Acid in Yeast. and DL-a-aminoadipic acid (AAA) were synthesized. in this laboratory by Dr. John A. Brockman, Jr., Dr.

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1 Biosynthesis of Lysine from a-ketoadipic Acid and oa-aminoadipic Acid in Yeast HARRY P. BROQUIST,' ARTHUR V. STIFFEY, AND ALBERTA M. ALBRECHT2 Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York Received for publication February 24, 96 Studies of the nutrition of certain lysine requiring mold mutants indicate that L-a-aminoadipic acid (AAA) or a-ketoadipic acid (KAA) are precursors of lysine. For example, Mitchell and Houlahan (948) found that certain lysine requiring mutants of Neurospora crassa would respond to either lysine or AAA, and Windsor (9) subsequently demonstrated that f-c'4-a-aminoadipic acid was completely incorporated into lysine in one such mutant. Lysine requiring mutants of Ophiostoma (Bergstrom and Rottenberg, 9) utilize either KAA or AAA for growth in lieu of lysine. The pathway of lysine biosynthesis in yeast has been studied by Strassman and Weinhouse (93); from the manner in which acetate is incorporated into lysine of the proteins of Torulopsis utilis, a scheme compatible with the idea that KAA and AAA could be precursors of lysine in yeast was proposed for the origin of KAA from carbohydrate metabolism. Current efforts to produce L-lysine commercially either by chemical or biological means such that it might be economically feasible to fortify certain foodstuffs deficient in this amino acid with L-lysine (e.g., see Howe, 97) prompted us to consider the possibility that microorganisms might be found having a high capacity to produce lysine when grown with adipic acid derivatives. After a brief screening program involving several hundred yeasts, molds, and bacteria, it became evident that many common yeasts, particularly certain strains of Saccharomyces and Torulopsis, have a marked capacity to convert KAA or AAA to lysine. Such lysine is found in the yeast cell but is apparently not bound to protein, as it is readily extracted from the cells with hot water; such yeasts may contain as much as 2 per cent of their dry weight as lysine. These findings have both practical and theoretical interest, for they suggest a possible means of producing lysine and lend strong support to an "adipic acid pathway" of lysine biosynthesis in yeast. A brief account of this work has appeared elsewhere (Broquist and Stiffey, 99). Present address, and address to which reprint requests should be sent: Department of Dairy Science, University of Illinois, Urbana, Illinois. 2 Present address: Institute of Microbiology, Rutgers University, New Brunswick, New Jersey. MATERIALS AND METHODS Adipic acid derivatives. a-ketoadipic acid (KAA) and DL-a-aminoadipic acid (AAA) were synthesized in this laboratory by Dr. John A. Brockman, Jr., Dr. Milon Bullock, and their associates. Microbiological procedures. Saccharomyces cerevisiae strains Y-8, Y-4, and Y-9 were obtained from Dr. Homer Tresnor of these laboratories. Torula utilis (Torulopsis utilis) strain Y-9 was obtained from Dr. Lynferd Wickerham, Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, Illinois. Commercial baker's yeast was purchased locally. These cultures were maintained on malt agar slants. Unless otherwise indicated in the tables, shake flasks experiments were carried out as follows: 24-hr malt agar slant cultures were suspended in ml sterile saline and.3 ml of this suspension used to inoculate ml of sterile media and test substance in 2-ml Erlenmeyer flasks. The flasks were placed on a reciprocating shaker and incubated for 72 hr at 28 C. The flasks were then steamed for min, readjusted to ml with water, centrifuged, and the clear supernatant assayed for L-lysine employing Leuconostoc mesenteroides strain P-6 using the medium and procedure recommended by Difco Laboratories, Inc. (93). All results are expressed as L-lysine monohydrochloride. The medium used in the shake flask studies was either a synthetic medium that has previously been described (Broquist, 97) and which contains ammonium sulfate as the principal form of nitrogen; or a crude cornsteep liquor medium consisting of cornsteep liquor, per cent; glucose, per cent; and ammonium sulfate,.38 per cent. The medium, plus appropriate additions, was adjusted to ph.3, dispensed, and autoclaved min at 2 C, cooled, and inoculated. Assay procedures. The yield of yeast was determined either by dry weight determinations of washed cultures or simply by centrifuging whole cultures in appropriately calibrated tubes from which the yield could be read directly. Glucose was measured by the method of Somogyi (94). A diet low in lysine, suitable for study of the growth response of chicks to lysine supplementation, was devised as follows: yellow corn, fine ground, 6 per cent; Downloaded from on January 24, 29 by guest

2 2 H. P. BROQUIST, A. V. STIFFEY, AND A. M. ALBRECHT [VOL. 9 sesame meal, per cent; cottonseed meal, per cent; Cerelose, 3 per cent; steamed bone meal, 2 per cent; mineral salts, 2 per cent; calcium carbonate, per cent; B-complex vitamins with Cerelose, per cent; vitamins A, D, and E in corn oil, per cent; and choline chloride,.2 per cent. One-day-old Silver Cross chicks were divided into groups of 2, placed in an electrically heated brooder, and fed the basal diet containing various supplements as indicated. The weight of the chicks and number of survivors were recorded at intervals during a 3-day assay period. RESULTS The percentage of molar conversion of KAA and AAA to lysine when certain yeasts are grown in synthetic or cornsteep liquor media under the conditions described, is illustrated in table. Experiment 3, table, may be regarded as typical, in that in general the efficiency of conversion of these intermediates to lysine is higher from KAA. Possibly this is related to a favored difference in permeability of the yeast cell to KAA rather than AAA. When KAA was added to the cornsteep liquor medium at levels of to mg per ml, it was converted to lysine to the extent of to 7 per cent by these yeasts. The efficiency of conversion was much less when the yeasts were grown in synthetic medium, probably an indication that nutritional factors, as yet unknown, in the crude medium are contributing to lysine biosynthesis from KAA and AAA. Experiment 4, table illustrates that a critical level of cornsteep liquor is required for optimum lysine formation from KAA. Although the yeast grew well at all concentrations of cornsteep liquor tested, the per cent level was inadequate for optimum lysine formation; 3 per cent and per cent cornsteep liquor favored lysine synthesis, whereas the 7 per cent level markedly inhibited synthesis. These effects of cornsteep liquor are of interest and require further study. The relationship of lysine biosynthesis to yeast growth and metabolism is given in figure. A series of shake flasks containing cornsteep liquor medium and mg per ml KAA was inoculated with Saccharomyces cerevisiae (Y-8) and duplicate flasks withdrawn from the shaker at various time intervals up to 2 hr of incubation. When this series of cultures was assayed for lysine content, for reducing sugar remaining in the medium, and for extent of yeast growth, the results were as plotted in figure. It can be seen that growth increased rapidly up to 36 hr with a concomitant drop in reducing sugar in the medium, and then leveled off for the remainder of the experiment. Lysine synthesis, however, proceeded much more slowly than the growth rate; for example, the maximum yield of lysine formed from KAA was not obtained until 84 hr, whereas only a third of this amount of lysine had been formed at 36 hr when growth had about reached a maximum. In the experiments described up to this point lysine assays were carried out on whole yeast cultures which had been steamed (cf. Materials and Methods). The experiment outlined in table 2 was designed primarily to determine whether lysine formed from KAA or AAA by growing yeast cultures was present in the spent medium or in the yeast cells. If the 48-hr data of table 2 are considered, for example, it can be seen that the lysine formed from the adipic acid intermediates is present only in heated cultures, demonstrating that it is present exclusively in the yeast cell, but it can be readily extracted from the cell by hot water. If the data with heated cultures are considered for the 3-day experimental period (table 2), further evidence is presented for a delayed synthesis of lysine from KAA and Expt No. it 4 TABLE Lysine biosynthesis from DL-a-aminoadipic acid and a- ketoadipic acid by yeast under various nutritional conditions Medium* 3% 3% 3% % 3% % 7% Adipic Acid Precursor mg/ml L-Lysine*HCI Produced From HOOC (CH2)3 CHCOOH Amt. AL mg/ml NH, Molar conversiont HOOC(CH2)3CCOOH Amt. mgml Molar conversion -/n a/ * = synthetic medium; = cornsteep liquor medium (cf. text, Methods and Materials). t Calculated on the assumption that only the L-enantio morph of DL-a-aminoadipic acid is utilized. t Saccharomyces cerevisiae (Y-8). Torulopsis utilis (Y-9). I/ Downloaded from on January 24, 29 by guest

3 BIOTHESIS OF LYSINE t \ I " Time -Hours Figutre. Relationships between cell growth, sugar utilization, and lysine biosynthesis by Saccharomnyces cerevisiae (Y-8) in corn steep liquior medium containing mg/ml a-ketoadipic acid. AAA in these cultures. The fact that lysine synthesized from KAA or AAA by yeast is found in the cell (table 2) should permit an unusually high production of lysine in yeast. This is illustrated by the data shown in table 3. When two different baker's yeasts were grown with or without KAA, there was no significant difference in the yield of yeast obtained (step 2), but the yeasts grown in the presence of KAA contained 7.3 per cent and 8.8 per cent of their dry weight as water soluble lysine (step 3). When the amount of lysine present in the cells as protein lysine is considered (step 4), it may be seen that the effect of growing commercial baker's yeast with KAA is to produce a yeast containing over 2 per cent of its total weight as lysine (step ). The assumption that the water soluble "lysine" in such cells, which supports growth of Leuconostoc mesenteroides in lysine-deficient medium, is authentic lysine was confirmed by paper chromatography and chick assay. Since the amount of "lysine" in yeast grown with KAA is so great in comparison to other amino acids present in heated yeast extracts, paper chromatography of water extracts of such yeasts and subsequent treatment of the chromatograms with ninhydrin gives only one predominant ninhydrin spot. This spot coincided with authentic lysine when an extract of highlysine yeast was chromatographed in four different solvent systems (table 4). Moreover, duplicate chromatograms of such yeast extracts when bioautographed with L. mesenteroides in lysine free media gave a growth zone agreeing with the position of free lysine. TABLE 2 Evidence for intracellular synthesis of lysine from adipic acid intermediates by Saccharomyces cerevisiae cultures 3 I- Lysine Content following Incubation for: S. cere- 24 hr 48 hr 72 hr visiae Adipic Acid Precursor Strain No No SI heat* c heat heat mglml mg/ml mg/ml mg/ml a-ketoadipate Y Y DL-a-AminlOadipate Y Experimental: Yeasts were grown in a series of 2-ml shake flasks in ml cornsteep liquor medium to which was added the indicated adipic acid precursor. Flasks were removed at daily intervals and the whole culture either steamed at C for min or not heated. The cultures were then centrifuged and the lysine content of the yeast supernatant determined by microbiological assay. * Treatment of culture prior to assay. When chicks were placed on a natural diet marginal in lysine content, the chicks did not give a growth response to the lysine precursors, KAA and AAA, at the levels tested (experiment A, table ); but yeast Downloaded from on January 24, 29 by guest

4 4 H. P. BROQUIST, A. V. STIFFEY, AND A. M. ALBRECHIT [VOL. 9 TABLE 3 Examples of the preparation of a "high lysine" bakers' yeast Step No. and Treatment of Yeast Cells Baker' yeast SI Grown With: Bakers' Yeast N 2 Grown With: No KAA mg/ml KAA No KAA mg/ml KAA. Whole yeast culture centrifuged; lysine content of supernatant determined by microbiological assay. 96 -y/ml 23 -y/ml 83 oy/ml 96 oy/ml 2. Cells from () resuspended in original volume with water, lyophilized and weighed. Yield of yeast g/l 6 g/l 6 g/l 7.3 g/l 3. Autoclaved an aliquot of yeast suspension (2 above), centrifuged and assayed supernatant for lysine. Lysine in yeast suspension, a/ml or as % dry weight of yeast... -y/ml;.9% 277 -y/ml; 7.3% 8 oy/ml;.2% 32 -y/ml; 8.8% 4. Insoluble cell debris from (3) was autoclaved 2 hr with 2. N HCI, and lysine determined on the hydrolyzate. Lysine content as 7% dry weight of yeast. 3.4% 3.7% 3.2% 3.9%. Total water soluble lysine plus protein lysine in yeast cells (3 plus 4) as % dry weight of yeast. 4.3%2.% 4.4% 22.7% Experimental: Two strains of bakers' yeast used commercially were grown in a series of 2-ml shake flasks in -ml cornsteep liquor medium either with or without a-ketoadipic acid (KAA). After 72 hr incubation, the yeast culture was treated as described in the table. TABLE 4 Identification of lysine in yeast by paper chromatography* Descending Rf Solvent System Chro- Pure Rf "Yeast Lysine" by: matog- Lysine NinhY- Bioraphy Nrinhy autog- Time drn raphy Pyridine-acetic acid-water (:3: by vol) Phenol-water (4: by vol) Butanol-water-acetic acid (2: 2:6, upper layer) Methylethylketone-acetic acidwater (9:2:3 by vol) * Paper chromatography on Whatman No. paper was carried out at 2 C in an all glass apparatus following the general procedure described by Block et al. (98). grown with the KAA precursor gave the expected growth response in terms of its content of water soluble lysine (experiment B, table ). The slight growth response of chicks to the control yeast grown in the absence of KAA probably reflects some growth response to lysine in the yeast proteins. Further work is required, however, to establish quantitatively whether the lysine produced from KAA by yeast growth is completely available to meet the chick's requirement for this amino acid. DISCUSSION The finding that Saccharomyces cerevisiae and Torulopsis utilis strains have an outstanding capacity for synthesis of lysine when grown in the presence of KAA or AAA provides evidence for an "adipic acid pathway" as a major route of lysine biosynthesis in yeast. These hr TABLE Growth response of chicks to lysine as supplied by yeast grown in a-ketoadipic acid (KAA) medium Avg Weight of Chicks and Expt No. Chick Group Supplement to Low Lysine Diet* Survivors at: days 7 days days 3 days g/kg g g g g A None 32 6" 76" 87" A 2 2 g L-lysine A 3 g L-lysine "2 7"2 322 A 4 g a-ketoadipic acid 2 6" "2 A g DL-a-aminoadipic acid B None B 2 4 g L-lysine 63 7 " 26" B 3 27 g dry yeast grown without KAA, contained.27 g L-lysinet "2 9"2 B 4 27 g dry yeast grown with KAA, contained 3.43 g L-lysinet "2 "2 Downloaded from on January 24, 29 by guest * A natural diet low in lysine in which the amino acids were obtained from yellow corn, sesame meal, and cottonseed meal; cf. text, Methods and Materials, for details. t As determined by microbiological assay. studies differ from the work with mold mutants discussed above in that these yeasts do not require KAA, AAA, or lysine for growth; and the amount of lysine that can be formed from these intermediates is far in excess of what would be expected for their metabolism. The experiments illustrated by figure and table 2 revealed that lysine biosynthesis from KAA markedly lagged behind yeast growth. These observations suggest

5 96] BIOTHESIS OF LYSINE that lysine synthesis might be carried out by resting cells; indeed, while this work was in progress a report appeared by Sagisaka and Shimura (97) showing that resting cells of Torulopsis utilis convert AAA to lysine in the presence of glucose and oxygen. Detailed studies of the biochemical transformations by which KAA and AAA are subseouently converted to lysine are in progress. It was shown (table 3) that when baker's yeast is grown with adequate KAA, it is possible to produce a yeast having more than 2 per cent of its dry weight as lysine, and such "yeast lysine" will meet the nutritional requirement of the chick for this amino acid (table ). By proper selection of a yeast strain that is efficient in the conversion of KAA to lysine and which possesses additional desirable characteristics including edibility, proficiency in synthesizing vitamins, etc., it might be possible to produce a yeast product of high nutritional value desirable for the fortification of certain foods. Alternatively, since the lysine formed from KAA is readily extracted from the yeast cells by hot water, the amino acid could be isolated from such extracts in pure form. KAA, if it could be produced cheaply, either by synthesis or by biological means, has a practical advantage over AAA as a lysine precursor in that it is not optically active, but can be utilized by yeast to carry out an asymmetric synthesis leading to L-lysine. SUMMARY When strains of Saccharomyces cerevisiae and Torulopsis utilis were grown for 2 to 3 days in shake flasks in crude cornsteep liquor medium containing to mg per ml a-ketoadipic acid (KAA), L-lysine was formed with an efficiency of to 7 per cent; similar results were obtained with higher levels of DL-a-aminoadipic acid (AAA). Such "lysine" was found in the yeast cells and had the following properties: (a) it was utilized as lysine for growth of lysine-deficient chicks; (b) it was readily extracted with hot water; (c) such water extracts were active for growth of Leuconostoc mesenteroides in lysineless media; and (d) paper chromatography of such extracts in diverse solvent systems gave zones indistinguishable from authentic lysine. When baker's yeasts were grown in KAA medium, yeasts were harvested having over 2 per cent of their total dry weight as lysine. It is suggested that such high lysine yeasts might be a desirable source of lysine for fortification of certain foods deficient in this amino acid. REFERENCES BERGSTROM, S. AND ROTTENBERG, M. 9 Utilization of a-ketoadipic acid by lysineless Ophiostoma mutants. Acta Chem. Scand., 4, 3. BLOCK, R. J., DURRUM, E. L., AND ZWEIG, G. 98 Paper chromatography and paper Electrophoresis, p. 23 Academic Press Inc., New York, New York. BROQUIST, H. P. 97 Evidence for the involvement of folic acid in histidine synthesis in microorganisms. Arch. Biochem. Biophys., 7, BROQUIST, H. P. AND STIFFEY, A. V. 99 Biosynthesis of lysine from adipic acid precursors by yeast. Federation Proc., 8, 98. Difco Laboratories, Inc. 93 Difco manual of dehydrated culture media and reagents, Ed. 9. Detroit, Michigan, p HOWE, E. E. 97 Lysine. Drug & Cosmetic Ind., 8, , 26, 32. MITCHELL, H. K. AND HOULAHAN, M. B. 948 An intermediate in the biosynthesis of lysine in Neurospora,. J. Biol. Chem., 74, SAGISAKA, S. AND SHIMURA, K. 97 Studies on lysine biosynthesis. Part. Lysine formation in yeast cells. Agr. Soc. of Japan, 3(2), -4. SOMOGYI, M. 94 A new reagent for the determination of sugars. J. Biol. Chem., 6, STRASSMAN, M. AND WEINHOUSE, S. 93 Biosynthetic pathways. III. The biosynthesis of lysine by Torulopsis utilis. J. Am. Chem. Soc., 7, WINDSOR, E. 9 a-aminoadipic acid as a precursor to lysine in Neurospora. J. Biol. Chem., 92, Downloaded from on January 24, 29 by guest