosynthetic regulation mechanism has also been elucidated at the enzymic level. Eisenberg and Krell (1969b) have reported that KAPA synthetase and DTB synthetase ofE. coli are almost completely repressed by the addition of 1ng/ml of biotin to the medium. Pai observed similar repression by biotin of DTB synthetase (Pai, 1969b) and DAPA aminotransferase (1971) ofE. coli. Izumi et al. (1973b)found KAPA synthetase ofB. sphaericus and B . subtilis was repressed by biotin. As shown in Fig. 4, they demonstrated that KAPA synthetase of B . sphaericus and DAPA aminotransferase of Brevibacterium divaricatum were repressed by the addition of 0.1 pg/ml of biotin to the medium (Izumi et al., 197313, 1975). Moreover, they found that, in contrast to the complete repression of DTB synthetase in B . megaterium by 0.25 pg/ml of biotin, as can be seen in Fig. 5, pimelyl-CoA synthetase was not repressed by even 1 pg/ml of biotin (Izumi et al., 1974). In this way, a strong repressive action of biotin has been demonstrated on all the enzymes between pimelyl-CoA and DTB and on part of the biosynthetic system between DTB and biotin. This repression is thought to be the main reason for the minute amounts of biotin produced by a large number of microorganisms.

158

YOSHIKAZU IZUMI AND KOICHI OGATA

IV. Preparation of Biotin and Its Vitamers

A. CHEMICALPROCESSES FOR PREPARATION OF BIOTIN In this section, the practical chemical processes for preparation of biotin are briefly described for the purpose of comparison with the microbial synthesis of biotin, although the latter is not yet competitive. Presently, the industrial preparation of biotin is being carried out by the method of Hoffmann-La Roche, Inc. (Goldberg and Sternbach, 1949;

0 d-Camphorsulfonate o f d- and Z - ( X I )

Resolution

(j)

(XII)

d-Camphorsulfonate o f &(XI)

(XIII)

I

(k) -y-y

HZC\s/CH-(CH

A00C2H5 > )-CH

(1)

&Biotin

‘COOC2H5

( X W

FIG. 6. Flow sheet of one chemical process for industrial preparation of biotin. (a) Benzylamine; (b) phosgene; (c) acetic anhydride; (d) Zn, a mixture of acetic acid and acetic anhydride; (e) H,S, HC1; (0 C,H,O(CH,),MgBr; (9) H,, Raney nickel; (h) HBr, acetic acid; (i) silver d-camphorsulfonate; (i) isopropanol; (k) sodium diethylmalonate; (1) HBr. From Coldberg and Sternhach (1949).

HoaOHCQ

R

e

C

H

3

0

0 x 0

O

HO

F

(1)

OB?

Ox'

Ez=benzyl

(11)

W

C

O

H

H -

0

Y

(VIII)

O

C

H

R=MCOOCH3

R

j H--

( I X ) R=H (X)

R=CH3S02

-H

N3

N3

(XI)

C

e

OR

HflCooCH3

S H ' H h 9 R

0

(111) R = w C O O C H ,

3 H@COOCH3 H R

0 OxO

062

(IV)

-

159

MICROBIAL PRODUCTION OF BIOTIN

(")

( V I ) R=H

r3 (VII) R=CH3S02

+ H-

+I

AcNH

__$

&Biotin

HNAc

Ac=CH3C0

(XI11

FIG. 7. Stereospecific synthesis of d-biotin from D-mannose. From Ohrui and Emoto (1975).

Gyorgy and Langer, 1968). As illustrated in Fig. 6, the synthesis is characterized by the use of a meso-diaminosuccinic acid derivative as a starting material, which contains two groups in the same spatial arrangement as the two amino groups present (in substituted form) in the biotin molecule, i.e., the meso-configuration in diaminosuccinicacid derivatives corresponding to the cis-structure of the two amino groups in a ring compound such as biotin. Moreover, since the resolution is carried out at the intermediate stage (XII-XIII), it permits the direct production of the optically active biotin, d-biotin and I-biotin, of which the d-form is naturally occurring and physiologically active. Recently, another efficient stereospecific total synthesis of d-biotin has been achieved by Ohrui and Emoto (1975).They used D-mannose as starting material. As shown in Fig. 7, D-mannose is converted to the aldehyde (11) through isopropyridenylation, benzoylation, selective isopropyridenylation, and periodate oxidation. Wittig reaction and hydrogenation of the aldehyde give the compound (IV) which has the side chain like biotin. Treatment of (IV) with NaOCH, in methanol, followed by the reduction of the resulting aldehyde (V) with NaBH, affords 0711) via (VI). Treatment of (VII) with NaS affords a tetrahydrothiophene derivative (VIII) which is converted via (IX) to (X). Treatment of (X) with NaN3 gives a diazido compound (XI). Catalytic reduction of the azido groups of (XI) in a mixture of methanol and acetic anhydride gives a diacetoamido derivative (XII). Treatment of (XII) with Ba(OH),, followed by the treatment with phosgene affords d-biotin. Thus, d-biotin is synthesized from D-mannose in good yield, because a fivemembered ring consisting of isopropyridene, which protects the hydroxyl groups of the sugar, is used to fix the molecular conformation during the intramolecular substitution reactions.

160

YOSHIKAZU IZUMI A N D KOICHI OGATA

B. MICROBIALSYNTHESIS OF BIOTINAND ITS VITAMERS 1. Biotin

Ogata et al. (1965a) and Iwahara et al. (1966~) examined the accumuation of biotin from pimelic acid and DTB by about 800 strains of stocked and isolated microorganisms. As a result, the maximum amount of biotin accumulated in the culture medium with 50-500 pg/ml of pimelic acid or SO pg/ml of DTB was about 500 ng/ml. The molds and Streptomyces tested generally accumulated larger amounts of biotin in their culture filtrates (Ogata, 1970a). In contrast, most of the bacteria and yeasts tested accumulated little biotin. Iwahara and Oguni (1973)have demonstrated that peptone and yeast extract have a remarkable promotive effect on the formation of biotin from DTB by various bacteria isolated from soil, and that one active ingredient in yeast extract is an iron salt. They reasoned that iron plays an important role in the biosynthesis of biotin. Yang et al. (1969a, 1971a)found that biotin was synthesized from BDC by the resting cells of B. sphaericus and R . glutinis. The conversion was markedly stimulated by addition of amino acids, especially alanine and glutamic acid, and under aerobic conditions. Under optimal conditions, about 6 pg/ml of biotin was formed from 10 pg/ml of BDC. Izumi et al. (1973e)found that biotin was synthesized from bisnorbiotin, a compound with two less carbon atoms in its side chain than biotin, by various microorganisms. Pseudomonas iodinum was the most effective convertor, giving 0.55 pg/ml of biotin from 1.0 pglml of bisnorbiotin. Similarly, they also observed the efficient conversion of bisnordethiobiotin to DTB. Ogino et al. (1974a,b) have demonstrated a useful method for industrial biotin production using an n-pardin-utilizing bacterium from a new com(dlpound, d-cis-tetrahydro-2-oxo-4-n-pentylthieno-(3,4d)-imidazoline TOPTI). TOPTI was chemically synthesized from N1,N3-dibenzyldl-cistetrahydrothieno-(3,4-d)-imidazoline-2,4-dione[compound (VII) in Fig. 61 through Grignard reaction with n-pentyl magnesium bromide, dehydration in the presence of acidic catalyst, catalytic hydrogenation, and debenzylation with concentrated HBr. In this method of producing biotin, n-pardin was used as carbon and energy source for cell synthesis with concurrent transformation (cooxidation) of TOPTI to biotin. First, n-pardin-utilizing microorganisms that cooxidize TOPTI were selected from natural sources. Of the 9 strains that could convert TOPTI to biotinol and biotin, 3 strains identified as Corynebacterium were the most excellent producers. The cooxidation products synthesized from d-TOFT1 were isolated from culture broth and identified as d-biotinol and d-biotin by infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The time course ofthe transformation showed that biotinol was initially produced before biotin began to

161

MICROBIAL PRODUCTION OF BIOTIN

dl-TOPTI

dl-Biotinol

dl- B iot in

FIG.8. Transformation of dl-cis-tetrahydro-2-0~04-~-pentylthieno-(3,4d)-imidazoline (dTOPTI) to biotin.

appear. From these results, the conversion ofTOPTI to biotin was assumed to occur via w-oxidation such as that of aliphatic hydrocarbon (Heydeman, 1960), as shown in Fig. 8. In a medium containing 2% n-paraffin and 0.2% urea, with addition of 50 mg/100 ml of dl-TOPTI after 24 hours of cultivation, maximum conversion of about 60% (32 mg/100 ml of dl-biotin) was obtained 96 hours after the addition of TOPTI. However, selective degradation of d-biotin occurred on prolonged incubation, leaving the h o m e r . Thus, to avoid such degradation, mutants that were incapable of assimilating n-paraffin and of degrading biotin, but capable of utilizing acetate, were derived. A mutant having onethirtieth of the ability of the parent strain to degrade biotin was cultivated on a medium containing 0.5%n-paraffin, 0.02% urea, 0.1% corn steep liquor, and 1% sodium acetate, and 0.8 mg/ml of dl-TOPTI was added after 15 hours cultivation. Under this condition, 0.65 mg/ml of dl-biotin was synthesized, i.e., maximum conversion increased to 80.5%. Ogino et al. (1974b) suggested that the enzyme system involved in the transformation might be inducible, from the fact that all the mutants obtained oxidized dt-TOPTI only when grown on a medium containing n-paraffin but not when grown on a medium containing acetate. In this manner, the unique method of Ogino et al. is characterized by microbial production of biotin with the use of a chemically synthesized substrate. 2.

DTB and Other Biotin Vitamers

Schopfer (1943) reported that the maximal yield of total biotin (referred to as biotin vitamers assayed with Saccharomyces cerevisiae) was about 0.020.03 pg/ml of the culture medium ofPhycumyces blakesleeanus after 7 days of growth. Eisenberg (1963), using the same mold, demonstrated that the addition of pimelic acid and the aeration of the culture increased the biotin yield 10- to 12-fold, with the average yield varying between 0.35 and 0.60 pglml of culture medium after 10-14 days of growth. On screening about 700 microorganisms, Ogata et al. (1965a) found that large amount of biotin vitamers were accumulated by Streptomyces, molds, and bacteria, in a medium with pimelic acid or azelaic acid. The main com-

162

YOSHIKAZU IZUMI A N D KOlCHl OGATA

ponent (60-95%) of the vitamers accumulated by these microorganisms was identified as DTB by anion exchange column chromatography, paper chromatography, and chemical analysis (Ogata et al., 196513).In particular, Bacillus sphaericus accumulated the vitamers in large amounts (150-200 pg/ml), and the dominant component was DTB (Iwahara et al., 1966a). Optimization experiments for maximum production of DTB by the bacterium indicated that peptone (1%)-soybean meal (10%) mixture as nitrogen sources was most effective. When the bacterium was cultivated on a medium containing this mixture and 0.5% casamino acid as nitrogen source, 2% glycerol as carbon source, and 1mg/ml of pimelic acid, the maximum amount of total biotin was about 150 pg/ml after 5 days of growth. In contrast, the accumulation of true biotin (referred to as biotin vitamers assayed with Lactobacillus plantarum) was less than 0.5 pg/ml under the cultural conditions tested. The DTB accumulated in the culture filtrate was isolated by active carbon treatment, and Dowex 1-f formate column chromatography, and crystallized from hot water. As a result, 30 mg of pure crystalline DTB was obtained from 3 liters of the culture medium containing 3 gm of pimelic acid (Iwahara et al., 1966a; Ogata, 1970a). Iwahara et al. (1967) also found that a large amount (20 pg/ml) of avidinuncombinable biotin vitamer was accumulated by a soil isolate, Bacillus cereus, and they identified it as KAPA. Subsequently, Eisenberg and Maseda (1970) obtained about 50 mg of KAPA in crystalline form from 100 gallons of culture medium of Penicillium chrysogenum. Tsuboi et al. (1966a) have screened many hydrocarbon-utilizing bacteria which accumulate biotin vitamers in hydrocarbon medium without exogenous precursors. Of more than 600 isolated strains, 35 strains accumulated over 100 ng/ml of biotin vitamers. A strain 0fPseudornona.s sp. which showed good assimilation of kerosine accumulated large amount of biotin vitamers, mainly DTB. Of various n-alkanes, n-alkenes, and glucose tested as carbon source, n-undecane a o r d e d the highest accumulation of biotin vitamers by this bacterium, and at the same time pimelic acid and azelaic acid were accumulated in the undecane culture broth (Tsuboi et al., 1966b). From this fact and the result that these two acids promoted accumulation of biotin vitamers, they assumed that n-undecane (Cll) might be converted to biotin vitamers via pimelic acid (C,) and azelaic acid (C,) through diterminal oxidation and P-oxidation mechanisms. This bacterium accumulated about 20 pg/ml of total biotin from kerosine in the presence of adenine (Tsuboi et al., 1967). This action of adenine on the accumulation of biotin vitamers is described in Section V,E. Yang et al. (1971b) found that various bacteria and yeasts could convert DAPA to other biotin vitamers during cultivation. I n particular, Pseudomonas graveolens and Saccharomyces marxianus accumulated large

MICROBIAL PRODUCTION OF BIOTIN

163

amounts of biotin vitamers, which responded to B . subtilis, from DAPA added to the medium. Bioautograms of culture filtrates during cultivation revealed that DAPA was first converted into DTB and that the DTB thus formed was converted into bisnordethiobiotin on longer incubation. Pseudomonas graveolens, which can convert DAPA to biotin vitamers in 60% yield under optimal conditions, was cultured in 15 liters of medium containing 1.5 gm of DAPA. After 6 days of cultivation, the biotin vitamers formed in the culture filtrate were purified by Dowex 1-=-formate column chromatography. As a result, 295 mg of d-DTB in crystalline form, together with bisnordethiobiotin in powder form, were obtained. In investigating fatty acid metabolism by microorganisms, Ohsugi et al. found that some strains which were able to utilize oleic acid (Ohsugi et al., 1972a; Ohsugi and Ishikawa, 1975), salicylic acid (Ohsugi et al., 1972b), or pelargonic acid (Ohsugi and Baba, 1975) as sole carbon source formed biotin vitamers in the culture broth, and the amounts of the vitamers accumulated were some micrograms per milliliter in each case. Ogata et al. (1966) have reported the isolation of bacterial strains that accumulate pimelic acid from azelaic acid. They obtained several strains of bacteria which showed heavy growth in a medium containing azelaic acid or acetic acid as a sole carbon source, but feeble growth in a medium containing pimelic acid. These strains accumulated pimelic acid in good yield from azelaic acid. Among these strains, a bacterium identified as Micrococcus sp. accumulated about 7.5 gmAiter of pimelic acid from 10 gmniter of azelaic acid in the culture filtrate after 22 hours growth.

V. Biotin Antimetabolites and Their Actions on Biotin Biosynthesis In fermentative production of amino acids, nucleotides, and nucleosides, methods using modified regulatory mutants that are insensitive to end product inhibition or to end product repression have been intensively developed in recent years (Abe, 1972; Ogata, 1975). In isolating regulatory mutants, strains resistant to amino acid and nucleic base analogs are derived, as in, e.g., L-threonine production by an a-amino-P-hydroxyvaleric acid-resistant mutant of Brevibactaium jZuvum (Shiio and Nakamori, 1970), L-histidine production by a thiazolealanine resistant mutant of Corynebacterium glutamicus (Araki et al., 1974), and inosine production by an 8azaguanine-resistant mutant of Bacillus sp. (Nogami et al., 1968). Since biotin biosynthesis is controlled via strong feedback repression by biotin, as described in Section 111, this control must be overcome in order to microbially produce large quantities of biotin and its vitamers. Some potent biotin antimetabolites have already been found. Therefore, it has become

164

YOSHIKAZU IZUMI A N D KOICHI OGATA

possible to use these antimetabolites for the derivation of regulatory mutants producing biotin and its vitamers.

A. ACTITHIAZIC ACID Actithiazic acid, or acidomycin (ACM), is an antibiotic that was independently isolated from the culture filtrates of Streptomyces sp. (Sobin, 1952), S. uirginiae (Grundy et al., 1952), S. lavendulae (Tejera et al., 1952), S . cinnumonensis (Maeda et al., 1952; Umezawa et al., 1953), and S. acidomyceticus (Ogata and Igarashi, 1954). The chemical structure of ACM was established as 4-thiazolidone-2-caproicacid (McLamore et al., 1952; Schenck and DeRose, 1952; Miyake et al., 1953). Its chemical synthesis has also been performed (Clark and Schenck, 1952). This antibiotic is most active against mycobacteria (Tejera et al., 1952; Grundy et al., 1952; Maeda et al., 1952). M . phlei was inhibited at 2.5 bglrnl and M. tuberculosis at 0.0625-0.125 pg/ml (Grundy et al., 1952). However, it exhibited no activity against tuberculosis in uivo (Sobin, 1952; Umezawa et al., 1953). The in vitro antibiotic activity was completely lost on addition of biotin to the test medium. Consequently, it was thought that the lack of activity in uivo might be due to the presence of biotin in the tissue. This loss of activity of ACM at concentrations of from 0.25 to lo00 pg/ml was accomplished by the addition of 0.064 p g ofbiotin per milliliter with M. tuberculosis (Grundy et al., 1952). Hamada et al. (1953)demonstrated that the competition ratio is 4 x lop4by the cylinder plate method using M. tuberculosis as test organism. Miyake (1953) studied the relationship between antitubercular activity and chemical structures of various synthetic compounds related to ACM. This results may be summarized as follows: (1)the side chain attached to the 2-position of the thiazolidone ring must contain a carboxyl or a group derivable from carbonyl for expression of antitubercular activity; the alcohol derivative has some antitubercular activity, but when the -CH,OH group is reduced to a methyl group, the activity disappears; (3)the side chain must be a straight pentamethylene chain for antitubercular activity; (4) the side chain is not the sole determinant of the activity; (5) modifications at the 2- and 3-positions of the thiazolidone ring cause disappearance of the activity; (6) a carbonyl group at the 4-position and a bivalent sulfur at the 1-position are essential. Subsequently, the antagonistic activity of biotin to the antitubercular activity of the active ACM derivatives shown in Fig. 9 was examined (Kawashima et al., 1956). These compounds inhibited the growth of aviantype tubercle bacilli at 0.32 to 1.6pg/ml. The inhibition caused by 10pg/ml of these compounds was lost on addition of 3.2 ng/ml of biotin in all cases.

MICROBIAL PRODUCTION OF BIOTIN

(I)

li'

(11)

(111)

165

R=COOH : Actithiazic acid RzCH20H R=COOCH3

FIG. 9. Actithiazic acid and its derivatives having antibiotin activity. From Kawashima et al. (1956).

Ogata et al. (1970, 1973a) found that the accumulation of biotin vitamers affected by addition of ACM to the culture medium, and investigated the action of ACM in relation to the pathway of biosynthesis of biotin vitamers in microorganisms. When ACM was added to the medium, the growth of most yeasts, molds, bacteria, and actinomycetes was not inhibited at all, but the amounts of DTB accumulated in the medium on incubation with pimelic acid were remarkably enhanced. Conversely, the amount of biotin formed when ACM was added dropped considerably. In the case of Bacillus sphaericus, the amount of DTB formed increased about five-fold by addition of 200 pglml of ACM, and reached a maximum of 350 pg/ml as shown in Fig. 10. As a result of investigation of the action of ACM on the biosynthesis of biotin (Ogata et al., 1973a), it is thought that ACM is not incorporated into the DTB molecule but that, because it suppresses formation of biotin by W ~ S remarkably

ACM added (pglrnl) FIG. 10. Effect of actithiazic acid (ACM) on the accumulation of biotin vitamers by Bacillus sphawicus. From Ogata et al. (1973a).

166

YOSHIKAZU IZUMI A N D KOICHI OGATA

inhibition of a part of the biosynthetic system of biotin from DTB, it releases the repression by biotin of the biosynthetic system of DTB from pimelic acid. Eisenberg (1973)also investigated the action of ACM using resting cells of E. coli and confirmed that ACM showed competitive inhibition of the biosynthetic system of biotin from DTB. Further, he found the enzyme activity of both DAPA aminotransferase and DTB synthetase to be considerably higher from cells cultured in ACM-supplemented medium.

B. a-DEHYDROBIOTIN a-Dehydrobiotin (aDHB) is an antibiotic produced by Streptomyces lydicus (Hanka et al., 1966, 1969). The antibiotic is active against a variety of gram-positive and gram-negative bacteria and fungi, such as Staphylococcus aureus, Sarcina lutea, Streptococcus pyogenes, E . coh, Proteus vulgaris, Salmonella pullorum, Candida albicans, Saccharomyces cerevisiae, and Penicillium oxalicum (Hanka et al., 1966). Minimum inhibitory concentration (MIC) of aDHB is 0.78 pg/ml for E. coZi (Pai, 1975).This antibiotic has a double bond conjugated with a carboxyl group (Hanka et al., 1966)(Fig. 11). The amount of aDHB produced in the culture increased on addition of biotin. The conversion of [14C]biotinto [14C]aDHBhas also been demonstrated using a growing culture of Streptomyces lydicus. These results suggest that aDHB is a product of biotin catabolism in S. lydicus (Hanka et aZ., 1969). More recently, the synthesis of aDHB has been established with the cyclic sulfonium salt [compound (XI) in Fig. 61 as starting material (Field et al., 1970, 1976). The antibacterial properties of aDHB are lost in the presence of biotin in synthetic media (Hanka et al., 1966). Eisenberg (1975) investigated the action of aDHB on the synthesis of the biotin biosynthetic enzymes in E. coli. Repression of the synthesis of both the DAPA aminotransferase and DTB synthetase was observed with increasing concentrations of aDHB, reaching a maximum between 10 and 80 ng/ml. However, only 73 and 81%repression was attained with aDHB for the aminotransferase and DTB synthetase, respectively, while repression of the synthesis of the two enzymes was essentially complete with 5 ng/ml of biotin under the same conditions.

A

N

N H

CH~-CH~-F=C-COOH H

FIG. 11. Structure of a-dehydrobiotin. From Hanka et al. (1966).

MICROBIAL PRODUCTION OF BIOTIN

167

Pai (1975) has isolated aDHB-resistant mutants from strains ofE. coli and classified them into two groups: dhb A and dhb B . The dhb B mutants overproduced biotin, and the levels of their biotin biosynthetic enzymes were elevated in comparison with those of the parent strains. One of the mutants accumulated 25 times as much extracellular biotin and 38 times as much intracellular biotin, and the specific activity of the mutant's DTB synthetase was 11times as high. Furthermore the biotin biosynthetic enzymes of dhb B mutants were not represented by 10 ng/ml of biotin. In the dhb A mutants, biotin biosynthetic activity was normal in that the amount of biotin and its precursors excreted in the culture medium and the levels of the biosynthetic enzymes were similar to the wild-type strain, and that the enzyme was subject to repression by biotin. From the findings that the ability of the mutant to take up biotin into the cell was reduced significantly and that aDHB, a competitive inhibitor of biotin uptake, was much less inhibitory to biotin uptake in the mutants than in the wild strain, dhb A mutants were suggested to be deficient in aDHB transport. Eisenberg et al. (1975) have also isolated four classes of aDHB resistant mutants of E. coli. One mutant group showed enhanced excretion levels of biotin vitamers (73 to 110 ng/ml of total biotin and 40 to 88 ng/ml of true biotin, compared with 4 ng/ml and 0 ng/ml, respectively, in the parent strains), derepressed levels of the biotin biosynthetic enzymes, and resistance to repression by biotin. A second class of mutants showed derepressed levels of the DTB synthetase enzyme. The other two mutant groups showed alterations in permeability; biotin uptake was also affected and growth on minimal media was poor, suggesting a generalized permeability defect.

c. a-METHYLBIOTINAND a-METHYLDETHIOBIOTIN During the isolation of aDHB produced by S. Zydicus, Hanka et al. (1972) isolated two more biotin antimetabolites from the fermentation liquors of this microorganisms: a-methylbiotin (aMB) and a-methyldethiobiotin (aMDB) (Fig. 12). [14C]Biotinand [14C]pimelicacid were not incorporated

aMDB

aMB

FIG. 12. Structure of a-methyldethiobiotin (aMDB) and a-methylbiotin (aMB). From Hanka et al. (1972).

168

YOSHIKAZU IZUMI A N D KOICHI OGATA

into these metabolites by the growing culture, and neither metabolite could satisfy the biotin requirement in Saccharomyces cerevisiae. Both compounds had strong antimicrobial activity against mycobacteria (MIC of aMDB, 0.23.12 pg/ml; MIC of aMB, 1.25-200 pg/ml). Furthermore, aMDB inhibited E . coli (MIC, 8 pg/ml) and B . subtilis (MIC, less than 0.5 pg/ml) cultivated in synthetic media. The antibacterial activities of both aMDB and aMB were reversed strongly by biotin. DTB also reversed the former, although less than biotin, but not the latter. Pimelic acid had no effect even at lo00 times the concentration of biotin or DTB. Further studies have not been carried out on the action of these antimetabolites on biotin biosynthesis. The chemical synthesis of racemic aMB was achieved with racemic N-blocked thiophanium salt [compound (XI) in Fig. 61 as a starting material (Martin et al., 1971). D. AMICLENOMYCINAND STELAVIDIN Amiclenomycin (AM) is produced by Streptomyces lavendulae (Okami et al., 1974). Its chemical structure was identified as ~-2-amino-4-(4'-amino2',5'-cyclohexadienyl)butyric acid (Fig. 13).The antibiotic inhibits growth .of mycobacteria including various resistant strains and tubercle bacilli (MIC, 3.1-6.25 pglml), but not other bacteria and fungi. The MIC value was significantly reduced when AM and ACM were added together: growth was completely suppresed even at one-eighth of their MIC (Kitahara et al., 1975). The action of AM alone or in combination with ACM was reversed by biotin at a concentration of 0.01 pg/ml, but not at 0.001 pglml. This reversal was also observed with DTB (0.1 pg/ml) and DAPA (1 pg/ml), but not with KAPA, pimelic acid, and other compounds involved in biotin biosynthesis. Furthermore, when Mycobacterium smegmutis was cultured in a medium containing pimelic acid and AM (2-6 pg/ml), significant inhibition of growth coupled with an appreciable increase of KAPA accumulation was observed. On the other hand, DTB was detected in the medium without AM, but not in that containing AM. This was also confirmed with B . sphaericus. From

4

HzaCHrCHrr-XlOH AM

/C-c,Hz HzN-C\H CHrCHrCHrCHrCOOH CHa KAPA

FIG. 13. Structure of amiclenomycin (AM). From Okami et al. (1974)

MICROBIAL PRODUCTION OF BIOTIN

169

FIG. 14. Structure of stravidin. From Baggaley et al. (1969).

these results, AM was thought to be a strong inhibitor of the DAPA aminotransferas e reaction. Enzymic investigation was carried out on the action of AM on biotin biosynthesis (Hotta et al., 1975). As expected, the DAPA aminotransferase from B . divaricatum was inactivated by AM, while AM exhibited no significant activity against the DTB synthetase from P . gruveoZens. In the aminotransferase reaction, the activity was reduced remarkably even when AM was added at a concentration of one-tenth of that of KAPA as substrate. Preincubation of the enzyme with AM resulted in the abrupt inactivation of the enzyme. However, on dialysis 46.1%of the enzyme activity was recovered from the inactivated form. Moreover, inhibition by AM decreased in proportion to the amount of KAPA, while increased amounts of SAM exhibited a tendency to enhance inhibition by AM of DAPA aminotransferase. Thus, AM was considered to exert its inhibitory action against DAPA aminotransferase by coupling with the KAPA binding site of the enzyme. It was also revealed that AM derivatives such as aromatized AM and y-phenylbutyrine had no antimicrobial activity. Stravidin, an antibiotic produced by Streptomyces avidinii, has a chemical residue similar to structure containing a 4-alkylcyclohexa-2,5-dienylamine AM (Fig. 14) (Baggaley et al., 1969). This antibiotic also inhibited biotin synthesis by susceptible organisms (Stapely et al., 1963; Chaiet et al., 1963; Miller, 1964). Although the action mechanism of this antibiotic has not been studied, it may exert an inhibitory action on DAPA aminotransferase similar to that of AM, judging from its structural resemblance to KAPA.

E. ADENINE Iwahara and Yoshikawa (1974)found that the synthesis of biotin from DTB by a biotin-forming bacterium was greatly accelerated by the addition of adenine sulfate to the culture medium. They investigated the action of adenine on biotin biosynthesis. The growth of the bacterium was strongly inhibited when adenine or adenosine was added to the medium, while other purine and pyrimidine bases and nucleosides had no effect on the growth. In order to reduce the growth inhibition by adenine sulfate (4 mg/ml), it was necessary to add biotin as well as thiamine and a purine or pyrimidine base or nucleoside, such as uracil, uridine, cytidine, guanine, guanosine, or

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YOSHIKAZU IZUMI A N D KOICHI OGATA

xanthine. Adenine added to the culture medium was partly converted to adenosine and hypoxanthine during the culture, and finally completely converted to hypoxanthine (Iwahara and Kanemaru, 1974). While adenine remained in the medium, a large amount of DTB (about40 pg/ml) was accumulated from pimelic acid, but biotin was not accumulated. After adenine had disappeared from the medium, the accumulation of biotin rapidly increased with culture time to reach about 400 ng/ml. Furthermore, Iwahara and Kanemaru (1975) investigated the mechanism of the function of adenine using intact cells. The biosynthesis of biotin from DTB by intact cells harvested from adenine-supplemented medium or from adenine-unsupplemented medium was strongly inhibited by the addition of adenine or adenosine. However, the intact cells harvested from adeninesupplemented medium had greater capacity to synthesize biotin from DTB than those harvested from adenine-unsupplemented medium. From these results, they suggested that adenine inhibits the biosynthesis of biotin from DTB, and that repression of biotin biosynthesis by biotin is released during the inhibition by adenine, resulting in the large accumulation of DTB. Subsequent disappearance of adenine in the late stage of culture results in normal biotin biosynthesis from DTB and in large accumulation of biotin. Tsuboi et al. (1967) have also found the accelerating effect of adenine on DTB accumulation by a hydrocarbon-utilizingPseudomonus.

VI. Biodegradation of Biotin and its Vitamers Brady et al. (1966)isolated a pseudomonad which grew on biotin as its sole carbon, nitrogen, and sulfur source and observed the total degradation of carbonyl-[l4C1biotinand ~arboxyl-['~C]biotin to 14C02by the cells and by a particulate preparation of this organism. In the particulate system, this degradation was promoted by addition of ATP, M g + , NAD, and CoA. Biotinrelated substances having a labeled carbonyl carbon atom were also degraded in the order biotin d-sulfoxide, most readily degraded, biotin, and biotin l-sulfoxide. Biotin sulfone, and oxybiotin were hardly degraded to C02. Those having a labeled carboxyl atom showed the same order of ease of degradation. Iwahara et al. (1966d)found that a large number of molds degrade DTB to form a product inactive toward S. cerevisiae, but having biotin activity toward B. subtilis. Next, they isolated the degradation product in crystalline form from the medium of Aspergillus oryzae, the organism showing the strongest degradation, and identified it as bisnordethiobiotin, a compound with two less carbon atoms in its side chain than DTB. Li et al. (1968a) also found that carbonyl-[14C]DTBwas degraded by A. niger; the degradation products were isolated in crystalline form and identified as bisnor-

171

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dethiobiotin and tetranordethiobiotin, a compound with side chain shorter by a further two carbon atoms. These results suggest that, as shown in Fig. 15, the degradation of DTB proceeds by P-oxidation. Yang et al. (1969b) tested a large number of yeasts and fungi, and found that several strains of genera Endomycopsis, Rhodotorula, and Penicillium showed strong degradation of biotin. Biotin was converted to compounds inactive toward Lactobacillus plantarum and S . cerevisiae and active toward B . subtilis. This conversion reached a maximum of 95%. Yang et al. (1969b) found by bioautography using B . subtilis that biotin d-sulfoxide and two unknown substances were formed from biotin. These were isolated in crystalline form by column chromatography (Yang et al., 1968, 1970% Ogata, 1970b). From physicochemical analyses, the two unknown compounds were identified as bisnorbiotin and bisnorbiotin sulfoxide, compounds having side chains shorter by two carbon atoms than biotin and biotin sulfoxide, respectively. Since biotin sulfoxide was not degraded by Endomycopsis and remained unchanged in the culture medium, biosnorbiotin sulfoxide is thought to be formed from bisnorbiotin but not from biotin sulfoxide. Ruis et al. (1968) examined the degradation of homobiotin and norbiotin labeled with I4C at the carbonyl or carboxyl group using a particulate preparation of a Pseudomonas sp. These compounds also underwent p-oxidation losing two carbon atoms each from the side chain; the degradation proceeds: homobiotin + norbiotin + trisnorbiotin. Similarly, Iwahara et al. (1968)also incubated a Pseudomonas sp. in medium supplemented with carbonyl[14C]biotin, and isolated radioactive bisnorbiotin, a-dehydrobisnorbiotin, tetranorbiotin, urea, and uracil from the culture filtrate. From these facts, they proposed that after two p-oxidations of the side chain of biotin (biotin + bisnorbiotin + a-dehydrobisnorbiotin + tetranorbiotin), urea is produced by degradation of the thiophane ring, and COz, and NH3 are finally produced by the action of urease. They considered that uracil is produced either from C 0 2 and NH3, or from further ring closure of a degradation product having a ureido fragment. Roth et al. (1970) and Im et al. (1970), using the same Pseudomonas sp., studied the degradation of ~arbonyl-[~~C]biotin I-sulfoxide and biotin d-sulfoxide, respectively. An unknown metabolite was isolated from the day 16 culture with biotin 1-sulfoxide and identified as

H3k

L(CHzbCOOH

Dethiobiotin

H3k

L(CH2)zCOOH

Bknordethiobiotin

&

LCOOH

Tetranordethobiotin

FIG. 15. Degradation of dethiobiotin by microorganisms.

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Biotin

Bisnorbiotin

Tet ranorbiotin

FIG. 16. Degradation of biotin by microorganisms.

P-hydroxybiotin 1-sulfoxide (Roth et al., 1970). Several metabolites were isolated from culture filtrates of the pseudomonad grown for 9 days on a medium containing biotin d -sulfoxide and identified as biotin, bisnorbiotin, tetranorbiotin, biotin 2-sulfoxide, biotin sulfone, and a new catabolite, bisnorbiotin sulfone, after purification (Im et al., 1970). Christner et al. (1964) observed the degradation of ~arboxyl-[~~C]biotin to 14C02by cell extracts of an isolated Pseudomonas sp. which grew on biotin as a sole carbon source, and by cell extracts of mammalian tissue. They found the presence of a biotin-activating enzyme (biotinyl-CoA synthetase), which is thought to participate in the first degradation reaction of biotin via p-oxidation. This enzyme forms biotinyl adenylate from biotin and ATP in the presence of M$+, and further forms biotinyl-CoA from biotinyl adenylate and CoA. All these results support thoroughly the presence of P-oxidation for the side chain of biotin in microorganisms as shown in Fig. 16.

VII. Conclusion Almost the complete picture of the enzyme system participating in the biosynthetic pathway from pimelic acid to DTB has been built up. Also the degradation of biotin has been elucidated. One of the most important tasks for future studies in biotin metabolism will be the elucidation of the enzymic system of biotin biosynthesis from DTB and its regulation mechanism. This elucidation may provide a clue to the problem of accumulating large quantities of biotin. In order to establish biotin fermentation, considerable efforts will have to be made to alter the strong regulation mechanism in biotin biosynthesis by mutation or by other means. Several effective biotin antimetabolites have been found and some mutants resistant to those antimetabolites show greater ability to synthesize biotin and its vitamers, although the amounts found are still very small. Therefore, further induction of such resistant mutants may make possible the microbial production of biotin and its vitamers. For this purpose, these antimetabolites have to be prepared effec-

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173

tively in large amounts, or other active antimetabolites which can be easily prepared have to be sought. One further means that might be effective for accumulating large quantities of biotin by alteration of the regulation mechanism is the induction of revertants from biotin auxotrophic mutants, similar to the revertants that can accumulate considerable amounts of amino acids. ACKNOWLEDGMENT

We are deeply indebted to Associate Professor Y. Tani of our laboratory for his helpful advice and encouragement during the course of this work and in the preparation of this manuscript.

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