BIOTIN BIOSYNTHESIS

I. BiOTIN YIELDS AND BIOTIN VITAMERS IN CULTURES OF PHYCOMYCES BLAKESLEEANUS

M. A. EISENBERGDepartment of Biochemistry, College of Physicians and Surgeons, Columbia University,

New York, New York

Received for publication 24 April 1963

ABSTRACTEISENBERG, M. A. (Columbia University,

New York, N.Y.). Biotin biosynthesis. I. Biotinyields and biotin vitamers in cultures of Phy-comyces blakesleeanus. J. Bacteriol. 86:673-680.1963.-The addition of pimelic acid to a well-aerated medium resulted in a 10- to 12-foldincrease in the biotin production of Phycomycesblakesleeanus. Azelaic acid also stimulated biotinproduction, but not to the same extent as didpimelic acid. A number of biotin analogues werefound to be inactive. Further enhancement of thebiotin yield could not be attained by replacingglucose and aspargine by other carbon and ni-trogen sources. Replacement cultures, however,proved to be equally as effective as growingcultures under the same conditions. The omissionof trace elements reduced the growth and biotinproduction. The "true" biotin was affected to agreater extent than the "total" biotin. Zinc andiron proved to be the essential trace metals. Inthe absence of zinc, both the growth and thetotal biotin production were markedly reduced.The omission of iron affected primarily the biotinproduction. P. blakesleeanus produces biotin,desthiobiotin, biotin-d-sulfoxide, biocytin, and anunknown biotin vitamer. The latter has beenidentified as an amino acid by electrophoreticanalysis. It is avidin-uncombinable, and doesnot support the growth of Lactobacillus arabinosus(L. plantarum) or Neurospora crassa.

Biotin is synthesized by a variety of microor-ganisms (Landy and Dicken, 1941), but in ex-ceedingly small quantities as compared withmost vitamins. Schopfer (1943), in his extensivestudy of biotin biosynthesis by the mold, Phy-comyces blakesleeanus, indicated this organism tobe one of the best sources for this vitamin. It wasfound that over 90% of the total biotin pro-duced by P. blakesleeanus appeared in the me-

dium. This extensive excretion of the vitaminoccurred after maximal growth had been attained.The biotin yield was markedly influenced by theconcentration of asparagine and vitamin B1 inthe medium, as well as by the addition of tracemetals.

Other investigators, in a series of studies withother microorganisms, demonstrated that the ad-dition of pimelic acid to the culture mediumenhances the yield of biotin and its vitamers. Asa result of these studies and of other observations,it has been postulated that pimelic acid is an in-termediate in biotin biosynthesis (Eakin andEakin, 1942; du Vigneaud, et al., 1942; Tatum,1945; Wright and Cresson, 1954). Recent evi-dence from isotopic studies suggests a directparticipation of pimelic acid in the synthesis ofbiotin and its vitamers (Eisenberg, 1962; Elsfordand Wright, 1962). Other compounds which havebeen shown to stimulate biotin production areazelaic and lipoic acids (Wright, Cresson, andDriscoll, 1955; Elsford and Wright, 1962). Thelatter compound exerts its effect only in thepresence of pimelic acid.

This report is part of a study on the mecha-nism of biotin biosynthesis. Since low biotinyields impose certain limitations on a study ofthis nature, the present investigation was under-taken to determine the optimal conditions formaximal biotin production with P. blakesleeanus.In addition, it was necessary to ascertain thechemical nature of the biotin vitamers producedby this organism.

MATERIALS AND METHODSCulture conditions. P. blakesleeanus (ATCC

6200) was maintained on Sabouraud agar slants(Difco). Spore suspensions were prepared fromcultures 2 to 3 weeks old. Sterile water was addedto the slant, and the spores were shaken loosewith a sterile inoculating needle. The suspensionwas centrifuged, and the spores were washed

673

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

EISENBERG

twice with distilled water. They were then acti-vated by placing the suspension in a water bathat 50 C for 15 min. A 0.5-ml amount of the sus-pension was utilized to inoculate 100 ml of me-dium of the following composition: glucose, 3.0g; L-asparagine, 150 mg; KH2PO4, 150 mg;MgSO4 7H20, 50 mg; thiamine hydrochloride,10 pug. The medium was prepared with warm tapwater or with distilled water to which was addedthe trace element mixture of either Hammer,Stewart, and Matrone (1943) or Schopfer (1943).The growth procedure, unless otherwise indi-cated, consisted of permitting the inoculatedcultures to remain at room temperature for 1 to2 days to permit sporulation. The culture wasthen placed in a shaker, kept in an incubator at30 C, and shaken at 70 to 120 rev/min.

Assays. The term "total" biotin as used in thistext encompasses the entire spectrum of biotinvitamers utilized by Saccharomyces cerevsae(ATCC 9896 or 7754) for growth. This includesbiotin, desthiobiotin, biocytin, biotin-d- andI-sulfoxides, diaminobiotin, diaminopelargonicacid, and a number of unknown vitamers. Theassay was carried out according to the methodof Hertz (1943). Neurospora crassa 1A was alsoutilized for "total" biotin determinations, butthe results were consistently lower than thoseobtained with the yeast assay. This strain waskindly furnished by Francis Ryan, and the assaywas performed according to the procedure ofHorowitz and Beadle (1943). The term "true"biotin will refer to those vitamers capable ofsupporting the growth of Lactobacillus arabinosus(ATCC 8014) and includes biotin, and biotin d-and i-sulfoxides. The general assay procedure ofWright and Skeggs (1944) was utilized, exceptthat the biotin content was determined by tur-bidity measurements.

Chromatography and electrophoresis. Ascending

TABLE 1. Effect of aeration on biotin production*Biotin yield

Shaking condition (jag/50 ml)Total True

None ............................ 6.2 0.58Twice daily..................... 6.8 0.55Continuous, 120 rev/min......... 22.2 1.60

* Cultures were grown for 6 days at room tem-perature in presence of 1 mg of pimelic acid andthe trace-metal mixture of Hammer et al. (1943).

paper chromatography was carried out on What-man no. 1 filter paper with n-butanol-water-aceticacid (60:15 :25) as the developing solvent. Paper-strip electrophoresis was performed in a SpincoDurrum cell at 300 v for 3 hr. The following buf-fer solutions were utilized in a final concentrationof 0.025 M: citrate, pH 3.0; acetate, pH 4.0 and5.0; phthalate, pH 5.9; phosphate, pH 7.5; andglycine, pH 8.5 to 10.5. Combined chromatog-raphy and electrophoresis were carried out onWhatman no. 3 MM filter paper. The materialwas first chromatographed in the solvent mixture(n-butanol-water-acetic acid) until the solventfront reached a height of 26 to 27 cm. The paperwas dried at 80 to 90 C, turned 900, and placedinto a Spinco electrophoresis rack. The paperwas carefully moistened with citrate buffer (pH3.0), and electrophoresis was carried out for 3 hrat 300 v.The bioautographic technique was used to de-

velop the chromatograms and the electrophore-tograms with S. cerevisiae and L. arabinosus astest organisms. The paper was left in contactwith the agar surface during the entire incubationperiod. The RF values obtained by this procedurecompared favorably with those reported in theliterature.

Reagents. The lactobacillus assay medium was aDifco product. Avidin, ion-exchange resins, L-meso-a , e-diaminopimelic acid, and all vitaminswere purchased from the California ChemicalFoundation, Los Angeles, Calif. d-Biotin, dl-des-thiobiotin, and all amino acids were obtained fromMann Research Laboratories, New York, N. Y.Pimelic acid was a product of Eastman KodakCo., Rochester, N. Y., and a-aminopimelic acidwas obtained from Nutritional BiochemicalsCorp., Cleveland, Ohio. Biotin-d- and l-sulfoxideswere kindly furnished by D. Melville. r ,,-Diami-nopelargonic acid was prepared by H. Gillespie,and 2,5-diketopimelic acid was a gift from S. W.Tannenbaum.

RESULTS

Aeration. To determine the effect of the degreeof aeration on the synthetic capacity of P.blacesleeanus, the organism was grown in bothstationary and shaken cultures. The latter wereeither shaken twice daily (to prevent sporangiaformation) or continuously at 70 to 120 rotationsper min. The data in Table 1 show a threefoldincrease in the biotin production as the result of

J. BACTERIOL.674

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

BIOTIN PRODUCTION BY P. BLAKESLEEANUS

continuously shaking the culture mediiincrease was the same for both the totalbiotin as measured by the yeast and L. aassays, respectively. It is also evidenttrue biotin constitutes only a small percthe total biotin under these conditions. 9of the trace metals on true biotin yieldealt with in more detail later.

Effect of acids. Schopfer (1943) foundincrease in biotin production on raisingperature from 18 to 25 C. A similar incobserved in going from 18 to 30 C, 1pimelic acid was present in the mediumfold increase was obtained (Fig. 1). Thipimelic acid on biotin yield was much i

nounced under optimal conditions ofTable 2 shows that a 10- to 12-fold irbiotin yield was obtained with a concen0.4 mg of pimelic acid per 100 ml (

Azelaic, suberic, and oleic acids were albut only azelaic acid proved to be acpimelic acid analogues, a-aminopimelicdiaminopimelic acid, and 2,5-diketopinwere without effect. Lipoic acid gave vE

14

12-

I0

0

4

2

2 4

DAYSFIG. 1. Effect of temperature and pime

total biotin yield. The cultures were grepresence of the trace element mixture of ial. (1948) and were shaken twice dailywere removed for analysis at times indiccircles and triangles, no pimelic acid; cloand triangles, 1 mg of pimelic acid.

um. Thisand true;rabinosusthat theentage ofrhe effectLd will be

a twofold;the tem-

TABLE 2. Effect of pimelic acid concentration onbiotin production

Concn Total biotin

mg/100 ml pg/1O0 ml0.0 2.60.2 18.90.4 26.31.0 26.5

rease was sults, and the maximal increase obtained wasbut when about 25%., a three- Effect of carbon and nitrogen sources. Sucroses effect of and maltose were found to support the growth ofmore pro- P. blaakesleeanus to the same extent as glucose,f growth but produced no further increase in biotin.icrease in Glutamic and aspartic acids, alanine, and glycine,Ltration of which had been previously shown to support theof media. growth of this organism (Leonian and Lilly,[so tested, 1940), were substituted for asparagine. Only intive. The the glutamic and aspartic acid media were theacid, a ,f- biotin yields comparable with the asparagineaelic acid, medium, and then only after 10 to 11 days ofariable re- incubation. In the earlier stages of growth (4 to

6 days), the asparagine medium was far superior.As 30° C Utilizing a medium of Leonian and Lilly (1940),

in which ammonium nitrate and glucose are sup-plemented with various Krebs cycle interme-diates, it was found that fumaric and citric acidswere the most effective, but again, neither sur-passed asparagine for yield.The possible role of pimelic acid as an interme-

diary in biotin biosynthesis suggested as a work-ing hypothesis the initial condensation of pimelicacid with a three-carbon precursor such as cys-teine or serine. When the growth medium wassupplemented with cysteine, in a concentration of

,a 30°C 1 to 2 mg per 100 ml of media, there was no in-180 C crease in biotin production, and increasing the

concentration caused a marked inhibition ofSo 18° C growth. Cystine, however, in the lower concen-

trations gave about 25% increase in biotin yield,| | and serine in concentrations of 5 to 25 mg gave10 12 variable results.

Effect of trace elements. In a number of experi-ments in which tap water replaced the trace ele-

lic acid on ment mixture of Hammer et al. (1943), betterwnmmierth growth and higher true biotin yields were ob-. Samples served. Similar results were also obtained withated. Open Schopfer's (1943) trace element mixture. To de-'sed circles termine the effect of trace elements on the true

biotin yield, Schopfer's (1943) trace element mix-

675oVOL. 86, 1963

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

TABLE 3. Effect of trace elements on biotin production

Biotin producedMixture

Total True Dry wt True Total* True*

j.g/100 ml Jg/100 ml mg %Complete ................... 16.3 4.44 717 24.2 22.7 6.2-B ..................... 15.2 4.53 707 29.2 21.5 6.4-Fe ..................... 12.9 2.51 680 19.4 19.0 3.7-Cu ..................... 13.9 3.28 687 23.6 20.2 4.7-Mn ..................... 13.9 4.40 695 31.6 20.0 Ii.3-Zn.............. 3.7 0.73 307 19.8 12.1 2.4-Mo................... 11.2 3.06 650 27.4 17.2 4.7+ Tap water ............. 15.7 5.44 748 34.4 21.1 7.3

* Expressed as m,g per mg of dry weight.

ture was utilized, and, omitting one element at atime, the results shown in Table 3 were obtained.The greatest change occurred when zinc was

omitted. The growth was reduced to less thanone-half, and the biotin yield was reduced about80%. The omission of iron, although havinglittle effect on growth, produced a 30% decreasein total biotin and a 44% decrease in true biotin.The omission of copper, manganese, molyb-denum, or boron produced little or no change inthe biotin yield.Replacement culture. It was shown by Schopfer

(1943) that P. blakesleeanus excretes biotin intothe medium for a considerable period of timeafter maximal growth has been obtained. How-ever, the rate of synthesis is reduced to about

TABLE 4. Effect of pH on biotin production inreplacement cultures

Total biotin (pg/5O ml) at True biotinpH (sg/50 ml)

3 days 8 days at 8 days

4.1 10.1 21.0 5.65.0 6.9 20.5 5.36.0 7.8 19.3 4.97.0 10.4 26.2 4.4

TABLE 5. RF value of media components as comparedwith known biotin vitamers

RF values

CompoundYeast assay LactobacillusYeastassay arabinosus assay

Media ............. 0.51, 0.63, 0.56, 0.79d-Biotin.0.53, 0.76 0.56, 0.79Biotin-d-sulfoxide 0.56 0.55dl-Desthiobiotin. 0.84 Not utilized

one-half during this time interval. This observa-tion has been confirmed in the present study. Onthe other hand, if one utilizes the replacement ofculture technique, rates of biotin synthesis al-most equivalent to those obtained with normalculture procedures can be obtained. Table 4shows the effect of altering the pH of the re-placement medium on biotin production. It canbe seen that there is a synthesis of biotin during8 days over a pH range of 4 to 7, comparablewith that of a growing culture.

Biotin vitamers. When the culture medium,after 7 to 10 days of growth, was chromato-graphed, bioautography revealed several compo-nents with biotin activity. Table 5 shows the RFvalues of these components as compared withbiotin and known biotin vitamers. The compo-nent with an RF value of 0.84 actually appearedas a fusion of two components. This was verifiedby the fact that, when the same material wasassayed with L. arabinosus, this component wasabsent, but a component with the RF value ofbiotin appeared. Since L. arabinosus cannotutilize desthiobiotin for growth, it would appearthat the compound with an RF value of 0.84 con-sists of both biotin and desthiobiotin. The pres-ence of biotin-d-sulfoxide with the RF value of0.56 was confirmed by the use of both assayorganisms. The component with an RF value of0.63 does not correspond to any of the knownvitamers reported in the literature and does notsupport the growth of L. arabinosus. This vitamerhas appeared consistently in all experiments un-der a variety of conditions. When the medium isconcentrated and chromatographed, additionalcomponents are sometimes revealed, amongwhich is biocytin.

Paper-strip electrophoresis at pH 3.0 usually

EISENBERG J. BACTERIOL.676

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

BIOTIN PRODUCTION BY P. BLAKESLEEANUS

revealed two components, one moving 7.0 to 8.0cm toward the cathode, and a second remainingnear the origin. Since biotin, desthiobiotin, andbiotin-d-sulfoxide remain near the origin at pH3.0, it was thought that this fast-moving compo-nent represented the unknown vitamer (Rp 0.63).That this indeed was the case is shown by theresults in Fig. 2 obtained from combined chroma-tography and electrophoresis. Component 1 isagain a composite of two compounds, represent-ing biotin and desthiobiotin. Compound 2 cor-responds to biotin-d-sulfoxide, and component 3,which gives only a weak spot, corresponds tobiocytin. Component 4 is the unknown vitamernoted previously. In the presence of avidin, onlycomponent 4 is present. At pH 5.9, electrophore-sis again indicated two components; one remain-ing near the origin, and containing the unknown,the second, consisting of biotin, desthiobiotin, andbiotin sulfoxide, moving 4.0 to 4.5 cm toward theanode.When attempts were made to utilize this tech-

nique to obtain the unknown component inlarger yields for further study, it was discoveredthat more than one component was frequentlypresent, depending upon the amount of materialapplied to the paper strip. This was determinedby cutting out the area containing the fast-mov-ing component from a large number of electro-phoretic strips run at pH 3.0, eluting with water,and then concentrating to a small volume. Thismaterial, when chromatographed, revealed usu-ally from one to three components by bioautog-raphy. One of these components had the RFvalue of the unknown compound (RF 0.63 to0.67) and was present in the highest concentra-tion. The other components were tentativelyidentified as biocytin and desthiobiotin. Eluatesobtained from control paper strips, which wererun in citrate buffer without the addition of themedium, revealed a component with an RF valuesimilar to desthiobiotin.

Since the unknown compound could representan intermediate in synthesis of biotin, it wasfelt that the electrophoretic data might affordsome clue as to its chemical nature. Although itcould not be readily separated by electrophoresisfrom other vitamers, the fact that it was theonly vitamer which did not combine with avidinenabled a more accurate determination of itsmobility over a wide pH range. This was ac-complished by adding avidin to the bioauto-graphic medium (Fig. 3). The mobilities in this

FIG. 2. Bioautogram of growth media after chro-matography and electrophoresis. Culture grown un-

der optimal conditions for 9 days and medium con-

centrated fourfold.

figure were corrected for endosmosis with the aidof glucose. It can be seen that the unknown com-

pound is positively charged below pH 5.9, andnegatively charged above pH 9.0. The mobilityof biotin is given for comparison.

Further information about the biotin vitamerswas also gained from their behavior on ion-ex-change chromatography. When the media were

placed onto a Dowex 50 H+ column (1.8 by 20cm), the initial effluent and the water washshowed considerable biotin activity. Chromato-graphic analysis indicated the presence of biotin,desthiobiotin, and biotin-d-sulfoxide. The columnwas then developed with either 1.0 N HCl, or a

mixture of ethyl alcohol, water, and concentratedammonia (1:1.0:1), and the fractions containingbiotin activity were pooled. Analysis of this mix-ture by chromatography and bioautography re-

vealed three components, none of which sup-

ported the growth of L. arabinosus. Among thesecomponents were biocytin and the unknownvitamer. It was found the the initial effluent andwater wash from a Dowex-1-acetate column con-

SOLVENT FRONT

02

COMPOUND Rf MOBILITYjX'I 0.78 - 2.5

2 0.54 - 2.7

0 3 0.41 -3.5

4 0.64 -10.0

677VOL. 861 1963

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

EISENBERG

CATHODE10-9-8-7-6-5-4-

o -1 c-

-J-o 2-

3-4-5-6-7-8-9-10-

ANODEI I I I I I I

2 3 4 5 6 7 8 9

pHFIG. 3. Comparison of the electrophoretic mo-

bility of the unknown vitamer with d-biotin.

tained biocytin and the unknown vitamer, whilethe biotin, desthiobiotin, and biotin-d-sulfoxideremained on the column. These compounds couldbe eluted with 1.0 to 1.5 N acetic acid. Somefractionation was attained, as the biotin-d-sulf-oxide was eluted somewhat earlier than the othertwo components.

DiscussioNThe maximal biotin yields obtained by Schop-

fer (1943) from the culture medium after 7 daysof growth varied from 0.6 to 0.8 Ag in 25 ml ofmedia. In the present studies, the addition ofpimelic acid and the aeration of the culture were

found to increase the biotin yield 10- to 12-fold,with the average yield varying between 20 to25 ,g in 100 ml of media. After 10 to 14 days, thebiotin yield varied from 35 to 60 MAg per 100 ml of

media. Replacement culture technique showedthat the mycelial mat can synthesize biotin at arate comparable with that of the growing culture.This may prove of value in isotopic experiments.None of the pimelic acid analogues proved effec-tive in enhancing biotin biosynthesis, thus fur-ther supporting the hypothesis of a direct role ofpimelic acid in biotin synthesis.The present attempts to further enhance the

biotin yields by altering the carbon and nitrogensources were unsuccessful. The fact that aspara-gine cannot be surpassed as a nitrogen sourcemay indicate a more direct role for this aminoacid in biotin biosynthesis. The small increaseobserved with added cystine may be attributed toa change in the percentage of the various vitamersappearing in the medium. Since yeast cannotutilize biotin-d-sulfoxide to the same extent asbiotin (Melville, Genghof, and Lee, 1954), adecrease in the percentage of the former wouldappear as an increase in total biotin. A similarexplanation was offered by Wright et al. (1955)to account for the discrepancy between their re-sults and those obtained by Eakin and Eakin(1942). It was thought that the presence of areducing agent would decrease the amount ofbiotin-l-sulfoxide which is normally formed byAspergillus niger. However, no evidence was pre-sented to verify this supposition. Although noattempt was made in the present study to exam-ine the distribution of vitamers in the presence ofcysteine, an extensive investigation was carriedout with lipoic acid. A small increase in biotin hasbeen occasionally observed with 0.5 mg of lipoicacid per 100 ml. However, chromatographicanalysis of these cultures revealed the completeabsence of biotin-d-sulfoxide. This observationwould support the explanation (Wright et al.,1955) of the action of reducing agents in prevent-ing biotin oxidation, and would also suggestbiotin-d-sulfoxide to be a normal metabolic prod-uct.When the trace element mixture is omitted

from the culture medium, not only are growth andtotal biotin production reduced, but the quanti-tative distribution of the biotin vitamers, particu-larly true biotin, is also altered. The absence ofzinc had the greatest effect on both growth andbiotin yield, suggesting a general decreased syn-thetic capacity of the organism. A similar effectof zinc on the growth of P. blake8leeanus wasobserved by Qdegard (1952), and, more recently,

678 J. BAcTIRIOL.

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

BIOTIN PRODUCTION BY P. BLAKESLEEANUS

Wacker (1962) indicated an impairment in pro-tein synthesis in zinc-deficient Euglena gracilis.The omission of iron, although not materiallyreducing the growth of the organism, had a morepronounced effect on the true biotin, suggesting apossible role in the formation of biotin fromdesthiobiotin.

P. blakesleeanus has been found in the presentstudy to produce a number of compounds pos-sessing biotin activity. These compounds havebeen shown by a variety of techniques to consistof biotin, desthiobiotin, biotin-d-sulfoxide, bio-cytin, and an unknown vitamer. Quantitativeestimates of these various vitamers have beendifficult to attain because of the inability atpresent to obtain complete separation of all ofthe components by any of the procedures used.It would appear that desthiobiotin comprises thelargest portion of the total biotin activity, andbiotin the next largest. Biotin-d-sulfoxide con-stitutes about 10 to 15%, the unknown vitamer5 to 15%, and biocytin 1 to 2% of the totalactivity.The electrophoretic-mobility study of the un-

known compound revealed the presence of bothan acidic and a basic group. The pKa value forthese two groups has been estimated to be ap-proximately 4.5 to 5.0 and 9.2 to 9.5, respectively.These values correspond to a carboxyl and aminogroup, thus identifying this compound as anamino acid. The pKa of the carboxyl groupwould further suggest a separation of charges inthis compound. This is supported by the factthat the electrophoretic mobility of the unknownresembles f-alanine and e-aminocaproic acid,much more than a-alanine. It has also been ob-served that the unknown compound moves moreslowly toward the cathode at pH 3.0 than doesdiaminopelargonic acid, indicating the presenceof only one free amino group. This compounddoes not support the growth of L. arabinosus inconcentrations which will support the growth ofyeast. It is also avidin-uncombinable, indicatingthe absence of the ureido structure. Its activitywith N. crassa has been difficult to ascertain as ithas not been possible to free it completely fromother components. However, a purified samplecontaining biocytin as a contaminant was assayedfor total biotin with both yeast and N. crassa. Inaddition, the amount of avidin-uncombinablematerial was also determined with the yeastassay. It was found that the N. crassa values were

about one-half of the yeast values, and the biotin-uncombinable material constituted about one-halfthe total biotin activity. This would indicate thatthe unknown, in the concentrations used, is un-available to N. crassa for growth.A compound with the same RF value has also

been reported in the culture media of Escherichiacoli (Dhyse and Hertz, 1958) and Corynebacteriumxerosis (Genghof, 1956), and was shown to beavidin-uncombinable. A compound with similarproperties has also been found (unpublished ob-servation) in the medium of a biotin-requiringmutant of Penicillium chrysogenum. It appears tobe present in much higher concentrations thandesthiobiotin, which also accumulates. Work isnow in progress to try to isolate this compoundfrom P. chrysogenum culture media in sufficientquantities to permit chemical characterizationand study of its role in the biosynthesis of biotin.

ACKNOWLEDGMENTS

The author wishes to acknowledge the tech-nical help of Susan Schwartz, Helle Puupil, andMarian Kaufman. This work was carried outunder National Institutes of Health grant AM40056-03.

LITERATURE CIrTED

DHYSE, F. G., AND R. HERTZ. 1958. The effect ofactithiazic acid on egg white-induced biotindeficiency and upon the microbial formationof biotin vitamers in the rat. Arch. Biochem.Biophys. 74:7-16.

DUVIGNEAUD, V., K. DITTMER, E. HAGUE, AND B.LANG. 1942. The growth stimulating effect ofbiotin for the diphtheria bacillus in the ab-sence of pimelic acid. Science 96:186-187.

EAKIN, R. E., AND E. A. EAKIN. 1942. A biosyn-thesis of biotin. Science 96:187-188.

EISENBERG, M. A. 1962. The incorporation of 1-7-C14-pimelic acid into biotin vitamers. Bio-chem. Biophys. Res. Commun. 8:437-441.

ELSFORD, H. L., AND L. D. WRIGHT. 1962. Studieson the biosynthesis of biotin. FederationProc. 21:467.

GENGHOF, D. S. 1956. Production of vitamers ofbiotin by Corynebacterium xerosis. Arch. Bio-chem. Biophys. 62:63-69.

HAMMER, K. C., U. S. STEWART, AND G. MATRONE.1943. Thiamine determination by the fungus-growth method and its comparison with othermethods. Food Res. 8:444-451.

HERTZ, R. 1943. Modification of the yeast-growth

VOL. 86, 1963 679

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

EISENBERG J. BACTERIOL.

assay method for biotin. Proc. Soc. Exptl.Biol. Med. 52:15-17.

HOROWITZ, N. H., AND G. W. BEADLE. 1943. Amicrobiological method for the determinationof choline by use of a mutant of Neurospora.J. Biol. Chem. 150:325-333.

LANDY, M., AND D. M. DICKEN. 1941. Biotin syn-thesis by microorganisms. Proc. Soc. Exptl.Biol. Med. 46.449-452.

LEONIAN, L. H., AND V. G. LILLY. 1940. Studies onthe nutrition of fungi. IV. Factors influencingthe growth of some thiamine fungi. Am. J.Botany 27:18-26.

MELVILLE, D. B., D. S. GENGHOF, AND J. M. LEE.1954. Biological properties of biotin d- and 1-

sulfoxides. J. Biol. Chem. 208:503-512.0DEGARD, K. 1952. On the physiology of Phy-

comyces blakesleeanus Burgeff. I. Mineral re-

quirements on a glucose-asparagine medium.Physiol. Plantarum. 5583-607.

SCHOPFER, W. H. 1943. La biosynthese de la bio-tine par un microorganisme autotrophe pour

cette vitamine (Phycomyces blakesleeanus). Unnoveau facteur de croissance pour la levure.Z. Vitaminforsch. 14:42-70.

TATUM, E. 1945. Desthiobiotin in the biosynthesisof biotin. J. Biol. Chem. 160:455-459.

WACKER, W. E. C. 1962. Nucleic acids and metals.III. Changes in nucleic acid, protein, andmetal content as a consequence of zinc defi-ciency in Euglena gracilis. Biochemistry1:859-865.

WRIGHT, L. D., AND E. L. CRESSON. 1954. Biotinl-sulfoxide. I. The occurrence of a previouslyunrecognized form of biotin in certain fer-mentation sources. J. Am. Chem. Soc. 76:4156-4160.

WRIGHT, L. D., E. L. CRESSON, AND C. A. DRIS-COLL. 1955. Biological precursors of biotinand/or biotin I-sulfoxide. Proc. Soc. Exptl.Biol. Med. 89:234-236.

WRIGHT, L. D., AND H. R. SKEGGS. 1944. Deter-mination of biotin with Lactobacillus ara-

binosus. Proc. Soc. Exptl. Biol. Med. 56:95-98.

680

on Decem

ber 21, 2020 by guesthttp://jb.asm

.org/D

ownloaded from