The Formation of Pyruvic and Dimethylpyruvic Acids by Aspergillus niger

K. Ramachandran and T. K. Walker From the College of Technology, University of Manchester, Manchester, England

Received July 26, 1951

INTRODUCTION

In a recent communication (8) the authors gave an account of the production of pyruvic acid (PA) and of dimethylpyruvic acid (DMPA) by Aspergillus niger when this mold was allowed to act upon xylose in Hida's medium (5). It was shown also that higher yields of these acids could be obtained when glycerol was employed as the substrate in place of the xylose and that the addition of sodium acetate to the glycerol medium caused a further considerable enhancement of the yield of both keto acids.

In further experiments it has now been discovered that glycolic acid and ethylene glycol, respectively, when added to the glycerol medium, give rise to increased yields of PA and DMPA, though the enhance- ment is not so pronounced as that effected by sodium acetate. On the other hand, sodium propionate entirely prevented the formation of both keto acids, while glyoxylic acid, even in small proportions, pre- vented the formation of PA and depressed that of DMPA in the gly- cerol medium. The effect of added ethanol was to lower the yield of PA without affecting significantly that of DMPA. The addition of sodium pyruvate produced an unexpected result, for it reduced the yield of DMPA to a mere trace.

The effect of glycine was tested also, in order to ascertain if the two- carbon acid, formed on deamination of this amino acid, coul d partici- pate in the synthesis of the keto acids. However, the only effect observed was a slight decrease in the yield Of DMPA.

Since both PA and DMPA had been obtained by use of glucose, xylose, and glycerol as substrates, the action of the mold on erythritol in Hida's medium was next examined, and again both keto acids were

195

196 K. RAMACttANDRAN AND T. K. WALKER

obta ined , t hough in traces only. However, addi t ion of sodium acetate to the e ry thr i to l m e d i u m again served to raise the yield of bo th products .

I n commenc ing a s t u d y of the react ions b y means of which glucose is conver ted to PA and to D M P A unde r the condi t ions first described

by Hida, we have carried out a few exper iments to ascer ta in the respec-

t ive effects of inhib i tors known to affect par t icu la r enzymic changes.

Those used were sodium cyanide (0.02 M), sodium fluoride (0.003 M), and sodium iodoacetate (0.002 M). At these respect ive concen t ra t ions

the cyanide largely suppressed the fo rmat ion of D M P A a nd effectively s topped t h a t of PA, while ne i ther of the two other agents produced

a n y depressant effect.

EXPERIMENTAL

Particulars of the Mold and of the Cultural Conditions Employed

In all the experiments A. niger strain N 1 was used. In many previous investi- gations this mold, presented to one of us (T. K. W.) by Prof. C. Neuberg 25 years ago, has shown itself to be an efficient producer of citric acid and to be in other ways enzymically very active. It was grown on Currie's medium at 30~ as described in our previous communication on the formation of PA and of DMPA and all the present series of experiments were conducted precisely in the manner detailed in the former paper. The methods employed in isolating, estimating, and identifying the products were also the same as those formerly adopted by us (8).

Expt. 1. The Respective Effects of Ethylene Glycol and of Acetic, Glycolic, Glyoxylic and Propionic Acids on the Formation of Pyruvic

and Dimethylpyruvic Acids from Glycerol

Felts of the mold were developed at 30~ from spores sown on a sterilized medium consisting of glucose (10 g./100 ml.) in Currie's salt solution (3). This medium was distributed in lots of 125 ml. between 30 conical flasks. Each flask was of 450 ml. capacity and was approximately 3 cm. in width at the neck. After incubation for 3 days the medium in all the flasks was replaced by sterile tap water, and after a further interval of 12 hr., during which the flasks were kept at 30~ the water was replaced by Hida's salt medium to which had been added, in the different cases, glycerol alone (5 % w./v.) as control, and glycerol (5 % w./v.) together with the second organic substrate, the latter in quantity to conform to the molecular ratio shown in Table I. The respective weights of these other components per 100 ml. were: sodium acetate 3.7 g., ethylene glycol 0.85 g., glycolic acid 1.5 g., glyoxylic acid 0.4 g., and sodium propionate 1.75 g. Each lot of these different media was distributed between five flasks (125 ml./fiask) and incubation at 30~ was then continued for a further period of 9 days, at which time the analyses were performed. It will be noted that the glycolic acid and glyoxylic acid were introduced in the free state, since the mold can assimilate them in this condition when they are at the concentrations adopted in

PYRUVIC AND DIMETHYLPYRUVIC ACIDS 197

TABLE I

The Respective Effects of Acetate, Ethylene Glycol, Glycolic Acid~ Glyoxylic Acid, and Propionic Acid on the Formation of PA and of DMPA from Glycerol

Substrate

Molecular ratio of glycerol to second substrate

Glycerol (5% w./v.) alone Glycerol d- acetate 3 : 1 Glycerol d- ethylene glycol 4:1 Glycerol d- glycolic acid 8:3 Glycerol d- glyoxylic acid 10:1

Glycerol -~ sodium propionate 3 : 1

Products PA DMPA

mg./l, mg./l. 57 316

1120 745 437 506

80 410 Not 221

estimated Nil Nil

this experiment. I t was necessary to employ the acetic and propionic acids in the form of salts in order to render them available to the mold. In the flasks containing pro- pionate, al though no keto acids were formed, the mold remained quite healthy. In the flasks containing glyoxylic acid i t was not possible to estimate pyruvic acid because the former acid interfered with the estimation.

Expt. 2. The Respective Effects of Acetate, Ethanol, and Pyruvate on the

Formation of Pyruvic and Dimethylpyruvic Acids from Glycerol

This experiment was conducted exactly as in the case of Expt. 1. Glycerol (5% w./v.) was used as control and, per 100 ml., the other components were sodium acetate 3.7 g., ethanol 1.25 g., and sodium pyruvate 1 g. The products were estimated on the 9th day after replacement of the initial medium. The results (Table II) indicate t h a t ethanol is either without effect or t ha t possibly it exercises an influence slightly prejudicial to the formation of bo th acids, while pyruvic acid as the sodium salt exerts an extremely detr imental effect, suppressing the formation of PA entirely and reducing tha t of D M P A to a very small quant i ty compared with tha t formed in the pyruvic acid-free control.

TABLE I I

The Respective Effects of Acetate, Ethanol, and Pyruvate on the Formation of PA and of DMPA from Glycerol

Substrate

Molecular ratio of glycerol to second substrate

Glycerol (5% w./v.) alone Glycerol ~ acetate 2 : 1 Glycerol -~ ethanol 2 : 1

Glycerol -~ pyruvate 6:1

Products PA DMPA

mg./l, rag.~1. 70 235

997 558 32 212

Not estimated 34

198 K. RAMACHANDRAN AND T. K. WALKER

Expt. 3. Formation of Pyruvic Acid and of Dimethylpyruvic Acid from

Glycerol in the Presence of Glycine

The technique employed was similar to t ha t adopted in Expts . 1

and 2. Glycerol was used a t the same concen t ra t ion as in the previous cases, glycine was added at 1.5 g/100 ml. to give a molecular rat io of glycerol: glycine of 5:2, and the molecular rat io of glycerol: ace ta te

was likewise ad ius ted at 5:2. Resul t s are shown in Table I I I . While in

Expt . 3 aceta te again exercised p ronounced s t imu la t i on of acid forma- t ion, glycine migh t be considered to be somewhat un favorab le to the fo rmat ion of D M P A .

TABLE III

The Respective Effects of Acetate and Glycine on the Formation of PA and of DMPA from Glycerol

Molecular ratio of glycerol to second Products

Substrate substrate PA DMPA m~. / l. mo.ll.

Glycerol (5% w./v.) alone 31 178 Glycerol -I- acetate 5 : 2 245 527 Glycerol + glycine 5:2 28 130

Expt. 4. Formation of Pyruvic and Dimethylpyruvic Acids from

Erythritol and from Erythritol Plus Acetate

Erythitol as substrate was tested under the conditions adopted in the previous experiments, the concentration of the erythritol in the medium being 2% (w./v.). At the end of 9 days only traces of the two keto acids could be detected in the metab- olism solution. When sodium acetate (2% w./v.) was added to the erythritol medium, both keto acids were formed in quantities which, while still very small, were yet sufficient to permit isolation, the yield of PA being 12 mg. and that of DMPA being 28 mg., in each case per liter.

Expt. 5. Formation of Pyruvic and Dimethylpyruvic Acids from Glucose

in the Presence of Cyani~te, Fluoride, and of

Iodoacetate, Respectively

The mold was first developed from spores sown, as in the previous experiments, on the solution of glucose in Currie's salt medium, but, in this experiment, conical flasks of 1.5 I. capacity were employed. These flasks were 4 cm. wide at the neck and each was charged with 250 ml. of medium. After 3 days at 30 ~ thick felts had formed in all the flasks and the medium was replaced by sterile water. After 12 hr.

PYRUVIC AND DIMETHYLPYRUVIC ACIDS 199

at 30 ~ the water was poured away and the several new media were introduced beneath the felts, each medium being divided between six flasks. These four media were the control consisting of glucose (5% w./v.) in Hida's solution of salts and the same with the addition, respectively, of sodium cyanide (0.02 M), sodium fluoride (0.003 M) and sodium iodoacetate (0.002 M). All the sets of flasks were then returned to the incubator and samples were withdrawn for examination after 3, 6, and 8 days. On the 8th day the liquid contents of each set of flasks were analysed and the results are shown in Table IV. I t should be mentioned that the distillates from the cultures

TABLE IV

The Respective Effects of 0.02 M Cyanide, 0.003 M Fluoride, and 0.002 M Iodoacetate, on the Formation of PA and of DMPA from Glucose

Substrate

Glucose (10% w./v.) alone Glucose -1- 0.02 M cyanide Glucose -I- 0.003 M fluoride Glucose -I- 0.002 M iodoacetate

Products PA DMPA

rag.//, mg./Z. 1119 712 Trace 167 1139 973 1099 804

containing fluoride and iodoacetate were each found to yield, in addition to both keto acids, a small quantity of a third substance which gave a derivative with 2,4-dini- trophenylhydrazine. This last derivative was insoluble in boiling sodium carbonate solution and also in ethanol and it melted at 285 ~ with decomposition. I t seems prob- able that it was the 2,4-dinitrophenylosazone of glyceraldehyde (or dihydroxyacetone) and its formation under these conditions will be further studied.

D I S C U S S I O N

Our or ig ina l sugges t ion (8) t h a t t he p r o d u c t i o n of D M P A f rom g lycero l b y A. niger occurs b y w a y of a sequence of changes which c o m m e n c e s w i th a c o n d e n s a t i o n b e t w e e n a molecu le of t r iose a n d one of ace t ic ac id led us to cons ider t h e f u r t h e r pos s ib i l i t y t h a t g l y c o l a t e or g l y o x y l a t e m i g h t t a k e the p lace of a c e t a t e in t h e p r e s u m e d conden- s a t i on w i th t r iose , because A. niger is k n o w n (2) to be c a pa b l e of effect ing t h e changes :

A c e t a t e --~ g lyco l a t e --* g l y o x y l a t e --* oxala te .

Th i s p r e s u m p t i o n is s u p p o r t e d b y t h e resu l t s of E x p t . 1 in which i t is shown t h a t in p resence of glycol ic ac id a n d also in t he presence of e t h y l e n e g lycol t h e y ie ld f rom g lycero l of b o t h P A a n d D M P A is a u g m e n t e d . T h e p a r t i c i p a t i o n of glycol ic ac id in t he syn thes i s of di - m e t h y l p y r u v i c ac id m i g h t be f o r m u l a t e d as fol lows:

200 K. RAMACHANDRAN AND T. K. WALKER

CH~'0H CH2"0H L I -m0

HO'CH2--CO+CH2(OH)--COOH "* HO'CH2--C(OH)--CH(OH)--COOH

CH~' OH CH2' OH I ! -H~.O

HO'CH2--C~C(OH)--COOH ~ HO'CH.~--CH CO'COOH

CH2 CH2 II C H ~-2H II HO'CH2--C--CO" OO ) HO'CH~--C--CH(OH).COOH --*

CH~ CH3 -toO [

HO'CH~-- H--CO'COOH ~ CH~C--CO.COOH +2H - )

CH3 CH3

CH2:C--CH(OH)'COOH ~ CH~--CH--CO'COOH

In the case of ethylene glycol one could suppose prior conversion of the latter to glycolaldehyde or to glycolic acid, followed by a reaction sequence leading to DMPA. The conversion of ethylene glycol to gly- colaldehyde has already been shown to occur in cultures of Fusarium lini Bolley (4) and also under the influence of Acetobacter acetigenum (Henneberg), Bergey et al. (7). Condensation of dihydroxyacetone with glycolaldehyde might be expected to give rise to the changes:

CH2OH CH~- OH I I +H~O

) HO.CH~--C=C(OH).CHO ~ HO.CH~--CH--CO.CHO oxidation-reduction

CH~" OH CH2 f H

HO.CH~--CH--CH(OH)--COOH -H20 HO--CH2--C--CH(OH)--COOH

and from this stage the reactions could be presumed to proceed as postulated in the case of glycolic acid, shown above.

The participation of glycolaldehyde in certain vital syntheses has been considered as likely in several instances, and the conversion of ethylene glycol to cellulose by way of glycolaldehyde, as demonstrated recently by Jowett, Kaushal, and Walker (6), was the first experi- mental realization of one of these syntheses. Recently, Birkinshaw and Morgan (1) have isolated the odorous substances methylheptenone (2-methylhept-2-en-6-one) and isobutyl acetate from the fungus Endo- conidiophora coerulescens (M~inch). In view of our results the suggestion may be made that this methylheptenone has its origin in reactions similar to those leading to DMPA, since triose and glycolaldehyde

P Y R U V I C AND D I M E T H Y L P Y R U V I C ACIDS 201

together with acetone could be regarded as possible ingredients for a methylheptenone synthesis. Further, isobutyl alcohol might be derived from DMPA by way of the changes:

CH~ CH~ CH~ I - c o 2 I +2H I

CH~--CH--CO--COOH ) CH~--CH--CHO ) CH~--CH.CH2.OH

followed by esterification with acetic acid.

In our previous communication on the formation of PA and of DMPA we expressed the view that when glucose is the substrate the zymase system of the mold first comes into operation and gives rise to triose and to pyruvic acid and that, from the latter, there is then produced acetic acid or some other 2-carbon metabolite. We assumed further that condensation between the latter and a molecule of triose would yield a 5-carbon acid which, by undergoing a sequence of changes, would be converted to DMPA. Now fluoride at its effective concen- tration inhibits enolase by forming a protein-Mg-fluorophosphate com- plex compound (12), and the addition of fluoride to media undergoing alcoholic fermentation causes accumulation of phosphoglyceric acid. Iodoacetate, even at low concentrations, is an inhibitor of glycolysis and, by its ability to attack the activating protein for phosphoglyceral- dehyde dehydrogenase (9), it can arrest the production of pyruvate in the normal Embden-Meyerhof scheme at an earlier stage than that at which fluoride operates to produce the same effect. Therefore, since the iodoacetate-resistant respiration of A. niger cannot involve the par- ticipation of phosphoglyceraldehyde dehydrogenase, it follows that the formation of PA in Expt. 5 must have been effected in some manner other than that by which it is produced from phosphoglyceric acid in the Embden-Meyerhof scheme. Possibly it arose by oxidation of meth- ylglyoxal formed from triose. Cyanide, the respiratory inhibitor used in Expt. 5, completely suppressed the formation of PA and considerably reduced the yield of DMPA. This may be an indication that an oxida- tion is involved in one of the preliminary steps leading to the formation of DMPA and that the mold is mainly aerobic in its action. The reason for the suppressiofl of formation of PA by the cyanide is not clear, especially in view of the fact that cyanide at the same concentration, namely 0.02 M, increases considerably both the assimilation of glucose and the formation of citric acid from glucose by the same strain of A. niger as was employed in the present investigations (11). Cyanide

202 K. RAMACHANDRAN AND T. K. WALKER

inhibits the functioning of many different enzymes and it is evident that its action on those of A. niger would repay investigation. On the whole, the evidence gained in Expt. 5 by the use of these three enzyme inhibitors, would seem to indicate that the formation of DMPA from carbohydrate by A. niger may proceed by more than one pathway, and this opinion is strengthened by an observation made very recently in this laboratory by A. N. Hall and J. W. Hopton working in association with one (T.K.W.) of us. They find that when A. niger is allowed to act upon ethyl acetoacetate or upon a mixture of the latter with acetate, in Hida's medium, pyruvic, dimethylpyruvic, and a-ketoglutaric acids are all formed. This discovery, which will be published in detail else- where, points to the formation of DMPA by way of a preliminary condensation between acetoacetic acid and acetate yielding

CH3- CO. CH~- COOH CH~. C. CH2. COOH

CH~. COOH H. COOH

a 6-carbon dicarboxylic acid. This, in turn, could give rise either by decarboxylation, to ~-methylcrotonic acid or, by decarboxylation and reduction, to ~-methyl-n-butyric acid, from either of which DMPA might be derived. In this connection it is of interest to compare recent investigations of Tatum and Adelberg (10) who, using labeled acetate, have demonstrated the syntheses of the carbon skeletons of isoleucine and valine from acetate.

SUMMARY

1. The formation both of pyruvic and dimethylpyruvic acids from glycerol by preformed felts of the mold Aspergillus niger, when placed on a glycerol solution containing NH~C1, KH2P04, and Na2SO3, is considerably enhanced when ethylene glycol is added to the medium, and is also enhanced, though to a less extent, by addition of glycolic acid. In neither case, however, is the increase of yield as great as are the increases produced by addition of sodium acetate to the culture fluid.

2. In similar conditions, the addition of ethanol" depresses the for- mation of pyruvic acid but has not much effect upon that of dimethyl- pyruvic acid.

3. Addition of sodium pyruvate to the glycerol medium reduces the formation of dimethylpyruvic acid to an insignificant quantity, while

PYRUVIC AND DIMETHYLPYRUVIC ACIDS 203

sodium propionate prevents entirely the formation of both pyruvic and dimethylpyruvic acids.

4. Aspergillus niger forms pyruvic and dimethylpyruvic acids in very small yields from erythritol in Hida's medium, and addition of acetate increases somewhat the yield of both acids.

5. Formation of both keto acids from glucose in Hida's medium is not influenced by addition thereto either of sodium fluoride (0.003 M) or of sodium iodoacetate (0.002 M). Addition of sodium cyanide (0.02 M) to this glucose medium reduces the yield of pyruvic acid to a trace and that of dimethylpyruvic acid to less than one-quarter of the normal yield.

6. Suggestions are made concerning the manner in which pyruvic acid and dimethylpyruvic acid may have been formed under the experi- mental conditions cited in this communication.

REFERENCES

1. BIRKINSgAW, J. H., AND MORGAN, E. N., Biochem. J. 47, 55 (1950). 2. CHALLENGER, F., SUBRAMANIAM, V., AND WALKER, T. K., J. Chem. Soc. 200,

3044 (1927). 3. CURRIE, J. N., J. Biol. Chem. 31, 15 (1917). 4. GOEPFERT, G. J., AND NORD, F. F., Arch. Biochem. 1, 289 (1942). 5. HIDA, T., J. Shanghai Sci. Inst. Sec. IV, 1, 201 (1935). 6. JowE~, P., KAUSHAL, R., AnD WALKER, T. K., Nature 167, 949 (1951). 7. K.~USHAL, R., AND WALKER, T. K., Nature 160, 572 (1947). 8. RAMACHANDRAN, K., AND WALKER, T. K., Arch. Biochem. Biophys. 31, 224

(1951). 9. RAPKINE, L., Biochem. J. 32, 1729 (1938).

10. TATUM, E. L., AND ADELBERG, E. A., J. Biol. Chem. 190, 843 (1951). 11. WALKER, T. K., Advances in Enzymol. 9, 569 (1949). 12. WAR~URG, O., AND CHRISTIAN, W., Naturwiseenschaften 29, 589 (1941).